The crystal structure of phenylpyruvate decarboxylase from Azospirillum brasilense at 1.5 Å resolution

Abstract

Phenylpyruvate decarboxylase (PPDC) of Azospirillum brasilense, involved in the biosynthesis of the plant hormone indole‐3‐acetic acid and the antimicrobial compound phenylacetic acid, is a thiamine diphosphate‐dependent enzyme that catalyses the nonoxidative decarboxylation of indole‐ and phenylpyruvate. Analogous to yeast pyruvate decarboxylases, PPDC is subject to allosteric substrate activation, showing sigmoidal v versus [S] plots. The present paper reports the crystal structure of this enzyme determined at 1.5 Å resolution. The subunit architecture of PPDC is characteristic for other members of the pyruvate oxidase family, with each subunit consisting of three domains with an open α/β topology. An active site loop, bearing the catalytic residues His112 and His113, could not be modelled due to flexibility. The biological tetramer is best described as an asymmetric dimer of dimers. A cysteine residue that has been suggested as the site for regulatory substrate binding in yeast pyruvate decarboxylase is not conserved, requiring a different mechanism for allosteric substrate activation in PPDC. Only minor changes occur in the interactions with the cofactors, thiamine diphosphate and Mg2+, compared to pyruvate decarboxylase. A greater diversity is observed in the substrate binding pocket accounting for the difference in substrate specificity. Moreover, a catalytically important glutamate residue conserved in nearly all decarboxylases is replaced by a leucine in PPDC. The consequences of these differences in terms of the catalytic and regulatory mechanism of PPDC are discussed.

Characterization of Phenylpyruvate Decarboxylase, Involved in Auxin Production of Azospirillum brasilense

“…The recombinant protein appeared as a tetrameric form in size-exclusion chromatography, which is consistent with other ThDP-dependent 2-keto acid decarboxylases (2,11,13) and the recently determined crystal structure of PPDC Ab , which shows the enzyme as a dimer of dimers (40). The kinetic analysis revealed quite surprising results.…”

“…Binding of the activator to ScPDC induces a conformational change in the enzyme: two (of the four) active sites transform from an open to a closed (ϭactive) form, which might be the molecular explanation for the substrate activation behavior (21,22). Since the pivotal cysteine in the proposed activation mechanism of ScPDC is not conserved in PPDC Ab (40), it will be interesting to unravel the activation mechanism used by the latter. Numerous sequences annotated as putative IPDCs and PPDCs are present in the NCBI database, but only for two has the activity of the purified enzymes been determined (PPDC Ab [this study] and IPDC Ec ).…”

“…Here, protonation of the enamine-carbanion intermediate is proposed to be mediated by a catalytic histidine residue in the active site (27). In PPDC Ab , it was proposed that the leucine, which is located underneath the carboxylate group of the lactyl-ThDP intermediate, increases the hydrophobic character of the active site and consequently stabilizes the zwitterionic intermediates (40). In view of this clear partitioning, site-directed mutagenesis studies at this and other amino acid residue positions (see below) are ongoing, to provide information on their role in catalysis and substrate activation.…”

Molecular Mechanism of Allosteric Substrate Activation in a Thiamine Diphosphate-dependent Decarboxylase

“…Protein Expression, Purification, and Preparation of the Complexes-The wild-type AbPPDC was cloned, expressed, and purified as described previously (7,12). During purification the enzyme activity was monitored with phenylpyruvate as substrate using the established coupled optical test with horse liver alcohol dehydrogenase and NADH (13).…”

“…The latter compound is a plant hormone, which is responsible for the plant growth promoting abilities of A. brasilense (10). AbPPDC shows high structural similarity to other 2-ketoacid decarboxylases from the pyruvate oxidase family (11), such as pyruvate decarboxylase (PDC) and indolepyruvate decarboxylase (IPDC) (12). Similar to several PDCs, the AbPPDC displays substrate activation with indolepyruvate and other substrates, characterized by sigmoidal v versus [S] plots (7).…”