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Applied Microbiology and Biotechnology

, Volume 102, Issue 15, pp 6479–6491 | Cite as

Structural and enzymatic characterization of acetolactate decarboxylase from Bacillus subtilis

  • Fangling Ji
  • Mingyang Li
  • Yanbin Feng
  • Sijin Wu
  • Tianqi Wang
  • Zhongji Pu
  • Jingyun Wang
  • Yongliang Yang
  • Song Xue
  • Yongming Bao
Biotechnologically relevant enzymes and proteins
  • 245 Downloads

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 1.5 Å 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 21 mM and a kcat value of 2.2 s−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 ~ 30 kcal/mol greater than that of (R)-acetolactate, indicating a more stable binding for (S)-acetolactate.

Keywords

Acetolactate decarboxylase Acetoin Bacillus subtilis Crystal structure 

Notes

Funding

This study was funded by the National Science Foundation of China (grant number 21506025) and Dalian University of Technology Science Foundation (grant number DUT16RC(4)12).

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2018_9049_MOESM1_ESM.pdf (89 kb)
Table S1 (PDF 88 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of Life Science and BiotechnologyDalian University of TechnologyDalianPeople’s Republic of China
  2. 2.Marine Bioengineering GroupDalian Institute of Chemical Physics, Chinese Academy of SciencesDalianPeople’s Republic of China
  3. 3.School of Food and Environment Science and EngineeringDalian University of TechnologyPanjinPeople’s Republic of China

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