NEBRASKA REDOX BIOLOGY CENTER EDUCATIONAL PORTAL

Nebraska Redox Biology Center Educational Portal


Glutaredoxins

Glutaredoxins are ubiquitous thioredoxin forld disulfide oxidoreductases which use glutathione as electron donor [ 1, 2, 3, 4 ]. Most of glutaredoxins have CxxC redox motifs in active site in which cysteines are separated by two other residues. One of cysteines in redox motif could be replased by serine or, in rare case, by another amino acid [ 1, 5, 6 ]. There are 117 solved glutaredoxin protein structure in Protein Data Bank at this time (June 2015).

3D structure of E. coli glutaredoxin.


Glutaredoxins reduces disulfide bonds via a dithiol and monothiol mechanism. The reduction of protein disulfides is going through a dithiol mechanism that requires both active site cysteines [ 5, 7, 8 ]. The reduction of glutathione-mixed disulfides can occur through a monothiol mechanism, which requires only one active site cysteine residue. In dithiol mechanism electrons are transferred from NADPH to glutathione reductase (GR), then to glutathione (GSH) and then to glutaredoxin (GRX), resulting in the GRX-mediated reduction of disulfides in target proteins. In monothiol mechanism reduction of glutathionylated proteins resulting in Grx-GSH conjugates formation deglutathionylation of target proteins. Glutaredoxins are also required for iron-sulfur cluster assembly and haem biosynthesis [ 7, 8, 9 ].

Reaction mechanism for dithiol and monothiol glutaredoxins.

Yeast Saccharomyces Cerevisiae Glutaredoxins:

Dithiol glutaredoxins

>gi|6319814|ref|NP_009895.1| Grx1 [Saccharomyces cerevisiae]
MVSQETIKHVKDLIAENEIFVASKTYCPYCHAALNTLFEKLKVPRSKVLVLQLNDMKEGADIQAALYEINGQRTVPNIYINGKHIGGNDDLQELRETGELEELLEPILAN

>gi|6320720|ref|NP_010801.1| Grx2 [Saccharomyces cerevisiae]
METNFSFDSNLIVIIIITLFATRIIAKRFLSTPKMVSQETVAHVKDLIGQKEVFVAAKTYCPYCKATLSTLFQELNVPKSKALVLELDEMSNGSEIQDALEEISGQKTVPN
VYINGKHIGGNSDLETLKKNGKLAEILKPVFQ

>gi|6323396|ref|NP_013468.1| Grx-like protein [Saccharomyces cerevisiae]
MSAFVTKAEEMIKSHPYFQLSASWCPDCVYANSIWNKLNVQDKVFVFDIGSLPRNEQEKWRIAFQKVVGSRNLPTIVVNGKFWGTESQLHRFEAKGTLEEELTKIGLLP


Mothiol glutaredoxins

>gi|6320303|ref|NP_010383.1| Grx3 [Saccharomyces cerevisiae]
MCSFQVPSAFSFNYTSYCYKRHQARYYTAAKLFQEMPVIEINDQEQFTYLTTTAAGDKLIVLYFHTSWAEPCKALKQVFEAISNEPSNSNVSFLSIDADENSEISELFEIS
AVPYFIIIHKGTILKELSGADPKEYVSLLEDCKNSVNSGSSQTHTMENANVNEGSHNDEDDDDEEEEEETEEQINARLTKLVNAAPVMLFMKGSPSEPKCGFSRQLVGILR
EHQVRFGFFDILRDESVRQNLKKFSEWPTFPQLYINGEFQGGLDIIKESLEEDPDFLQHALQS

>gi|6321022|ref|NP_011101.1| Grx4 [Saccharomyces cerevisiae]
MTVVEIKSQDQFTQLTTTNAANKLIVLYFKAQWADPCKTMSQVLEAVSEKVRQEDVRFLSIDADEHPEISDLFEIAAVPYFVFIQNGTIVKEISAADPKEFVKSLEILSNA
SASLANNAKGPKSTSDEESSGSSDDEEDETEEEINARLVKLVQAAPVMLFMKGSPSEPKCGFSRQLVGILREHQIRFGFFDILRDENVRQSLKKFSDWPTFPQLYINGEFQ
GGLDIIKESIEEDPEYFQHALQ

>gi|6325198|ref|NP_015266.1| Grx5 [Saccharomyces cerevisiae]
MFLPKFNPIRSFSPILRAKTLLRYQNRMYLSTEIRKAIEDAIESAPVVLFMKGTPEFPKCGFSRATIGLLGNQGVDPAKFAAYNVLEDPELREGIKEFSEWPTIPQLYVNK
EFIGGCDVITSMARSGELADLLEEAQALVPEEEEETKDR

>gi|6320193|ref|NP_010274.1| Grx6 [Saccharomyces cerevisiae]
MIPSNKRNARILSITTLLLLLVFFVAQNANFLTVEIKEETSKAFSTNMDNMAGGSSREYAAMPTSTTNKGSSEVDEEINEIKQKVGLQQPIASVDDSLSAIKNDKGSRITK
AFNVQKEYSLILDLSPIIIFSKSTCSYSKGMKELLENEYQFIPNYYIIELDKHGHGEELQEYIKLVTGRGTVPNLLVNGVSRGGNEEIKKLHTQGKLLESLQVWSDGKFSV
EQREKPSNN

>gi|6319488|ref|NP_009570.1| Grx7 [Saccharomyces cerevisiae]
MAIVINKRNVRVLVITNLLLIVVFFVLRNSNASVNESITTHHPDSLVTFDNSGNAPGTHQSVHDTVNTQDKEAEEVDKNSGDAEFDAAAEYNKIMEQSPMIVFSKTGCPYS
KKLKALLTNSYTFSPSYYVVELDRHEHTKELQDQIEKVTGRRTVPNVIIGGTSRGGYTEIAELHKNDELLDSFKKWSDGAFTVKANSQSESA



Measurement of glutaredoxin activity. The major and most commonly used assay is based on reduction of disulfides in target molecule in presense of glutathione, NADPH and glutathione reductase [ 10 ]. The reaction rate can be followed spectrophotometrically by decrease in absorbanse as result of NADPH oxidation:

2GSH + X-S-S-X +GRX → GSSG + 2 XSH ;

GSSG + NADPH + H+ → 2GSH + NADP+

Numerous commertially avaliable assays are besed on similar reactions with fluorescently labeled disulfide substrate [ 11 ].

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