Structural insights into conformationally-gated reactions catalyzed by thiamine pyrophosphate-dependent enzymes

Abstract

Thiamine pyrophosphate (TPP)-dependent enzymes utilize TPP as a biological carbanion to initiate reactions on substrates with carbonyl carbon(s), such as pyruvate and 2-oxoglutarate, two central metabolites. This thesis presents structural analyses of three TPP-dependent enzymes. Most interestingly, each mechanism of three proteins studied contains a gated step that is drastically accelerated by 3-5 orders of magnitudes through conformational changes. 1-deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the conversion of pyruvate and D-glyceraldehyde 3-phosphate (D-GAP or G3P) into DXP, an essential precursor of isoprenoids, vitamin B1, and vitamin B6 (pyridoxal phosphate, PLP) in bacterial pathogens. Human adapts different pathways to access those essential metabolites; thus, selective inhibition of DXP synthase has been considered to be a possible approach for antibiotic therapies.

Surprisingly, decarboxylation of pyruvate by DXP synthase is accelerated by 600-fold in the presence of DGAP, the molecular basis of which was unknown. Here we present crystallographic snapshots at different stages of the DXP synthase reaction using the well-studied DXP synthase from Deinococcus radiodurans in order to evaluate the molecular basis of the D-GAP-based rate enhancement and afford a deeper mechanistic understanding that will further pharmaceutical campaigns. Pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) reversibly oxidize 2-oxoacids and coenzyme A (CoA) into CO 2 and acyl-CoA. This enzyme family (2-oxoacid:ferredoxin oxidoreductase, OFOR) is essential in three of seven of the known pathways of biological CO 2 fixation and has consequently been a focus for biological engineering. In addition to TPP, OFORs bind [4Fe-4S] clusters that mediate electron transfers between reaction intermediates and external ferredoxins.

Remarkably, CoA binding accelerates electron transfer from the reaction intermediate to an enzyme-bound [4Fe- 4S] cluster by as much as 100,000-fold. In this work, we resolved the long-standing question of how CoA achieves this dramatic rate acceleration by acquiring the first structure of an OFOR with CoA bound. The structures of PFOR, and later OGOR, revealed the binding mode of CoA and showed dramatic conformational changes accompany CoA binding. Comparisons between PFOR and OGOR further explain the molecular basis for substrate specificity in the OFOR family. Our findings are expected to facilitate future efforts for bioengineering CO 2 fixation pathways.