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Indexed by:期刊论文

Date of Publication:2018-01-01

Journal:Applied microbiology and biotechnology

Included Journals:PubMed、SCIE

Volume:102

Issue:15

Page Number:6479-6491

ISSN No.:1432-0614

Key Words:Acetolactate decarboxylase; Acetoin; Bacillus subtilis; Crystal structure

Abstract:Acetoin is an important physiological metabolite excreted by microbes. Its functions include avoiding acidification, participating in regulation of the NAD+/NADH ratio, and storing carbon. Acetolactate decarboxylase is a well-characterized anabolic enzyme involved with 3-hydroxy butanone (acetoin). It catalyzes conversion of the (R)- and (S)-enantiomers of acetolactate to generate the single product, (R)-acetoin. In addition to the X-ray crystal structure of acetolactate decarboxylase from Bacillus brevis, although the enzyme is widely present in microorganisms, very few atomic structures of acetolactate decarboxylase are reported. In this paper, we solved and reported a 1.5A resolution crystal structure of acetolactate decarboxylase from Bacillus subtilis. Dimeric assembly is observed in the solved structure, which is consistent with the elution profile conducted by molecular filtration. A zinc ion is coordinated by highly conserved histidines (191, 193, and 204) and conserved glutamates (62 and 251). We performed kinetic studies on acetolactate decarboxylase from Bacillus subtilis using circular dichroism, allowing the conversion of acetolactate to chiral acetoin for real-time tracking, yielding a Km value of 21mM and a kcat value of 2.2s-1. Using the two enantiomers of acetolactate as substrates, we further investigated the substrate preference of acetolactate decarboxylase from Bacillus subtilis by means of molecular docking and dynamic simulation in silico. The binding free energy of (S)-acetolactate was found to be ~30kcal/mol greater than that of (R)-acetolactate, indicating a more stable binding for (S)-acetolactate.