Glutamate 636 of the Escherichia coli Pyruvate Dehydrogenase-E1 Participates in Active Center Communication and Behaves as an Engineered Acetolactate Synthase with Unusual Stereoselectivity

Abstract

The residue Glu 636 is located near the thiamine diphosphate (ThDP) binding site of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), and to probe its function two variants, E636A and E636Q were created with specific activities of 2.5 and 26% compared with parental PDHc-E1. According to both fluorescence binding and kinetic assays, the E636A variant behaved according to half-of-the-sites mechanism with respect to ThDP. In contrast, with the E636Q variant a K d,ThDP ‫؍‬ 4.34 M and K m,ThDP ‫؍‬ 11 M were obtained with behavior more reminiscent of the parental enzyme. The CD spectra of both variants gave evidence for formation of the 1,4-iminopyrimidine tautomer on binding of phosphonolactylthiamine diphosphate, a stable analog of the substrate-ThDP covalent complex. Rapid formation of optically active (R)-acetolactate by both variants, but not by the parental enzyme, was observed by CD and NMR spectroscopy. The acetolactate configuration produced by the Glu 636 variants is opposite that produced by the enzyme acetolactate synthase and the Asp 28 -substituted variants of yeast pyruvate decarboxylase, suggesting that the active centers of the two sets of enzymes exhibit different facial selectivity (re or si) vis à vis pyruvate. The tryptic peptide map (mass spectral analysis) revealed that the Glu 636 substitution changed the mobility of a loop comprising amino acid residues from the ThDP binding fold. Apparently, the residue Glu 636 has important functions both in active center communication and in protecting the active center from undesirable "carboligase" side reactions.The Escherichia coli pyruvate dehydrogenase complex (PDHc) 1 plays a pivotal role in carbohydrate utilization by bacterial cells according to the Reaction 1.The first subunit of the complex, pyruvate dehydrogenase (PDHc-E1) is a dimer of two identical subunits of molecular mass of 99,474 Da each and has thiamine diphosphate (ThDP) as a cofactor in each of the two active centers as depicted in Scheme 1. The three-dimensional structure of the E. coli PDHc-E1 has been solved in complex with ThDP (2) and with thiamine 2-thiazolone diphosphate (ThTDP) (3), an analog of ThDP with C2 ϭ O substitution in place of C2 ϭ H. In the crystal structure of PDHc-E1 with ThTDP, the residue Glu 636 (Glu 636 PDHc-E1) participates in an extensive hydrogen bonding network with amino acid residues in the active site important for binding of ThTDP and probably binding of reaction intermediates as well ( Fig. 1) (3). We carried out substitution at this position, resulting in the E636A and E636Q PDHc-E1 variants. A variety of tools were used to study the kinetic and structural/mobility consequences of the substitutions, leading us to conclude that the residue Glu 636 in PDHc-E1 has multiple functions: participation in monomer-monomer interactions; affecting the mobility of some key loops located near the active center; and protecting the active site from undesirable "carboligase" side reactions. The carboligase side products, acetoin and acetolacta...

Efficient coupling of catalysis and dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex

“…Our results regarding loop movement refer to isolated E1ec component. For the overall reaction of the complex, reductive acetylation is probably rate limiting (11). In pyruvate decarboxylase (catalyzing the sequence of reactions as E1ec through decarboxylation) from both Zymomonas mobilis and Saccharomyces cerevisiae, product release is the rate-limiting step (29).…”

“…In contrast, such a plot for E401K was linear and displayed a much larger slope (0.8 Ϯ 0.1). Our earlier studies signaled that the rate-limiting transition state in E1ec catalysis involves steps through formation of LThDP (i.e., predecarboxylation steps) (11). Consistent with this observation, similar values are observed for k cat for pyruvate (3.2 Ϯ 0.3 s Ϫ1 ) and the rate of formation of PLThDP (k 1 obs ϭ 3.6 Ϯ 0.2 s Ϫ1 ) at ϭ 1.0 in E1ec (SI Table 2 and Fig.…”

The Pyruvate Dehydrogenase Complexes: Structure-based Function and Regulation

“…Solution NMR evidence of nonequivalence of two active centers in E1h and E1ec was obtained from H/D exchange kinetics at C2-H of E1-bound ThDP (42,43) and by analysis of covalent ThDPbound intermediates in E1ec and E1h catalysis (54,61). A "proton wire" mechanism (62, 63) with a hydrated "tunnel" of acidic residues and water molecules was suggested to enable direct communication for proton shuttling between two ThDP N1Ј atoms.…”

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

“…Variants of 2-ketoacid decarboxylases can be engineered to enhance rates of production of carboligase products. Examples include the E636A E1p variant of E. coli pyruvate dehydrogenase (k cat ϭ 1 s Ϫ1 ) and the D28A variant of Saccharomyces cerevisiae pyruvate decarboxylase (k cat ϭ 0.05 s Ϫ1 ), which behave like acetolactate synthase (31), and the E473Q variant of Zymomonas mobilis pyruvate decarboxylase, which catalyzes the enantioselective conversion of pyruvate and benzaldehyde to (R)-phenylacetylcarbinol at 2.5 s Ϫ1 (32). This comparison suggests that HOAS is indeed catalytically competent toward C-C bond-forming reactions and even surpasses the engineered variants when AcCoA activation is taken into account.…”