Inligting

Hoekom is hierdie oksoglutaraat dehidrogenase en nie oksoglutaraat dekarboksilase nie?

Hoekom is hierdie oksoglutaraat dehidrogenase en nie oksoglutaraat dekarboksilase nie?



We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Ek het na die oksoglutaraat-dehidrogenase-kompleks gekyk en die reaksiemeganisme vir sy E1-TPP-meganisme gesien, wat lei tot die vorming van 'n gestabiliseerde karbanion-tussenproduk.

Die meganisme begin met 'n protonasie, maar aangesien die "doel" van die ensiem is om die karbanion-tussenproduk te vorm, lyk dit vir my of die dekarboksilering die sleuteldeel van die reaksie is. So hoekom is dit 'n dehidrogenase-gekataliseerde reaksie en nie 'n dekarboksilase-gekataliseerde een nie?


Ek is nuut in sitroensuursiklus en ek beoordeel hierdie vraag op grond van my vorige kennis van biochemie en organiese chemie. Ek het na verskeie proteïendatabasisse gekyk en skokkend genoeg lyk dit of oksoglutaraatdehidrogenase die korrekte naam daarvoor is.( Sien: Uniprot, PDB 1, PDB 2) Alhoewel die nomenklatuur van biologiese ensieme soms nie vir my reg klink nie, glo ek hierdie benaming is geskik vir hierdie molekule. Kyk eers na hierdie definisies:

(Definisies van Oxford Languages ​​/ Google)

Dehidrogenase: 'n ensiem wat die verwydering van waterstofatome uit 'n bepaalde molekule kataliseer, veral in die elektrontransportkettingreaksies van selrespirasie in samewerking met die koënsieme NAD en FAD.

(Wikipedia)

Dehidrogenase (ook genoem DH of DHase in die literatuur) is 'n ensiem wat aan die groep oksidoreduktase behoort wat 'n substraat oksideer deur 'n elektronaannemer, gewoonlik NAD+/NADP+ of 'n flavienkoënsiem soos FAD of FMN, te reduseer.

(Wikipedia)

Dekarboksilering is 'n chemiese reaksie wat 'n karboksielgroep verwyder en koolstofdioksied (CO2) vrystel. Gewoonlik verwys dekarboksilering na 'n reaksie van karboksielsure, wat 'n koolstofatoom uit 'n koolstofketting verwyder

Hier is hoekom die gebruik van "dekarboksilase" in die naam van die ensiem verkeerd is:

Herroep amilase (Amylase-Wiki) . Daardie ensiem verbruik amilose as substraat en produseer maltose daaruit. Dit klink vreemd as mens dit "Demaltase" noem net omdat dit maltose uitdeel. In plaas daarvan, in hierdie geval, speel verbruik van amilose 'n meer belangrike rol in vertering.

Volgens bogenoemde definisies, die "doel" hier is bloot die beweging van H+ en die elektron na FAD (om FADH2 te genereer) en dan na NAD+ (Om NADH te genereer), nie die verwydering van CO2 nie. Dit is net 'n neweproduk wat voortspruit uit die meganisme. sien: Reaksie

Hier is hoekom die gebruik van "dehidrogenase" in die naam van die ensiem korrek is: Kyk bietjie hierna . Die hele proses behels die beweging van een proton vanaf tiamindifosfaat en een van CoA-SH en ook 2 elektrone van twee SH-bindings (ek het die waterstof vir jou opgespoor. Trek self die elektrone na! :) ) Herroep die konsepte van karboniel funksionele groep uit organiese chemie. Een manier om enige reaksies op karboniele uit te voer, is om 'n suur katalise wat die meeste van die kere is wat H+ uit H2SO4 of HCl ontstaan ​​het. Kyk bietjie hierna. Sien ook Morrison-boyd organiese chemie of Vollhardt se This H+ Aktiveer die karboniel funksionele groep, wat dit makliker laat deelneem aan nukleofiele substitusie. Die byvoeging van H+ tot Karboniel suurstof is omkeerbaar en daarom is dit belangrik om hierdie ewewig te optimaliseer. Die aangeduide ensiem werk op hierdie ewewig om die maksimum opbrengs te verkry. Boonop kan dit TPP veroorsaak om 'n H+ te verloor. Hierdie twee prosesse (die verwydering van H+ van TPP en die byvoeging van H+ tot oksogultaraat ) kan nie met behulp van die ensiem gedoen word nie, vandaar die naam Dehidrogenase vir die ensiem.


Hoekom is hierdie oksoglutaraat dehidrogenase en nie oksoglutaraat dekarboksilase nie? - Biologie

Heterogeniteit van die mitochondriale proteoom in plante onderlê fundamentele verskille in die rolle van hierdie organelle in verskillende weefsels. Ons het die mitochondriale proteoom wat uit 'n nie-fotosintetiese selkultuurmodel geïsoleer is, kwantitatief vergelyk met meer gespesialiseerde mitochondria wat uit fotosintetiese lote geïsoleer is. Verskille in ongeskonde mitochondriale respiratoriese tempo met verskeie substrate en aktiwiteite van spesifieke ensieme verskaf 'n agtergrond van die funksionele variasie tussen hierdie mitochondriale populasies. Proteomika-vergelykings het 'n diepgaande insig gegee in die verskillende bestendige-toestand oorvloede van spesifieke mitochondriale proteïene. Hierdie data gekombineer het die verhoogde vlak van die fotorespiratoriese apparaat en die komplekse wisselwerking daarvan met glikolaat-, sisteïen-, formiaat- en eenkoolstofmetabolisme getoon, asook die afname van geselekteerde dele van die trikarboksielsuursiklus, veranderinge in aminosuurmetabolisme gefokus op 2- oksoglutaraatgenerering, en afbraak van vertakte kettingaminosure. Vergelykings met mikroskikking analise van hierdie weefseltipes het 'n positiewe, ligte korrelasie tussen mRNA en mitochondriale proteïen oorvloed getoon, 'n strenger korrelasie vir spesifieke biochemiese weë, maar meer as 78% konkordansie in rigting tussen veranderinge in proteïen en transkripsie oorvloed in die twee weefsels. Oor die algemeen het hierdie resultate aangedui dat die meerderheid van die variasie in die plantmitochondriale proteoom in die matriks voorgekom het, die konstitutiewe aard van die respiratoriese apparaat uitgelig het, en die verskille in substraatkeuse en/of beskikbaarheid tydens fotosintetiese en nie-fotosintetiese metabolisme getoon het.

Gepubliseer, MCP Papers in Press, 1 April 2008, DOI 10.1074/mcp.M700535-MCP200

Hierdie werk is ondersteun deur Grant CE0561495 van die Australian Research Council (ARC) deur die Centers of Excellence Program en deur Wes-Australië se staatsregeringssteun aan die Centre for Computational Systems Biology. Die koste van publikasie van hierdie artikel is gedeeltelik deur die betaling van bladsykoste gedek. Hierdie artikel moet dus hiermee gemerk word "advertensie” in ooreenstemming met 18 U.S.C. Artikel 1734 uitsluitlik om hierdie feit aan te dui.

Alle mikroskikkingdata is beskikbaar by ArrayExpress onder toegangsnommer E-ATMX-31.


EC Explorer

1.2.1 Met NAD + of NADP + as aanvaarder (740 organismes) 1.2.2 Met 'n sitochroom as aanvaarder (12 organismes) 1.2.2.1 formaat dehidrogenase (sitochroom) (3 organismes) 1.2.2.2 piruvaatdehidrogenase (sitochroom) 1.2.2.3 formaat dehidrogenase (sitochroom-c-553) 1.2.2.4 koolstofmonoksieddehidrogenase (sitochroom b-561) (9 organismes) 1.2.3 Met suurstof as aanvaarder (136 organismes) 1.2.4 Met 'n disulfied as aanvaarder (87 organismes) 1.2.4.1 piruvaatdehidrogenase (asetiel-oordragend) (55 organismes) 1.2.4.2 oksoglutaraatdehidrogenase (suksiniel-oordragend) (39 organismes) 1.2.4.3 geskep 1972, geskrap 1978 1.2.4.4 3-metiel-2-oksobutanoaatdehidrogenase (2-metielpropanoiel-oordragend) (24 organismes) 1.2.5 Met 'n kinoon of soortgelyke verbinding as aanvaarder (43 organismes) 1.2.7 Met 'n yster-swaelproteïen as aanvaarder (148 organismes) 1.2.98. ongedefinieerd (12 organismes) 1.2.99 Met onbekende fisiologiese aanvaarders (24 organismes) 1.2.99.1 geskep 1961, geskrap 1984 1.2.99.2 koolstofmonoksied dehidrogenase (akseptor) 1.2.99.3 aldehieddehidrogenase (pirrolokinolien-kinoon) 1.2.99.4 formaldehied dismutase 1.2.99.5 formielmetanofuran dehidrogenase 1.2.99.6 karboksilaatreduktase (11 organismes) 1.2.99.7 aldehieddehidrogenase (FAD-onafhanklik) (6 organismes) 1.2.99.8 gliseraldehieddehidrogenase (FAD-bevattend) (4 organismes) 1.2.99.9 formaat dehidrogenase (koënsiem F420) 1.2.99.10 4,4&prime-diapolikopenoaat sintase (4 organismes) 1.16.1 Met NAD + of NADP + as aanvaarder (172 organismes) 1.16.3 Met suurstof as aanvaarder (73 organismes) 1.16.9 Met 'n koperproteïen as aanvaarder (3 organismes)

Inleiding

Sintetiese fosfonaatanaloë van piruvaat en 2-oksoglutaraat (OG) is spesifieke inhibeerders van die reaksies, gekataliseer deur sleutel regulatoriese ensieme, onderskeidelik die tiamiendifosfaat (ThDP)-afhanklike piruvaatdehidrogenase (PDH) en 2-oksoglutaraatdehidrogenase (OGDBunik) (OGD). et al., 2015, 2016 Artiukhov et al., 2016 Bunik, 2017). In hierdie analoë vervang die fosfonaatgroep die karboksielgroep wat die dekarboksilering ondergaan. Die interaksie van 2-oksofosfonate met verwante dehidrogenases lei meestal tot die nie-splitsbare tussenprodukte, wat die ensiemoorgangstoestand naboots (Kluger en Pike, 1979 Wagner et al., 2019). Die stywe, dog omkeerbare, binding van die fosfonate aan hul verwante dehidrogenases maak voorsiening vir spesifieke inhibisie van 2-oksosuur dehidrogenase in vivo (Bunik et al., 2013 Artiukhov et al., 2016).

Die bekende OGDH-Gekodeerde OGDH is 'n sleutelensiem in die mitochondriale trikarboksielsuur (TCA) siklus, waarvan die mutasies wat die funksie benadeel, onversoenbaar is met lewe (Bunik, 2017). Daarteenoor is die fisiologiese betekenis van die DHTKD1-gekodeerde isoënsiem 2-oksoadipaat dehidrogenase (OADH), wat oksidatiewe dekarboksilering van 2-oksoadipaat kataliseer (OA, Figuur 1A), die algemene tussenproduk van die lisien- en triptofaankatabolisme, is baie minder voor die hand liggend, aangesien die DHTKD1 mutasies bly dikwels ongemerk. Boonop, hoewel daar aanvaar word dat OADH as 'n komponent van die 2-oksoadipaat dehidrogenase multi-ensiemkompleks funksioneer, analoog aan die een wat deur OGDH gevorm word, is ons onlangse identifikasie van die DHTKD1-gekodeerde isovorme van OADH in soogdierweefsels het ons vorige voorspelling van die volgorde-analise ondersteun (Bunik en Degtyarev, 2008), dat OADH ook aktief kan wees in die geïsoleerde toestand, wat die nie-oksidatiewe dekarboksilering kataliseer (Boyko et al., 2020) . Die rol van so 'n funksie in ontgifting van aldehiede (Bunik en Fernie, 2009) kan die assosiasie van die disreguleerde onderlê. DHTKD1 uitdrukking met diabetes, vetsug en kanker (Lim et al., 2014 Wu et al., 2014 Kieᐫus et al., 2015 Plubell et al., 2018 Timmons et al., 2018).

Figuur 1. Strukture van die dikarboksiel-2-oksosure (A) sowel as die ooreenstemmende fosfonaatanaloë (B) en hul membraandeurlaatbare voorlopers (C), in hierdie werk bestudeer.

Ten einde die fisiologiese betekenis van die reaksies wat deur die isoensieme van 2-oksoglutaraatdehidrogenase gekataliseer word, gekodeer deur die OGDH en DHTKD1 gene, in vivo, is 'n homoloë reeks van die fosfonaatanaloë van dikarboksiel-2-oksosure (Figuur 1B) gesintetiseer (Artiukhov et al., 2020), naamlik die suksiniel (SP), glutariel (GP) en adipoiel (AP) fosfonate. Sellulêre eksperimente het spesifieke werking van die verbindings op metabolome getoon, wat saamval met hul inhibisie van die OGDH- en OADH-reaksies, gekataliseer deur die gedeeltelik gesuiwerde OGDH en OADH. 'n Aantal ander ensiematiese reaksies, wat 2-oksosure gebruik, of hul strukturele analoë, word nie deur die fosfonate beïnvloed nie (Artiukhov et al., 2020).

Hierdie belowende bevindinge regverdig 'n gedetailleerde studie van die molekulêre meganismes onderliggend aan die spesifieke werking van die fosfonaat-inhibeerders op OADH en OGDH in vivo. Om die mees selektiewe inhibeerders te ontwikkel in vivo, is ons huidige werk daarop gemik om die binding van die homoloë fosfonaatanaloë van dikarboksiele 2-oksosure aan OADH en OGDH te vergelyk. Ons gebruik inhibisiekinetika om die binding te kwantifiseer, en ontleding van beskikbare strukture om die molekulêre oorsprong van die interaksie-spesifisiteit te identifiseer. Die kwantifikasies verkry deur die kinetiese studie toon 'n goeie ooreenkoms met die strukturele data wat beskikbaar is vir die twee iso-ensieme en die bekende OGDH-komplekse met die fosfonate (Wagner et al., 2019). As gevolg hiervan word molekulêre basis van die selektiewe regulering van die OGDH- of OADH-isoensieme deur die sintetiese fosfonaat-inhibeerders geopenbaar, wat nuwe kennis verskaf oor die gerigte regulering van die teiken-ensieme in selle en organismes. Terwyl SP, wie se inhibisie van OGDH vir die eerste keer sowat drie dekades gelede gepubliseer is (Bunik et al., 1992), is nou goed erken as 'n spesifieke en doeltreffende inhibeerder van OGDH in vivo, ons ontwikkeling van 'n soortgelyke inhibeerder van OADH open nuwe maniere om die swak verstaanbare biologiese rol van hierdie isoënsiem te bestudeer. Verder kan farmakologiese instrumente om spesifiek die OADH-funksie te beïnvloed van terapeutiese belang wees, aangesien regulering van die DHTKD1 uitdrukking word waargeneem in 'n aantal patologiese toestande, insluitend diabetes, vetsug en kwaadaardige transformasie.


Bespreking

Ensieme van Mtb se CCM, insluitend sommige wat glikolise, glukoneogenese, die TCA-siklus en die glioksilaat-shunt onderhou, lewer 'n belangrike bydrae tot die patogeen se virulensie (5 ⇓ -7, 23, 35, 36). Bewyse kom na vore dat sommige van hierdie ensieme nie bloot die metaboliese rolle speel waarvoor hulle geannoteer is nie, maar die patogeen kan beskerm deur deel te neem aan antioksidante of antinitroksidatiewe verdediging. So 'n rol is eers gesien met DlaT (23, 37) en Lpd (5), en later met die isocitraat liases (38). Hier het ons HOAS by daardie lys gevoeg en twee vier-komponent peroksidase sisteme, HOAS/DlaT/AhpD/AhpC en AceE/DlaT/AhpD/AhpC, geïdentifiseer wat peroksidatiese werking kan onderhou met alternatiewe bronne van elektrone behalwe NADPH en NADH, naamlik , α-KG en piruvaat. Ten slotte het ons gehelp om die rol van die KDHC in Mtb te verduidelik.

HOAS kan in vitro in KDHC funksioneer. Dit lyk egter of KDHC self 'n beperkte rol speel in Mtb se TCA-siklus tydens groei op glikolitiese of vetsuurkoolstofbronne. In die teenwoordigheid van KDHC is daar min vloei van koolstof deur die nodus van die TCA-siklus wat KDHC beheer (4) en verwydering van sy E1 het min of geen impak op Mtb se groei in daardie toestande nie, in ooreenstemming met die lae KDHC ensiematiese aktiwiteit wat waargeneem is. hier en elders (15) en die minimale effek van HOAS-skrapping op metabolisme wanneer verbypadpaaie soos die glioksilaat- en GABA-shunts beskikbaar is. Miskien SucCoA sintetase rekeninge vir die voldoende van SucCoA vir biosintetiese doeleindes (39). Daarteenoor het ons drastiese metaboliese en groeifenotipes waargeneem wanneer HOAS-tekorte Mtb in glutamaat gekweek is. Onvolledige metabolisme van glutamaat/α-KG in HOAS-tekorte Mtb het gelei tot intrasellulêre en ekstrasellulêre ophoping van potensieel toksiese aldehiede, waarvan een, SSA (40), groei opgehef het wanneer dit ekstrasellulêr bygevoeg is.

Waarom het aldehiede intrasellulêr en ekstrasellulêr opgehoop toe HOAS-tekorte Mtb met ekstrasellulêre glutamaat aangebied is? WT Mtb kan eksponensieel vermeerder met glutamaat as sy enigste koolstofbron. Ekstrasellulêre glutamaat het egter gekeer dat Mtb repliseer as HOAS afwesig of onaktief was. Glutamaat gaan die TCA-siklus binne deur oksidatiewe deaminering na α-KG of as suksinaat via die GABA-shunt. Metabolomiese analise het 'n glutamaat-afhanklike opbou van α-KG en SSA in beide Δhoas en Δhoas::E956Ahoas. SSA kan ontstaan ​​uit HOAS-gekataliseerde nie-oksidatiewe dekarboksilering van α-KG of as 'n produk van die GABA-shunt in HOAS-tekorte stamme. SSA kan geoksideer word tot suksinaat deur SSADH's GabD1 en GabD2 (rv0234c en rv1731) (13). GabD1 word geïnhibeer deur hoë konsentrasies van substraat SSA (41) sowel as deur glioksilaat (42). Nie net kon glutamaatmetabolisme die GABA-shunt dryf nie, maar HOAS-tekort kan lei tot 'n toename in glioksilaat deur 'n afname in die beskikking daarvan deur die HOAS-reaksie. Verhoging van SSA kan dus die gesamentlike impak van twee effekte van HOAS-tekort weerspieël: verhoogde aktiwiteit van die GABA-shunt en inhibisie van SSADH-aktiwiteit deur verhoogde glioksilaat. Die toksiese potensiaal van SSA illustreer die beginsel dat intermediêre metabolisme nie net molekules produseer wat lewe onderhou nie, maar ook lewensgevaarlike molekules, soos getoon vir Mtb, wat ook vertakte ketting α-ketosure, propionaat, maltose 1-fosfaat of gliserolfosfaat ophoop wanneer die relevante metaboliese weë deur chemiese of genetiese middele ontwrig word (5, 8, 43 ⇓ ⇓ ⇓ –47).

Behalwe opeenhoping van groei-inhiberende SSA, kan ander meganismes verantwoordelik wees vir of bydra tot die onderdrukkende effek van glutamaat op groei van HOAS-tekorte Mtb, wat in kontras staan ​​met die vermoë van WT Mtb om verskillende substrate, insluitend glutamaat, te kokataboliseer vir optimale groei (4). In sommige bakterieë speel intrasellulêre metaboliete 'n seinrol in koolstofbenutting. Byvoorbeeld, in Escherichia coli, verhoogde α-KG inhibeer ensiem I van die fosfotransferase stelsel, blokkeer glukose opname (19), en benadeel cAMP sintese, ontlok kataboliet onderdrukking (48). Boonop kan aldehiede wat as elektrofiele optree, verbygaande addukte vorm met uitgesoekte Lys-reste om ensiemaktiwiteite te beheer en CCM-metabolisme te herlei (49).

Behalwe vir dismutasiereaksies waarin H2O2 of O2 − dien as beide 'n oksidant en 'n reduktant, voorheen beskryf stelsels van ensiematiese ontgifting van reaktiewe suurstoftussenprodukte en RNI's is uiteindelik afhanklik van reduseer ekwivalente van NADH of NADPH, waarvoor CCM die hoofbron is. Lpd, NADH en NADPH is egter almal bekende teikens van RNI's (50 ⇓ -52). Deur elektrone direk vanaf CCM-metaboliete te trek, en Lpd te omseil, kan verdedigingstelsels wat HOAS of AceE gebruik as belangrike komplemente tot die Lpd-afhanklike stelsel optree.

Die omsetgetal vir die PNR/P-komplekse wat hier beskryf word, was laer as wat 'n mens sou verwag vir 'n lewensreddende verdediging. Ons spekuleer dat die toestande wat in vitro gebruik is, nie die toestande in die ongeskonde sel voldoende saamgevat het nie. Ons kon 'n peroksidase-reaksie demonstreer, maar reagens-peroksinitriet het KoA so vinnig vernietig dat ons nie 'n PNR-reaksie kon demonstreer nie. Nietemin, die vier-ensiemstelsel wat óf HOAS óf AceE bevat, was in staat tot sikliese vermindering van AhpC, en AhpC is getoon om peroksinitriet te verminder in 'n toetsstelsel wat nie van CoA afhanklik is nie (31).

Die peroksidase-reaksie waaraan HOAS deelneem blyk fisiologies relevant te wees tydens Mtb se infeksie van die muis. Die rol van HOAS in die voorkoming van toksisiteit van glutamaat anaplerose kan ook vir virulensie vereis word, maar so 'n rol kon nie deur die huidige studies geëvalueer word nie. Deur HOAS-tekorte Mtb aan te vul met 'n aktiewe plekpuntmutant, het ons vasgestel dat die rol van HOAS in beide gevalle katalities is, nie bloot struktureel nie. HOAS-tekort het nie vatbaarheid verhoog vir die ander fisiologiese spanning wat getoets is of gelei tot aminosuur-auksotrofie nie (21, 39).

Kortom, onder die toestande wat hier en vroeër bestudeer is (23) in vitro en in muise, gebruik Mtb die E1- en E2-komponente van sy KDHC nie vir groei nie, maar vir verdediging teen voedingswanbalans en gasheer-immuunchemie.


Pediatriese Neurologie Deel III

Abstrak

Piruvaat dehidrogenase en piruvaat karboksilase tekort is die mees algemene afwykings in piruvaat metabolisme. Diagnose word gemaak deur ensiematiese en DNS-analise na basiese biochemiese toetse in plasma, urine en CSF.

Pyruvaat dehidrogenase het drie hoof subeenhede, 'n bykomende E3-bindende proteïen en twee komplekse regulerende ensieme. Die algemeenste is tekortkominge in PDH-E1α. Daar is 'n spektrum van kliniese voorstellings in E1α-tekort, wat by seuns wissel van ernstige neonatale melksuurdosis, Leigh-enkefalopatie, tot latere aanvang van neurologiese siektes soos intermitterende ataksie of distonie. Wyfies is geneig om 'n meer eenvormige aanbieding te hê wat lyk soos nie-progressiewe serebrale gestremdheid. Neuroradiologiese abnormaliteite soos corpus callosum agenesis word meer gereeld by meisies gesien, basale ganglia en middelbreinversteurings by seuns.

Tekortkominge in die ander subeenhede is ook beskryf, maar in 'n kleiner aantal pasiënte.

Pyruvaat-karboksilase-tekort het drie kliniese fenotipes. Die infantiele tipe word hoofsaaklik gekenmerk deur ernstige ontwikkelingsagterstande, mislukking om te floreer en aanvalle.

Die tweede tipe word gekenmerk deur neonatale aanvang van ernstige melksuurdosis met rigiditeit en hipokinesie. 'n Derde vorm is skaarser met intermitterende episodes van melksuursidose en ketoasidose. Neuroradiologiese bevindings soos sistiese periventrikulêre leukomalasie is beskryf.


Materiale en Metodes

Reagense en gereedskap tabel

Reagens/hulpbron Verwysing of bron Identifiseerder of katalogusnommer
Eksperimentele modelle
B. diazoefficiens USDA 110 spc4 Regensburger en Hennecke (1983) NA
E coli BL21 pET42b::gabT2 Hierdie studie BC3422
E coli BL21 pET42b::gabD9 Hierdie studie BC3421
E coli BL21 pET42b:: gabD6 Hierdie studie BC4312
E coli BL21 pET42b::odcB Hierdie studie BC4308
E coli BL21 pET42b:: gabD8 Hierdie studie BC4294
E coli BL21 pET42b::argI2 Hierdie studie BC3424
E coli BL21 pET42b::speB Hierdie studie BC4265
E coli BL21 pET42b::speB2 Hierdie studie BC4266
E coli BL21 pET42b::argD Hierdie studie BC4270
E coli BL21 pET42b::dataA Hierdie studie BC4292
E coli BL21 pET42b::aspC Hierdie studie BC3417
E coli BL21 pET42b::ilvB1 Hierdie studie BC3419
E coli BL21 pET42b ::gabD7 Hierdie studie BC4296
E coli BL21 pET42b::gabD1 Hierdie studie BC3420
E coli BL21 pET42b::odcA Hierdie studie BC4269
E coli BL21 pET42b::argI1 Hierdie studie BC3423
E coli BL21 pET42b::aatB Hierdie studie BC4293
E coli BL21 pET42b::gabT3 Hierdie studie BC4271
S. meliloti CL150 (R1021 pstC + ecfR1 + ) Schlüter et al ( 2013 ) BC2175
S. meliloti CL150 nifD::Tn5-233 Lang et al (2018) CL309
S. meliloti CL150 dctAB::aacC1 Hierdie studie BC4081
S. meliloti CL150 argI2:: aacC1 Hierdie studie BC3455
S. meliloti CL150 satABC::aacC1 Hierdie studie BC3451
S. meliloti CL150 ureGFE::aacC1 Hierdie studie BC4083
S. meliloti CL150 aspC::aacC1 Hierdie studie BC3457
S. meliloti CL150 argI1::aacC1 Hierdie studie BC3453
S. meliloti CL150 argI1::smR argI2::aacC1 Hierdie studie BC3766
S. meliloti CL150 artABCDE::aacC1 Hierdie studie BC3459
S. meliloti CL150 amtB::aacC1 Hierdie studie BC4085
M. truncatula Jemalong gew Pecrix et al (2018) NA
M. truncatula Jemalong lss Schnabel et al ( 2010 ) NA
G. maks kultivar Williams NA
Oligonukleotiede en volgorde-gebaseerde reagense
PCR primers Hierdie studie Datastel EV5
Chemikalieë, ensieme en ander reagense
4-Aminobutaansuur Sigma-Aldrich A2129-10G
4-Guanidinobottersuur Sigma-Aldrich G6503-1G
Agmatien Sigma-Aldrich 101443-1G
Aminobutanaal Hierdie werk NA
Guanidinobutanal Hierdie werk NA
L-arginien Fluka 11010
L-arginien (13C) Sigma-Aldrich 643440-100MG
L-arginien (15N) Sigma-Aldrich 643440-100MG
L-Citrulline Sigma-Aldrich C7629-1G
L-Ornithine Sigma-Aldrich O8305-25G
NAD + Sigma-Aldrich N1636
Putrescine Sigma-Aldrich P5780
Pyruvaat Sigma-Aldrich P2256-100G
Suksinaat Sigma-Aldrich 150-90-3
Succiniese semialdehied Hierdie werk NA
Swaelsuur Sigma-Aldrich 258105
Natriumhipochloriet oplossing VWR BDH7038
Beperkingsensiem Spel New England Biolabs R0133
Beperkingsensiem MfeI New England Biolabs R0589
Ander
GC6850 gaschromatograaf instrument Agilent Technologies
6550 akkurate massa kwadrupool tyd-van-vlug Agilent Technologies
Agilent HILIC Plus RRHD kolom Agilent Technologies
5500 QTRAP drievoudige-kwadrupool massaspektrometer AB Sciex
HisTrap FF kru kolom GE Healthcare GE11-0004-58

Materiale en Metodes

Bakteriese stamme, verbouing en groeitoestande

Sinorhizobium meliloti ras CL150 (Rm1021, pstC + ecfR1 + ) (Schlüter et al, 2013) is by 30°C in LB-bouillonmedium met 5 g NaCl per liter gekweek. Escherichia coli is in LB-bouillon by 37°C gekweek. Waar nodig is groeimediums aangevul met antibiotika teen die volgende konsentrasies: gentamisien, 10 μg/ml vir E coli en 30 μg/ml vir S. meliloti wanneer gekweek in LB streptomisien, 200 μg/ml en ampicillien, 50 μg/ml.

Plantverbouing en inentingtoetse

Medicago truncatula WT Jemalong-sade is vir vyf minute met 70% etanol aan die oppervlak gesteriliseer en deeglik met water afgespoel. Sade is met sagte roering vir ten minste vier uur met twee waterverversings ingedrink en verder oornag by kamertemperatuur sonder lig ingedrink. Na imbibering is sade met water gewas en vir 24 uur by 30°C ontkiem. Saailinge is in 'n steriele perlietsubstraat (Isoself, Knauf) binne 300 cm 3 swart plastiekpotte (Greenhouse, Elho) geplant. Plante is gekweek in plantgroei rakke by kamertemperatuur met 'n beheerde 16-uur dag en 8-uur nag siklus. Gedurende die ligsiklus het elke pot 2500 lumen ontvang met 36 W Fluora 77 OSRAM gloeilampe. Plante is outomaties natgemaak met 'n druppelbesproeiingstelsel (Micro-drup-stelsel, Gardena Art. 8311-20 Art. 1407-20) met 3-dae intervalle met 80 ml 10% BNM oplossing (Ehrhardt) et al, 1992). Potte is tydens groei met 'n deursigtige PET-silinder bedek. Drie dae na ontkieming is plante geënt met 20 ml van a S. meliloti selkultuur met 'n seldigtheid van 'n OD600nm van 0,05 hersuspendeer in 1 mM MgSO4. Die inokulasietydpunt is as dag nul na-inokulasie (dpi) beskou.

Glysien maks kultivar Williams sade is oppervlak-gesteriliseer met 'n was in 100% etanol vir 5 minute gevolg deur 'n was in 35% H2O2 vir 15 min. Daarna is die sade deeglik met water afgespoel. Sade is ontkiem in 0.8% agarplate vir 24–48 uur by 25°C in die donker. Saailinge is geplant op 200 cm 3 bruin glas flesse gevul met geoutoklaveerde vermikuliet (Vermisol) en 100 ml Jensen media (Vincent, 1970). Na oorplanting is plante ingeënt met B. diazoefficiens (1 ml OD600 nm van 0,01). Die tydpunt van inenting is beskou as dag nul na-enting (dpi). Plante is in plantgroeikamers by 28°C gekweek met 'n beheerde 16-uur dag- en 8-uur nag siklus soos voorheen beskryf (Göttfert et al, 1990). Na 5 dpi is plante elke 3 dae met steriele water natgemaak.

Fenotipiese karakterisering van plante om simbiotiese stikstofbinding te bepaal

Simbiose fenotipes soos nodulasie frekwensie, plant droë gewig en nitrogenase aktiwiteit is bepaal in M. truncatula WT plante 8 weke na-enting met S. meliloti geenuitwissingsmutante en wildtipe beheerstamme. Nitrogenase aktiwiteit van M. truncatula is bepaal deur asetileenreduksie na etileen te meet soos voorheen gerapporteer. Plante is in 50 cm 3 verseëlde flessies geplaas waarin asetileen tot 'n finale konsentrasie van 2% ingespuit is. Etileenproduksie is gemeet na 3 uur en 5 uur se inkubasie op 'n analitiese gaschromatograaf instrument (GC6850, Agilent Technologies). Die etileen agtergrond is gemonitor en sistematies verwyder uit elke meting. Om die gemiddelde etileenproduksie per nodule te bepaal, is etileenproduksie genormaliseer deur nodule getal en duur van asetileen inkubasie. Nodulasiefrekwensie is bepaal deur al die sigbare nodules per plant handmatig te tel. Vir droëgewigbepaling is plantlote gedroog by 85°C vir ten minste 20 uur voor individuele plantgewigmeting (ABS 80-40 N, KERN). Metings van replikas is gemiddeld en genormaliseer na die S. meliloti CL150 WT verwysingsstam. Nitrogenase aktiwiteit, plant droë gewig en nodulasie frekwensie is bepaal vir ten minste 22 onafhanklike plante vir elke getoets S. meliloti druk.

Sinorhizobium meliloti Bakteroïede ultrastruktuur bepaling deur skandeerelektronmikroskopie

Medicago truncatula nodules is 10 weke na-inokulasie (wpi) geoes, in die lengte oopgesny en versamel in 'n houer met fikserende buffer (2,5% glutaaraldehied, 2% formaldehied in 0,15 M Na-kakodilaat met 2 mM CaCl)2). Die nodule-bevattende houer is vir ten minste 30 minute aan vakuum onderwerp om die nodules te laat sink, gevolg deur mikrogolffiksasie in vars fikseermiddel (PELCO BioWave Pro +) en gewas met fikserende buffer. Nodules is gekleur deur opeenvolgende inkubasie met (i) 2% OsO4, 1,5% K4Fe(CN)6 in 0,15 M Na-Cacodylate met 2 mM CaCl2 (ii) 1% tiokarbohidrasied (iii) 2% OsO4 (iv) 1% uranilasetaat en (v) Waltons lood aspartaat monsters is tussen elke stap met fikserende buffer gewas. Monsters is deur 'n gegradeerde etanolreeks (25%, 50%, 75%, 100%, droë etanol) gedehidreer en met droë asetoon gewas. Monsters is in 'n gegradeerde reeks Epon/Araldite in droë asetoon (25%, 50%, 75%, 100%, 100%) geplaas en toegelaat om te polimeriseer by 60°C vir 3 dae. Ultradun snitte (100 nm) is oorgedra na Si-wafer-skyfies en afgebeeld op 'n FE-SEM FEI Magellan 400i wat werk by 1.8 kV en 0.8 nA met 'n 20 nm pixelgrootte deur terugstrooi-elektronopsporing.

Substraat spesifieke stimulasie van nitrogenase-aktiwiteit in geïsoleerde bakterieë

Sinorhizobium meliloti en B. diazoefficiens bakterieë is geïsoleer uit M. truncatula en G. maks wortelnodules, onderskeidelik, volgens die volgende prosedure. Plantwortels is 3 weke geoes (G. maks) of 10 weke (M. truncatula) na-inokulasie met wild-tipe B. diazoefficiens 110spc4-stam (Regensburger & Hennecke, 1983) en S. meliloti CL150, onderskeidelik, en spoel deeglik met water af. Onder anaërobiese toestande (92% N2, 8% H2), knoppies (250 mg tot 1 g nat gewig) is fyngemaak in PBS (10 mM Na2HPO4, 1,8 mM KH2PO4, 137 mM NaCl, 2,7 mM KCl, pH 7,4) en deur drie lae gaas gefiltreer om puin te verwyder. Bakteroïedsuspensies (2 ml wat ooreenstem met 4 × 10 6 bakteroïedselle) is by 15-ml verseëlde flesse gevoeg om verwysingsmetings van nitrogenase-aktiwiteit in geïsoleerde bakterieë in die teenwoordigheid van plant-ru-ekstrak te verkry. Om die stimulasie van nitrogenase-aktiwiteite na die byvoeging van substrate te evalueer, is bakteroïedsuspensies twee keer met PBS onder anerobiese toestande gewas en deur sentrifugering by 1 000 gewas. g. Herwonne bakteroïede (4 × 10 6 selle) is hersuspendeer in 2 ml induksie media (2 μM biotien, 1 mM MgSO4, 42,2 mM Na2HPO4>, 22 mM KH2PO4, 8,5 mM NaCl, 21 nM CoCl2, 1 μM NaMoO4 pH 7.0) en by 15-ml verseëlde flesse gevoeg. Induksiemedia is aangevul met óf 7.4 mM suksinaat óf 5 mM arginien of albei substrate. Asetileen en suurstof is bygevoeg tot 'n finale konsentrasie van onderskeidelik 5% en 0.01% in die kopspasie van elke fles. Nitrogenase-aktiwiteit is vir elke monster bepaal deur die reduksie van asetileen na etileen na 1 en 3 uur tydpunte te meet. Etileenproduksie is met gaschromatograaf (GC6850, Agilent Technologies) opgespoor. Aktiwiteite is genormaliseer deur nodule nat gewig. Verder is relatiewe aktiwiteite bereken deur die basislynaktiwiteit van gewasde bakteroïedmonsters sonder substraataanvulling af te trek. Gerapporteer B. diazoefficiens en S. meliloti aktiwiteit is die resultaat van onderskeidelik 17 en 4 onafhanklike voorbereidings.

Substraat spesifieke stimulasie van ATP-produksie in geïsoleerde bakterieë

Bradyrhizobium diazoefficiens Bakteroïede is geïsoleer vanaf 3 weke na-geïnteer G. maks wortelnodules. Nodules (1 g nat gewig) is fyngemaak in PBS (10 mM Na2HPO4, 1,8 mM KH2PO4, 137 mM NaCl, 2,7 mM KCl, pH 7,4) en gefiltreer deur drie lae gaas om puin te verwyder. Bakteroïedsuspensie is deur sentrifugering teen 2 500 verpille g, en die supernatant (nodule-ekstrak) is gestoor vir latere gebruik. Bakteroïedsuspensie (5 × 10 8 > selle) is twee keer met PBS gewas en hersuspendeer in 1 ml induksiemedia (2 μM biotien, 1 mM MgSO4, 42.2 mM Na2HPO4, 22 mM KH2PO4, 8,5 mM NaCl, 21 nM CoCl2, 1 μM NaMoO4 pH 7.0). Om ATP-generering deur aërobiese respirasie te vermy, is die bakteroïedsuspensie onder anaërobiese toestande geplaas (92% N2, 8% H2) om die res van die prosedure uit te voer. Bakteroïede (200 μL) is geïnkubeer in 2 mL nodule-ekstrak (verkry aan die begin van die protokol) of induksiemedia sonder aanvullings of aangevul met óf 7.4 mM suksinaat óf 5 mM arginien of albei substrate. ATP-inhoud is bepaal vir elke monster (met 'n 1:10 verdunning) deur ATP-afhanklike lusiferasereaksie (BacTiter-Glo Microbial Cell Viability Assay, Promega). Luminesensie van luciferase-aktiwiteit is gekwantifiseer met Victor3 multi-etiket plaatteller (PerkinElmer).

Transposon mutagenese

Om hiperversadigde transposonmutantbiblioteke te genereer in S. meliloti, 'n voorheen beskryfde Tn5 mutagenese prosedure vir Caulobacter crescentus is aangepas (Christen et al, 2011). Kortliks, die Tn5 aflewering ColE1 plasmied pTn5_gent_14N (Christen et al, 2016) is vervoeg van 'n E coli SM10 skenker stam in a S. meliloti CL150 ontvanger stam. Afsonderlike transposon mutant biblioteke is gegenereer en groei gekies op ryk medium (LB). Vir elke toestand is 'n totaal van sestien onafhanklike vervoegings uitgevoer en replikaatbiblioteke is gemerk met agt strepieskodes Tn5 afgeleides. Transposon invoegingsmutante is geselekteer op LB aangevul met gentamisien en streptomisien. Plate is vir 2 dae by 30°C geïnkubeer, en transposonmutantbiblioteke van elke plaat is afsonderlik saamgevoeg, aangevul met 10% v/v DMSO (Sigma-Aldrich), en in 96-put diepputplate by -80° gestoor. C vir verdere verwerking.

In planta selection of transposon mutant libraries

Transposon mutant pools were selected for infection and nodule formation in legume plants. For nodulation experiments, a Medicago truncatula lss super-nodulator mutant was used (Schnabel et al, 2010). Seeds were treated with concentrated H2SO4 (Sigma-Aldrich) for 5 min, thoroughly rinsed with sterile water, then sterilized with 7% NaClO (VWR chemicals) for 3 min, and again rinsed with sterile water. The seeds were then imbibed with gentle agitation for four hours with regular water changes and then incubated overnight in the dark at room temperature. After imbibition, seeds were rinsed with sterile water, placed in deep petri plates, and inverted for 24 h at 30°C to allow for the downward growth of the seedling roots. After removing seed coats, groups of 25 seedlings were planted on large square plates (Genetix) containing 1.2% buffered nodulation medium (Ehrhardt et al, 1992 ) supplemented with 0.1 nM aminoethoxy vinyl glycine. Altogether, 4,500 M. truncatula lss plant seedlings were grown at 22°C with a controlled 16-h day and 8-h night cycle (2500 Lumens using Osram Fluora L36 W/77 bulbs). Five days post-germination, M. truncatula lss seedlings were flood-inoculated with S. meliloti Tn5 mutant reference libraries initially selected on rich media conditions (LB). During inoculation experiments, input libraries were kept independent from each other. S. meliloti transposon input mutant pools were inoculated from 96-well storage plates, grown overnight, washed, and resuspended to an OD600 nm = 0.05 in 10 mM MgSO4. Plant roots were then aseptically inoculated with 5 ml of a dilute bacterial suspension, followed by removal of the excess bacterial suspension.

Recovery of transposon mutant libraries from nodules

After 6 weeks post-inoculation, nodules were harvested. To recover transposon mutants capable of infecting root nodules, a two-step surface sterilization protocol was employed. Nodule material was washed with 1% SDS and then treated with 70% ethanol for 5 min, followed by rinsing with sterile water. Next, nodules were treated for 3 min with 0.2% NaClO (VWR chemicals) followed by three rinses with sterile water. The surface-sterilized nodules were crushed in cold PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) and filtered through three layers of gauze to remove debris. The filtered suspension containing bacteroids was plated on LB supplemented with gentamicin and streptomycin and grown for 2 days at 30°C. The recovered S. meliloti colonies were pooled and arrayed in 96-well plates and stored at −80°C for subsequent use.

Data analysis and mapping of transposon insertion sites

Raw sequencing data processing and read alignment were performed using a custom sequence analysis pipeline based on Python, Biopython (Cock et al, 2009 ), bwa (Li & Durbin, 2010 ), and MATLAB routines as previously described (Christen et al, 2016). Adapter sequences were detected using Python string comparison with a 15 bp search window. Demultiplexing into the different TnSeq selection experiments was performed according to a defined barcode sequence tag internal to the arbitrary primer sequence. Reads were aligned onto the S. meliloti 1021 NCBI reference genome (NC_003047, NC_003037, NC_003078 Barnett et al, 2001 Finan et al, 2001 Galibert et al, 2001 ) using bwa-07.12 (Li & Durbin, 2010 ). Insertion datasets were correlated with the genome annotation to analyze global insertion statistics and calculate transposon insertion occurrence and distributions within each annotated GenBank feature of the S. meliloti 1,021 genome (Galibert et al, 2001 )> as previously described (Christen et al, 2016 ).

Gene essentiality analysis across selection conditions

For k smaller than (n−i)/2 and n not to exceed 100, p2 was numerically calculated by calculating the number (h) of compositions of length i + 1> for n where each part does not exceed k by brute force. For k smaller than (n−i)/2 and n equals or exceeding 100, we approximated p2 by sampling the composition space by random simulation and counting the occurrences of compositions of length i + 1 for n where each part does not exceed k.

Construction of targeted gene deletions in Sinorhizobium meliloti

Sinorhizobium meliloti deletion mutants were generated by replacing the native gene with the aacC1 gene conferring gentamycin resistance using a one-step double homologous recombination procedure as detailed in Ledermann et al ( 2016 ). Flanking DNA regions covering 750 bp upstream and downstream of a target gene were PCR amplified (Dataset EV5) and subsequently fused to a central gentamicin resistance gene using splicing by overlapping extension PCR or Gibson assembly (Gibson et al, 2009 ) to produce gene replacement cassettes. The gene replacement cassettes were cloned via SpeI and MfeI into the pNPTS138 plasmid, which is non-replicative in S. meliloti and confers kanamycin resistance. Cloned plasmids were sequence confirmed and conjugated into S. meliloti strain CL150. Recombinants were selected on LB media supplemented with gentamicin and subsequently screened for kanamycin sensitivity. Single gene deletion strains were confirmed by PCR and sequencing. A similar deletion strategy was employed for construction of the double arginase mutant (∆argI1argI2) with a gene replacement cassette for argI1 carrying a spectinomycin marker gene. The resulting plasmid was conjugated into S. meliloti CL150 ∆argI2, and the double arginase double mutant strain was screened for kanamycin sensitivity before verification of constructed deletion by PCR and sequencing.

13 C arginine isotope tracing in Bradyrhizobium diazoefficiens bacteroids

Bradyrhizobium diazoefficiens bacteroids were isolated under anaerobic conditions from three weeks post-inoculated G. max root nodules according to Sarma and Emerich ( 2005 ) and Delmotte et al ( 2010 ). Nodules (10 g wet weight) were crushed in PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4). The homogenate was passed through four layers of cheesecloth (pre-moistened with PBS) into a sterile centrifuge tube, re-extracted several times with buffer, and centrifuged at 400 g for 10 min. The resulting pellet was resuspended twice in PBS and centrifuged at 8,000 g vir 20 min. The pellet was dispersed into the extraction buffer (2 ml/g original weight of the nodule) and was layered onto a pre-equilibrated Ludox gradient consisting of 25% (10 ml) Ludox and 75% PBS (30 ml). The gradient tubes were centrifuged in an SW-28 rotor at 10,000 g for 35 min at 4°C in a Beckman L8–55 ultracentrifuge. The bacteroid layer with a density of 1.09 g/ml was collected. The bacteroid pellet was suspended in distilled water and collected by centrifugation at 10,000 g. For labeling assays, bacteroids were resuspended in 10 ml volume in PBS to a final OD of 4.0. After the addition of succinate (5 mM) and 13 C arginine (5 mM), bacteroids were incubated at RT under microaerobic condition (0.1% v/v) and samples of 250 μl were taken at regular time intervals, filtered on a PVDF 0.45 μl membrane and immediately washed with 1.0 ml H2O Chromasolv. The filter with the cell pellet was extracted in 3 ml (40% MeOH, 40% acetonitrile and 20% H2O) at −20°C for 1 h, stored at −80°C, and finally dried in a SpeedVac. The metabolite extracts were resuspended in 100 μl MilliQ water, and metabolites were analyzed using a HILIC method. 5 μl of metabolite extract was injected on an Agilent HILIC Plus RRHD column (100 mm × 2.1 mm × 1.8 μm Agilent). The gradient of mobile phase A (10 mM ammonium formate and 0.1% formic acid) and mobile phase B (acetonitrile with 0.1% formic acid) was as follows: 0 min, 90% B 2 min, 40% B 3 min, 40% B 5 min, 90% B and 6 min, 90% B. The flow rate was held constant at 400 μl min −1 . Metabolites were detected on a 5500 QTRAP triple-quadrupole mass spectrometer in positive mode with MRM scan type (AB Sciex, Foster City, CA). The raw data were processed and analyzed by custom software using MATLAB (MathWorks).

15 N arginine isotope tracing in Bradyrhizobium diazoefficiens bacteroids

Bradyrhizobium diazoefficiens bacteroids were isolated from 3 weeks post-inoculated G. max root nodules under anaerobic conditions as described above. Bacteroids were resuspended (1 ml/g nodule wet weight) in 2 ml of tracing media (2 μM biotin, 1 mM MgSO4, 42.2 mM Na2HPO4, 22 mM KH2PO4, 8.5 mM NaCl, 21 nM CoCl2, 1 μM NaMoO4 pH 7.0, 10 mM NH4Cl, 7.4 mM succinate, and 5 mM 15 N arginine). Bacteroid suspensions were incubated at room temperature under microaerobic conditions (0.1% v/v). Aliquots (20 μl) of the enzymatic reaction were sampled over the time series, and reaction was blocked by adding 180 μl of ice-cold methanol. Relative metabolite abundances were determined by non-targeted flow injection analysis as described previously (Fuhrer et al, 2011). Mass spectra were recorded in negative-ionization profile mode from m/z 50 to m/z 1,000 on an Agilent 6550 accurate-mass quadrupole time-of-flight instrument with a frequency of 1.4 spectra/s using the highest resolving power (4 GHz HiRes). The source gas temperature was set to 225°C, with 11 l min −1 drying gas and a nebulizer pressure of 20 psig. The sheath gas temperature was set to 350°C, and the flow rate was 10 l min −1 . Electrospray nozzle and capillary voltages were set at 2,000 and 3,500 V, respectively.

Purification of recombinant proteins

Coding sequences of interest were amplified by PCR (Dataset EV5) and cloned into pET42 expression plasmids inframe to a C-terminal (His)6-tag (6xHis). The resulting vectors were sequence-verified and electroporated into BL21 rosetta pLys strains. E coli BL21 harboring the expression vectors were grown at 30°C in LB medium containing chloramphenicol (20 mg/l) and kanamycin (30 mg/l). When the cultures reached an OD600nm of 0.4, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 M. After the addition of IPTG, the culture was grown for 2–4 h more at 30°C to induce the expression of the recombinant proteins. Grown cells were harvested by centrifugation at 5,095 g for 10 min at 4°C. The resulting pellet was either shock frozen with liquid N2 and stored at −80°C or immediately used for protein extraction and purification. To purify the recombinant proteins, cells were resuspended in NPI-buffer (20 mM sodium phosphate, 500 mM NaCl, pH7.4) supplemented with 10 mM imidazole and disrupted using a French press (SLM Instruments Inc.). Lysates were cleared by centrifugation at 17,000 g for 15 min at 4°C to remove cell debris. The supernatant was loaded on HisTrap FF crude column (GE Healthcare) previously equilibrated with NPI-buffer supplemented with 10 mM imidazole. The column was washed twice with NPI-buffer supplemented with 10 mM and 20 mM imidazole, respectively. The purified enzymes were eluted with NPI-buffer supplemented with 250 mM imidazole, concentrated by ultrafiltration with Amicon Ultra-4 centrifugal filters (Merck Millipore), and dialyzed against NPI-buffer containing 10% (v/v) glycerol. Protein concentration was determined using the Pierce BCA Protein Assay kit (Thermo Scientific). Protein samples were shocked frozen with liquid N2 and stored at −80°C until use.

Chemical synthesis of succinate semialdehyde, 4-aminobutanal, and 4-guanidinobutanal

The arginine transamination metabolic network contains a set of 13 intermediates. Thereof, three intermediates succinate semialdehyde, 4-aminobutanal, and 4-guanidinobutanal are not commercially available and were chemically synthesized for use in subsequent enzyme characterization studies as aldehyde dehydrogenase substrates.

Succinate semialdehyde was synthesized according to the procedure as previously described (Bruce et al, 1971 ). In a 15 ml Falcon tube, a solution of monosodium glutamate (169 mg, 1 mmol) in 5.0 ml distilled water was exposed to a gentle flow of N2 for 5 min. An equimolar amount of chloramine T (227 mg, 1 mmol) was added to the solution and dissolved by heating the solution to 60°C. After further incubation at 60°C for an additional 15 min, the mixture was cooled to 25°C and adjusted to pH 2.0 with concentrated HCl and degassed. Reaction by-products were crystallized by placing the mixture on ice and removed by filtration. The aqueous phase was extracted with diethyl ether 3 times, organic phases were combined, and the water fraction was discarded. Diethyl ether was evaporated yielding an aqueous solution of 1% of the starting material and a concentration of approximately 2 M succinic semialdehyde.

4-guanidinobutyraldehyde was prepared from L-arginine according to Tanaka et al ( 2001 ). The procedure follows a similar reaction scheme as detailed for the synthesis of succinic semialdehyde with the following modifications. As starting material, arginine hydrochloride (210 mg, 1 mmol) was dissolved. Upon addition of chloramine T (227 mg, 1 mmol), the mixture was adjusted to a pH 6.5 with 1 N HCl (50 μl) and heated to 60°C. Prior extraction with diethyl ether, the solution was adjusted to pH 13.5 with 10 N NaOH.

4-aminobutyraldehyde was prepared by the hydrolysis of 1.0 ml of 0.5 M 4-aminobutyraldehyde diethyl acetal (Sigma-Aldrich). 172 μl 4-aminobutyraldehyde diethyl acetal was dissolved in 2 ml H2O, and 1 N HCl was added to acidify to pH 3.0. After incubation for 30 min at 30°C, the reaction mixture was titrated to pH 10.0 with 1 N NaOH. The crude reaction product 4-aminobutanal was extracted from the water phase with five consecutive extraction steps using 1 ml diethyl ether. Ether fractions were combined, and the solvent was removed by evaporation. The reaction product 4-aminobutyraldehyde was obtained as a colorless liquid of 40.0 mg mass.

Enzyme characterization and activity assays

Transaminases were assayed for enzymatic activity according to the following procedure. A total of 30 μg purified enzyme were added to 200 μl reaction mixture containing 1 mM arginine, 1 mM ornithine, 1 mM citrulline, 1 mM agmatine, 1 mM putrescine, 1 mM 4-guanidinobutanoate, 1 mM 4-aminobutanoate, 10 mM of pyruvate, 1 mM MgCl2, 1 mM MnCl2, and 100 μM pyridoxal phosphate in 50 mM PBS pH 7.4 followed by incubation at 25°C. Aliquots (20 μl) of the enzymatic reaction were sampled over the time series, and reactions were blocked by adding 180 μl of ice-cold methanol. Ureohydrolases were assayed for enzymatic activity according to the following procedure. A total of 30 μg purified enzyme were added to 200 μl reaction mixture containing 1 mM arginine, 1 mM agmatine, 1 mM 4-guanidinobutanoate, 10 mM of pyruvate, 1 mM MgCl2, 1 mM MnCl2, and 100 μM pyridoxal phosphate in 50 mM PBS pH 8.0, previously incubated at 25°C for 30 min with 0.15 ng/μl purified AspC followed by heat inactivation at 65°C for 5 min to generate 5-guanidino-2-oxopentanoate (GOP) and guanidinobutanal, from arginine and agmatine transamination, respectively. Ureohydrolase reactions were done at 25°C. Aliquots (20 μl) of the enzymatic reaction were sampled over the time series, and reaction was blocked by adding 180 μl of ice-cold methanol. Decarboxylases were assayed for enzymatic activity according to the following procedure. A total of 30 μg purified enzyme were added to 200 μl reaction mixture containing 1 mM arginine, 1 mM ornithine, 1 mM citrulline, 10 mM of pyruvate, 1 mM MgCl2, 1 mM MnCl2, 100 μM pyridoxal phosphate, and 500 μM thiamine pyrophosphate in 50 mM PBS pH 8.0, previously incubated at 25°C for 30 min with 0.15 ng/μl purified AspC followed by heat inactivation at 65°C for 5 min to generate 5-guanidino-2-oxopentanoate (GOP) from arginine transamination. Decarboxylase reactions were done at 25°C. Aliquots (20 μl) of the enzymatic reaction were sampled over the time series, and reactions were blocked by adding 180 μl of ice-cold methanol. Substrate consumption and product formation of transaminases, ureohydrolases, and decarboxylases reactions were determined by non-targeted flow injection MS analysis as described previously (Fuhrer et al, 2011). Dehydrogenase activities were assayed from cell lysates of BL21 rosetta pLys strain expressing S. meliloti dehydrogenases according to the following procedure. Cell lysates, corresponding to 20 μg of dehydrogenase enzymes, were added to a substrate mixture containing 1 mM succinate semialdehyde or 1 mM 4-guanidinobutanal or 1 mM 4-aminobutanal in 10 mM PBS pH 10.0 supplemented with 1 mM NAD + . Dehydrogenase reaction was determined by the conversion of NAD + into NADH + H + , which was measured by the increase in absorbance at 340 nm.

Biochemical reconstitution of the catabolic arginine transamination network

To reconstruct a functional catabolic arginine transamination network in vitro, a multienzyme assay comprising 14 purified enzymes was established. A reaction buffer containing of 500 μM thiamine pyrophosphate, 100 μM pyridoxal phosphate, 2 mM NAD, 1 mM MgCl2, and 1 mM MnCl2 in 50 mM PBS pH 8.0 was prepared. Purified enzymes (25 μg each) from the catabolic arginine transamination network (AspC, AatB, ArgD, GabT2, DatA, ArgI1, SpeB, SpeB2, IlvB1, OdcA, OdcB, GabD1, GabD6, and GabD7) were added one by one and gently mixed into the reaction mixture. After the addition of all enzymes, 20 mM pyruvate and 2 mM arginine were added and gently mixed. As a control, the enzyme mix was incubated in a reaction buffer lacking arginine and pyruvate. Aliquots (15 μl) of the enzymatic reaction were sampled over the time series, and reaction was blocked by adding 135 μl of ice-cold methanol. Substrate consumption and product formation of enzymatic reactions were determined by non-targeted flow injection MS analysis as described previously (Fuhrer et al, 2011 ).

Data beskikbaarheid

The datasets produced and presented in this study are available as Datasets EV1–EV5.


Yet α-lipoic acid functions to increase acetylcholine twice, first through pyruvate decarboxylase — along with thiamine — and then again via choline acetyltransferase.

Acetylcholine also increases strength by creating the action potential of nerves controlling the muscles.

This had been a good study yet simple and brief. The two authors had, however, done a follow-up which had confirmed the initial observation and had helped elucidate some finer details:

This study had used the exact same materials and methods as the former, yet had included dialysis equipment and a few other reagents.

They had found that after the purified enzyme had been dialyzed it’d lost it’s activity, implying that some necessary factor had been removed.

‘Dialysis of the preparation for 24 h caused almost complete loss of activity.’ ―Haugaard

The enzyme choline acetyltransferase had no known cofactor at that time, so this had come somewhat as a surprise.

Yet despite its initial inactivation, the enzyme had regained function upon the addition of dihydrolipoic acid:

They had also tested some other biological “reducing agents” for activity, yet glutathione, vitamin C, and NADH all failed to increase acetylcholine.

This rules out the possibility that dihydrolipoic acid was simply acting as a nonspecific electron donor, although no electrons are consumed in the process of acetylcholine synthesis.

‘It can be seen that the activity of the enzyme is quite low compared to the activity that can be reached after addition of dihydrolipoic acid.’ ―Haugaard

The authors had eventually come to the conclusion, and perhaps rightly so, that lipoic acid had been acting a coenzyme — its well-established function in five other enzymes.

In two of the five known lipoic acid enzymes — d.w.s. pyruvate decarboxylase and acetoin dehydrogenase — an series of enzyme-bound dihydrolipoic acid molecules carry acetyl groups from thiamine to coenzyme A.


ENVIRONMENTAL EFFECTS ON RESPIRATORY PROCESS

A large number of measurements have been made concerning gas exchange (i.e. rates of photosynthesis, respiration and transpirations) of different plants growing under contrasting conditions ( Lambers, Chapin & Pons 2008 ). These measurements have yielded a mass of experimental results, some of which have been previously discussed. The enormous variety of alternative respiratory substrates and metabolic pathways makes plant respiration remarkably flexible especially in response to changing environmental circumstance. For instance, it has been shown that oxygen isotope discrimination during plant respiration seems to be independent of temperature over the range of temperature normally encountered during growth ( Macfarlane et al. 2009). These authors also observed that there is a relatively large temperature dependence of the respiration rate, suggesting that there was little substrate limitation to respiratory rate in the leaves of healthy plants ( Macfarlane et al. 2009). Thus, it seems reasonable to assume that enzyme capacity is the main limitation of respiratory rate, and the reduction state of the ubiquinone pool varies little or none with measurement of temperature.

It has been suggested that higher temperatures reduce net carbon gain by increasing plant respiration more than photosynthesis. In fact, the light-saturated photosynthesis rate of C3 crops such as wheat and rice is at a maximum for temperatures from about 20–32 °C, whereas total crop respiration shows a steep non-linear increase for temperatures from 15 to 40 °C, followed by a rapid and nearly linear decline ( Porter & Semenov 2005 ). Although the stimulation of C3 photosynthesis by growth at elevated atmospheric [CO2] can be somewhat predicted with confidence, the nature of changes in respiration remains uncertain ( Leakey et al. 2009b ). The primary reason for uncertainty is that the mechanisms of plant respiratory responses to elevated [CO2] are not fully understood ( Gifford 2003 Leakey et al. 2009b ). In fact, the results observed in the literature are somehow contradictories and have shown that plant respiration may increase as much as 37%, decrease as much as 18%, or even not change at all with increased [CO2] (e.g. ( Drake et al. 1999 Gifford 2003 Leakey et al. 2009a). In a recent free air carbon dioxide enrichment (FACE) study where soybean was grown at elevated [CO2] (550 ppm), the stimulated (37%) rate of night-time respiration was associated with the additional carbohydrate available from enhanced photosynthesis at elevated CO2 ( Leakey et al. 2009a). Although at the leaf and plant scales, stimulated respiration at elevated [CO2] may reduce net carbon balance, it is possible, nevertheless, that such stimulation could facilitate increased yield by providing greater energy for export of photoassimilate from source organs to sink tissues. However, the precise role of plant respiration in augmenting the sink capacity remains fragmented ( Gonzalez-Meler, Taneva & Trueman 2004 ).

Considerable research effort has additionally been directed towards the adaptive responses of respiratory metabolism to low oxygen concentrations. An important environmental stress condition that rapidly leads to the depletion of molecular oxygen within plant organs is flooding or water logging of the soil ( Bailey-Serres & Voesenek 2008 ). The most immediate effect on soil flooding is a decline in the oxygen concentration and a consequent decrease in aerobic root respiration leading to a restriction in ATP production. Furthermore, low availability of oxygen to plant cells can also occur under optimal growth conditions, because of the relatively high resistance to diffusion of oxygen through plant tissues ( van Dongen et al. 2011). Steep oxygen gradients have been observed in various plant tissues such as roots, stems, seeds or tubers ( Armstrong et al. 1994 Geigenberger et al. 2000 van Dongen et al. 2003 Borisjuk & Rolletschek 2009 Zabalza et al. 2009). Moreover, during development, local oxygen concentrations can vary, depending on the metabolic activity of the tissue ( van Dongen et al. 2003 Benamar et al. 2008). Therefore, the metabolic responses to low oxygen are directly involved in optimizing the plant's energy status while consuming as little oxygen as possible ( van Dongen et al. 2011 ).

It is well known that both metabolic and anatomical adjustments are important strategies in order to allow plants to cope with spatial and temporal variations of the oxygen availability. The major structural change is an increased formation of aerenchyma to lower the resistance to oxygen diffusion into the respiring tissue ( Drew, He & Morgan 2000 Jiang et al. 2010). From a metabolic perspective, the hypoxic responses includes the down-regulation of a suite of energy-, and therefore, oxygen-consuming, metabolic pathways ( Geigenberger 2003 ). Examples of such metabolic adaptations to hypoxia include the down-regulation of storage metabolism ( Geigenberger et al. 2000 ), the switch from invertase to sucrose synthase routes during sucrose hydrolysis ( Bologa et al. 2003 Huang, Colmer & Millar 2008 ) and the inhibition of mitochondrial respiration ( Gupta, Zabalza & van Dongen 2009 Zabalza et al. 2009). It seems reasonable to assume that these responses are already initiated before oxygen becomes limiting as a substrate for respiration. Therefore, it has been suggested that these metabolic changes are important components of the survival strategy as they considerably extend the period of hypoxia that a plant can withstand ( van Dongen et al. 2011 ).

Limited water availability, on the other hand, impairs plant growth and is one of the main issues of future climate changes ( Ciais et al. 2005 Loreto & Centritto 2008 ). Several studies on the effect of severe drought stress on respiratory pathways have revealed contrasting results, as respiration remained unaltered in soybean ( Ribas-Carbo et al. 2005 ), increased in wheat ( Bartoli et al. 2005 ), and decreased in bean and pepper ( Gonzalez-Meler, Matamala & Penuelas 1997 ). Nevertheless, the effects of mild to moderate water stress were relatively small on the mitochondrial activity of several key TCA cycle enzymes in two CAM species ( Herppich & Peckmann 2000 ). However, changes in the in vivo activities of the cytochrome oxidase (COX) and alternative oxidase (AOX) pathways, measured with the oxygen isotope fractionation technique that has been demonstrated to be the most reliable technique for the studies of electron partitioning between the two main respiratory pathways ( Ribas-Carbo et al. 1995 Day et al. 1996 ), have been reported by Ribas-Carbo and colleagues ( Ribas-Carbo et al. 2005 Flexas et al. 2006). In their study on soybean ( Ribas-Carbo et al. 2005 ), a decrease in COX activity was detected in leaves during severe drought stress, while AOX activity increased. Accordingly, despite complex I dysfunction and hence altered redox balance, the CMSII mutant seems to be able to adjust its photosynthetic machinery during and after drought stress to reduce photo-oxidation and to maintain the cell redox state and the ATP level ( Galle et al. 2010). Notwithstanding, identifying whether, and to what extent, plant species-specific factors and/or experimental conditions affect in vivo respiratory pathways, particularly the TCA cycle, under drought stress, awaits further studies.

It is clear, however, that although there have been a range of studies analysing changes in respiratory rates in response to light, temperature and CO2 ( Day et al. 1985 Atkin et al. 1997 Scheurwater et al. 2000 Kruse, Rennenberg & Adams 2011 ), our knowledge of the environmental impact on plant respiration and the TCA cycle remains fragmented. Although the global response of respiration is well characterized, the specific response of the TCA cycle enzymes and intermediates has only been described in a limited number of conditions. Nevertheless, the molecular, enzymatic and metabolic responses were observed in the case of biotic stress such as to moderately low nitrogen ( Tschoep et al. 2009 ), low carbon ( Gibon et al. 2006, 2009 Osuna et al. 2007 Usadel et al. 2008b ), low potassium ( Armengaud et al. 2009 ), small decreases in temperature ( Usadel et al. 2008a ) and water deficit ( Hummel et al. 2010 ), and this was interpreted as an adaptive response to maintain carbon flux through the TCA cycle. Moreover, a robust link between circadian-clock function and metabolic homeostasis in the TCA cycle was recently suggested ( Fukushima et al. 2009). Further studies are clearly needed to explore the interactions of mitochondrial non-phosphorylating pathways with photosynthetic processes and cell homeostasis under stressful conditions.

In summary, it is evident that there are several modes of regulation of the TCA cycle activity. For instance, proteomic studies have indicated that the TCA cycle enzymes (aconitase, succinyl-CoA ligase isocitrate, malate, pyruvate and succinate dehydrogenases) are potential targets for redox regulation ( Balmer et al. 2004). These results, associated with the allosteric properties of succinyl CoA ligase ( Studart-Guimarães et al. 2005 ) and with the ability to assess free, as opposed to bound, NADH levels ( Kasimova et al. 2006 ) when coupled with observations that glycolitic enzymes are functionally associated to the outer mitochondrial membrane ( Giege et al. 2003 ), suggest that many aspects of the regulation of TCA cycle remain to be elucidated. Additionally, there is a wealth of evidence suggesting that the TCA cycle is inhibited in the light as well as being transcriptionally down-regulated however, it is also equally clear that respiration remains active at considerable levels in illuminated leaves ( Atkin et al. 2000a Nunes-Nesi et al. 2008). Therefore, it seems likely that the physiological purpose for regulation is not the control of respiration op sigself but of other metabolic processes mediated by respiratory metabolism.


Perspektiewe

In assessing the merits of FBA as a tool to predict net CO2 evolution rates of plant tissues, we have been restricted by the number of tissue types and environmental conditions for which experimentally constrained metabolic flux maps are available as a point of comparison/validation. This is mainly because of the limited number of experimental systems that are suitable for the more tractable steady-state stable isotope MFA approach. In particular, because flux quantification in photosynthetic tissues requires the more challenging analysis of labelling time-courses, there is a shortage of quantitative flux maps for such tissues. So, although there have been several detailed FBA studies of photosynthetic tissues of higher plants (Montagud et al. 2010 de Oliveira Dal'Molin et al. 2010b Chang et al. 2011 Saha, Suthers & Maranas 2011 Nogales et al. 2012 ), at the time of writing, the only comparable experimental flux map of a sufficiently large-scale metabolic network is for the cyanobacterium Synechocystis (Young et al. 2011). Although the requirement for calculation of fluxes from dynamic labelling patterns is both experimentally and computationally more demanding, there are several groups that have been developing the necessary experimental and analytical tools for flux analysis in leaves in the light (Huege et al. 2007 Hasunuma et al. 2010 Keerberg et al. 2011 Lattanzi et al. 2012 ) and it is likely that flux maps will emerge in due course. Recently, a major step forward towards this goal was made with the publication of an analysis of the dynamic label redistribution of label from 13 CO2 supplied to Arabidopsis rosette leaves, from which a small set of fluxes were calculated (Szecowka et al. 2013). This paper establishes the experimental, analytical and mathematical frameworks that will allow a more systematic analysis of metabolic network fluxes in leaves and will facilitate the assessment of FBA for predicting CO2 evolution profiles in the dominant tissues of higher plants.

In summary, it is clear that FBA has the potential to predict fluxes through the CO2-consuming and CO2-generating processes in plant tissues, and based on existing work, it should be capable of predicting how the CO2 evolution profile will change in response to environment. Given the increasing interest in FBA as a tool to examine plant metabolic networks and the acceleration of sequencing of diverse plant genomes, there is every reason to expect that a more sophisticated, species-specific prediction of plant net CO2 evolution could ultimately be incorporated into higher-level ecosystem models.