The structures of pyruvate oxidase fromAerococcus viridanswith cofactors and with a reaction intermediate reveal the flexibility of the active-site tunnel for catalysis

Abstract

The crystal structures of pyruvate oxidase from Aerococcus viridans (AvPOX) complexed with flavin adenine dinucleotide (FAD), with FAD and thiamine diphosphate (ThDP) and with FAD and the 2-acetyl-ThDP intermediate (AcThDP) have been determined at 1.6, 1.8 and 1.9 A resolution, respectively. Each subunit of the homotetrameric AvPOX enzyme consists of three domains, as observed in other ThDP-dependent enzymes. FAD is bound within one subunit in the elongated conformation and with the flavin moiety being planar in the oxidized form, while ThDP is bound in a conserved V-conformation at the subunit-subunit interface. The structures reveal flexible regions in the active-site tunnel which may undergo conformational changes to allow the entrance of the substrates and the exit of the reaction products. Of particular interest is the role of Lys478, the side chain of which may be bent or extended depending on the stage of catalysis. The structures also provide insight into the routes for electron transfer to FAD and the involvement of active-site residues in the catalysis of pyruvate to its products.

Biological and Chemical Adaptation to Endogenous Hydrogen Peroxide Production in Streptococcus pneumoniae D39

“…The average activity is 0.32 ± 0.04 µmol·min −1 ·mg −1 protein (dashed line) and is independent of H 2 O 2 over this concentration range. (C) Structure of Aerococcus viridans (pyruvate oxidase [AvPOX] subunit [1V5G; 69% identity, 82% similarity to S. pneumoniae SpxB]) (94) highlighting the FAD and TPP cofactors, the latter as the 2-acetyl-thiamine diphosphate (2-Ac-TDP) reaction intermediate, the Mg(II) ion, and the approximate position of two Cys (C148 and C447, based on S. pneumoniae residue numbering) not found in A. viridans pyruvate oxidase. C447 is quite close to the active site, while C148 is sulfonylated in cells (Table S2D).…”

A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

“…cerevisiae ( Sc PDC, Swissprot: P06169, PDB: 2VK8 [6]), was used as the reference sequence for numbering all proteins of the decarboxylase superfamily. In addition, 22 functionally and structurally relevant residues in the sequence of Sc PDC were annotated as described in literature [2,5,28,30,31,37-39] (Additional file 1: Table S1). These positions include the highly conserved active site residues E51 (standard numbering) [40-43], the conserved HH motif in PDCs (H114/H115) [28], the GDGX motif 443–446 and the Mg 2+ binding site N471 [39], as well as more variable regions such as the S -pocket residues P26, G27, I476, and Q477 [29-31] and the start and end position of the three decarboxylase domains, the PYR, PP, and the TH3 domain [2].…”

Determination of Pre-Steady-State Rate Constants on the Escherichia coli Pyruvate Dehydrogenase Complex Reveals That Loop Movement Controls the Rate-Limiting Step

“…In related oxidative decarboxylases such as pyruvate oxidase and pyruvate ferredoxin oxidoreductase the enamine is oxidized by enzyme-bound cofactors such as FAD or FeS clusters, respectively, and the resultant acetyl moieties are transferred to phosphate or CoA acceptor groups, respectively. This allows accumulation of 2-acetylthiamin diphosphate (AcThDP) in the latter enzymes when the acceptors are absent 23–25 . However, during the overall PDHc pathway, since the lipoamide serves both functions and AcThDP does not appear to accumulate at significant levels as shown by Frey et al, 26 this hinders pre-steady state studies aimed at determining the rates of oxidation.…”