High Resolution Crystal Structures of the Acetohydroxyacid Synthase-Pyruvate Complex Provide New Insights into Its Catalytic Mechanism

摘要

abstract_textpAcetohydroxyacid synthase (AHAS) is the first enzyme in the biosynthesis pathway of the branched-chain amino acids, catalyzing the condensation of pyruvate with another molecule of pyruvate or with 2-ketobutyrate, to produce 2-acetolactate or 2-acetohydroxybutyrate, respectively. The catalytic subunit of the dimeric enzyme has thiamin diphosphate (ThDP), a divalent metal ion, flavin adenine dinucleotide (FAD), and two molecules of oxygen (O-2(I) and O-2(II)) as cofactors. Here, crystal structures of Saccharomyces cerevisiae AHAS in complex with pyruvate provide novel insights into the mechanistic features of this enzyme including: i) The precise position taken by pyruvate molecules as they enter the active site (i.e. prior to catalysis occurring) with conformations suitable for the transfer of electrons to/from O-2(I) and FAD; ii) The formation of ternary donor-acceptor-O-2(I) complexes and iii) The location of O-2(II) relative to the substrate showing that it plays a critical role in the organization of substrate for catalysis. These structural data, accompanied by electron paramagnetic resonance evidence that a radical is produced during AHAS catalysis, lead to the proposal that FAD and O-2 are involved in an indirect one-electron redox cycle. In this mechanism, the spatial configurations of O-2 and FAD in the active site can allow electrons to be exchanged with the substrates and catalytic intermediates to satisfy and control the overall AHAS catalyzed reaction./p/abstract_text