Mechanistic Studies of 1-Deoxy-D-Xylulose-5-Phosphate Synthase from Deinococcus radiodurans

Abstract

The non-mevalonate dependent (NMVA) pathway for the biosynthesis of isopentenyl pyrophosphate and dimethylallyl pyrophosphate is the sole source of these terpenoids for the production of isoprenoids in the apicomplexan parasites, in many eubacteria, and in plants. The absence of this pathway in higher organisms has opened a new platform for the development of novel antibiotics and anti-malarials. The enzyme catalyzing the first step of the NMVA pathway is 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). DXPS catalyzes the thiamine pyrophosphate- and Mg (II)-dependent conjugation of pyruvate and D-glyceraldehyde-3-phosphate to form 1-deoxy-D-xylulose-5-phosphate and CO2. The kinetic mechanism of DXPS from Deinococcus radiodurans most consistent with our data is random sequential as shown using a combination of kinetic analysis and product and dead-end inhibition studies. The role of active site amino acids, identified by sequence alignment to other DXPS proteins, was probed by constructing and analyzing the catalytic efficacy of a set of targeted site-directed mutants.

Active Site Histidines Link Conformational Dynamics with Catalysis on Anti-Infective Target 1-Deoxy-d-xylulose 5-Phosphate Synthase

“…We have recently reported the first ligand-bound crystal structures of Deinococcus radiodurans DXP synthase that provide further support for this hypothesis. 16 Consistent with mutagenesis results, 19,21,22 the structure of DXP synthase with PLThDP bound shows that H51 and H304 (analogous to E. coli DXP synthase residues H49 and H299, respectively) are within hydrogen bonding distance of the phosphonyl group of PLThDP in the closed conformation (Figure 2A,C). By extension, H51 and H304 are predicted to interact with and stabilize the carboxyl group of the predecarboxylation intermediate LThDP.…”

“…18 These results suggest a role for the flexible EX1 regions in catalysis and hint at residues that may link conformational dynamics to the unique catalytic mechanism of DXP synthase. Two active site histidine residues (H49 and H299 on E. coli DXP synthase) proposed to be important for pyruvate binding and catalysis 19,21,22 reside within (H49) and adjacent to (H299) EX1 regions 1 and 3, respectively, suggesting they may play additional roles in coordinating the pyruvate-dependent conformational change to a closed form.…”

X-ray crystallography–based structural elucidation of enzyme-bound intermediates along the 1-deoxy-d-xylulose 5-phosphate synthase reaction coordinate

“…Two key differences between DXPS and other ThDP-dependent enzymes include its ability to stabilize the pre-decarboxylation intermediate, LThDP, and the requirement for ternary complex formation in catalysis, enabled in part by a large active site (15,23). Several DXPS enzymes have been shown to follow a unique random sequential mechanism (15,18,21,48). Similar to other DXPS enzymes, DrDXPS displays DXP-forming activity (Table S1 and Fig.…”

“…2, inset; 3A; and S8A). His-434, predicted to be involved in pyruvate binding (48,49), and N4Ј of ThDP are positioned to form hydrogen bonds with the C2␣-hydroxyl of PLThDP, and Phe-398 stacks against the pyrimidine ring of the ThDP. His-51 and His-304, also predicted to be critical for catalysis in DXPS enzymes (48 -50), are positioned to make favorable electrostatic interactions with the phosphonate of PLThDP (Fig.…”

Toward Understanding the Chemistry and Biology of 1-Deoxy-d-xylulose 5-Phosphate (DXP) Synthase: A Unique Antimicrobial Target at the Heart of Bacterial Metabolism

“…Hints of a conformational change upon d -GAP binding are evident by CD 18,25 and fluorescence binding experiments. 18–20 Stopped-flow CD analysis of d -GAP-induced LThDP decarboxylation on DXPS reveals a 20 – 30 msec delay between d -GAP addition and the exponential decrease in LThDP, 18,25 possibly arising from a conformational change reorienting LThDP for decarboxylation. Additional evidence of d -GAP-induced conformational change comes from tryptophan fluorescence experiments performed by us on E. coli DXPS 18 , and by the Merkler lab where titration of D. radiodurans DXPS with d -GAP resulted in a Stokes shift of tryptophan fluorescence, indicating local environmental tryptophan changes upon d -GAP binding.…”