Abstract
Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.
Highlights
We present a series of crystal structures of the ThDPdependent phenylpyruvate decarboxylase in complex with different substrates and an analogue of a covalent reaction intermediate
Substrate and Covalent Intermediate Complexes of PPDC—A standard procedure to obtain detailed structural information on the catalytic mechanism of an enzyme is to solve the structure of slow mutants with trapped substrates or covalent reaction intermediates
In a different approach we used unreactive analogues of the coenzyme to trap intermediates in a thiaminedependent enzyme. 3dThDP is such an unreactive analogue of Thiamine diphosphate (ThDP), in which the single nitrogen atom of the thiazolium ring is replaced by a carbon [23]
Summary
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]. Complexes of PPDC with the inhibitors 3-deaza-ThDP (PPDC-3dThDP) and 2-(1-hydroxyethyl)-3-deaza-ThDP (PPDC2HE3dThDP) were subsequently obtained by incubating PPDC (purified without addition of ThDP) with 1 mM 3dThDP or 2HE3dThDP for 48 h at 4 °C. Complete exchange of ThDP with the analogues was confirmed by total loss of activity with phenylpyruvate as a substrate. The tertiary complexes with phenylpyruvic acid (PPDC-3dThDP-PPA) and 5-phenyl-2oxovaleric acid (PPDC-3dThDP-POVA) were obtained
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