A structural phylogeny for understanding 2-oxoacid oxidoreductase function

Abstract

2-Oxoacid:ferredoxin oxidoreductases (OFORs) are essential enzymes in microbial one-carbon metabolism. They use thiamine pyrophosphate to reversibly cleave carbon-carbon bonds, generating low potential (~ −500 mV) electrons. Crystallographic analysis of a recently discovered OFOR, an oxalate oxidoreductase (OOR), has provided a second view of OFOR architecture and active site composition. Using these recent structural data along with the previously determined structures of pyruvate:ferredoxin oxidoreductase, structure-function relationships in this superfamily have been expanded and re-evaluated. Additionally, structural motifs have been defined that better serve to distinguish one OFOR subfamily from another and potentially uncover novel OFORs.

A Reverse TCA Cycle 2-Oxoacid:Ferredoxin Oxidoreductase that Makes C-C Bonds from CO2

“…As described above, the shortening of the helix-loop motif following Cys60b effectively pulls the helix of the motif away from the active site, allowing for the binding of the larger succinyl-CoA or 2-oxoglutarate. Revisiting the sequence analysis previous published, 15 we find that shorter loop lengths correlate to larger substrates as a general trend in the OFOR family. The helix-loop motifs of OGOR, VOR, and IOR are all shorter than that of OOR and PFOR ( Figure S16), and the substrates of OGOR, VOR, and IOR (2-oxoglutarate, 2-oxoisovalerate, and indolepyruvate) are all larger than that of OOR or PFOR (oxalate and pyruvate).…”

“…In summary, these data (in addition to previously available structural and biochemical data) 7,15,23,24 reveal how changes in the length of a loop, or positioning of a charged residue, or installation of second layer interactions can dramatically impact substrate preference or even reaction direction. We find similar mechanistic strategies co-opted by different family members and merged with other strategies to afford altered reactivities.…”

“…Arg303a and Thr227a, which are conserved 2-oxoacid binding residues in all structurally characterized CoA-dependent OFORs, 15 are also found in MmOGOR ( Figure 3B). MmOGOR has a second Arg in the active site, Arg63b from the helix-loop motif, which is 10.7 Å from the C2 of TPP.…”

“…Divided by substrate specificity, OFORs can be categorized into 2-oxoglutarate:ferredoxin oxidoreductase (OGOR), 9 pyruvate:ferredoxin oxidoreductase (PFOR), 10 2-oxoisovalerate:ferredoxin oxidoreductase (VOR), 11 indopyruvate:ferredoxin oxidoreductase (IOR), 12,13 and oxalate oxidoreductase (OOR) 6,14 (Figure S2). Notably, OFORs adapt different oligomeric states ( Figure 1C) and are mostly coenzyme A (CoA)-dependent except for OOR, 15 which is specific for cleaving oxalate into CO 2 without CoA. 6,14 Although PFOR and OGOR from the rTCA cycle engage in CO 2 fixation, on many occasions, OFORs catalyze the reversal reaction to oxidize a 2-oxoacid and provide low-potential electrons for downstream reactions, including sulfate reduction, 16 dinitrogen reduction, 17 and aromatic compound reduction.…”

“…However, OGOR enzymes found in the rTCA largely are composed of enzymes that differ from other OFORs in that they lack the ferredoxin domain (domain V) that harbors two [4Fe-4S] clusters ( Figure 1C). 15 In other words, bioinformatics predicts that an OGOR is an OFOR with only one [4Fe-4S] cluster, which should be insufficient for the two-electron process. We have turned to investigating the structural and biochemical properties of a native rTCA OGOR, as well as its physiologically relevant Fd, in order to understand how an OFOR can be biased toward CO 2 reduction and how electron transfer chemistry may operate in support of the rTCA cycle.…”