Double Duty for a Conserved Glutamate in Pyruvate Decarboxylase: Evidence of the Participation in Stereoelectronically Controlled Decarboxylation and in Protonation of the Nascent Carbanion/Enamine Intermediate,

Abstract

Pyruvate decarboxylase (PDC) catalyzes the nonoxidative decarboxylation of pyruvate into acetaldehyde and carbon dioxide and requires thiamin diphosphate (ThDP) and a divalent cation as cofactors. Recent studies have permitted the assignment of functional roles of active site residues; however, the underlying reaction mechanisms of elementary steps have remained hypothetical. Here, a kinetic and thermodynamic single-step analysis in conjunction with X-ray crystallographic studies of PDC from Zymomonas mobilis implicates active site residue Glu473 (located on the re-face of the ThDP thiazolium nucleus) in facilitating both decarboxylation of 2-lactyl-ThDP and protonation of the 2-hydroxyethyl-ThDP carbanion/enamine intermediate. Variants carrying either an isofunctional (Glu473Asp) or isosteric (Glu473Gln) substitution exhibit a residual catalytic activity of less than 0.1% but accumulate different intermediates at the steady state. Whereas the predecarboxylation intermediate 2-lactyl-ThDP is accumulated in Glu473Asp because of a 3000-fold slower decarboxylation compared to that of the wild-type enzyme, Glu473Gln is not impaired in decarboxylation but generates a long-lived 2-hydroxyethylThDP carbanion/enamine postdecarboxylation intermediate. CD spectroscopic analysis of the protonic and tautomeric equilibria of the cocatalytic aminopyrimidine part of ThDP indicates that an acidic residue is required at position 473 for proper substrate binding. Wild-type PDC and the Glu473Asp variant bind the substrate analogue acetylphosphinate with the same affinity, implying † This work was supported in part by Grant 0126FP/0705M from the Ministry of Education at Saxony-Anhalt and the DFG-funded Göttingen Graduate School for Neurosciences and Molecular Biosciences (both to K.T.). At Rutgers University, the research was supported by National Institutes of Health Grant GM050380 (to F.J.). ‡ The refined model and corresponding structure factor amplitudes have been deposited in the Protein Data Bank of the Research Collaboratory for Structural Biology as entry 3OE1.© American Chemical Society * To whom correspondence should be addressed. Phone: +49-551-3914430. Fax: +49-551-395749. ktittma@gwdg.de. # Current address: Institute for Microbiology and Genetics, Georg-August-University Göttingen, Göttingen, Germany NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 September 21. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript a similar stabilization of the predecarboxylation intermediate analogue on the enzyme, whereas Glu473Gln fails to bind the analogue. The X-ray crystallographic structure of 2-lactyl-ThDP trapped in the decarboxylation-deficient variant Glu473Asp reveals a common stereochemistry of the intermediate C2α stereocenter; however, the scissile C2α-C(carboxylate) bond deviates bỹ 25-30° from the perpendicular "maximum overlap" orientation relative to the thiazolium ring plane as commonly observed in ThDP enzymes. Because a reactant-state stabiliza...

Defining critical residues for substrate binding to 1‐deoxy‐d‐xylulose 5‐phosphate synthase – active site substitutions stabilize the predecarboxylation intermediate C2α‐lactylthiamin diphosphate

“…The structure of the stable LThDP analog phosphonolactyl ThDP on PDHc E1 in the V‐conformation shows an almost perpendicular C2α–Pβ bond . An additional study on Zymomonas mobilis pyruvate decarboxylase may also provide insights into the long‐lived LThDP intermediate on DXP synthase. In this case, LThDP accumulates on E437D pyruvate decarboxylase, and the scissile C2α–C(carboxylate) bond deviates significantly from the perpendicular orientation required for efficient decarboxylation.…”

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

“…Ile469 is, however, located in a region of the enzyme which may be sensitive to changes. It is positioned adjacent to Glu468, important in catalytic activity, Ile467, involved in substrate recognition, and Ile471, crucial for substrate positioning (Pohl et al, 1998;Siegert et al, 2005;Meyer et al, 2010). Figure 1).…”

“…, 2010) and supports the interpretation that PDCs requires a protonated residue for efficient binding of the substrate. The ionizable group is thought to be the aminopyrimidine ring of the ThDP coenzyme (Meyer et al, 2010). The GoxPDC enzyme displayed normal Michaelis Menten kinetics with pyruvate as the substrate and was not subject to allosteric substrate activation as has been seen in PDCs from plants, yeasts and the bacterium S. ventriculi (Konig et al, 2009).…”

Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity

“…This geometry is thought to promote decarboxylation by allowing maximum overlap of π electrons of the thiazolium ring and the p-orbital of the scissile bond [25,45]. The conformation has been observed in the X-ray structures of all the first tetrahedral intermediates, and intermediate analogues [25,42,[46][47][48]. Further, Ser26 has been proposed to assist in the decarboxylation step by carrying out a nucleophilic attack on carboxylate group of MThDP [40].…”

“…To see how this was achieved we compared the binding of the LTHDP to PDC and to BFDC T377L/A460Y. Previously, the ZmPDC E473D structure in complex with LThDP was solved [42]. In this structure the methyl substituent of the lactyl moiety is within 5 Å of 11 atoms evenly distributed across seven different residues.…”