Structural and enzymatic characterization of acetolactate decarboxylase from Bacillus subtilis
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.
KeywordsAcetolactate decarboxylase Acetoin Bacillus subtilis Crystal structure
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.
- Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L-W, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr, Sect D: Biol Crystallogr 66(2):213–221. https://doi.org/10.1107/s0907444909052925 CrossRefGoogle Scholar
- Crout DHG, Littlechild J, Mitchell MB, Morrey SM (1985) Cheminform abstract: stereochemistry of the decarboxylation of α-acetolactate (2- hydroxy-2-methyl-3-oxobutanoate) by the acetolactate decarboxylase. J Chem Soc, Perkin Trans 1 16(4):2271–2276Google Scholar
- Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, Wang X, Murray LW, Arendall WB III, Snoeyink J, Richardson JS, Richardson DC (2007) MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res 35:W375–W383. https://doi.org/10.1093/nar/gkm216 CrossRefPubMedPubMedCentralGoogle Scholar
- Drake AF, Siligardi G, Crout DHG, Rathbone DL (1987) Applications of vibrational infrared circular-dichroism to biological problems-stereochemistry of proton-exchange in acetoin (3-hydroxybutan-2-one) catalyzed by acetolactate decarboxylase. J Chem Soc, Chem Commun 24:1834–1835. https://doi.org/10.1039/c39870001834 CrossRefGoogle Scholar
- Fisher Z, Prada JAH, Tu C, Duda D, Yoshioka C, An HQ, Govindasamy L, Silverman DN, McKenna R (2005) Structural and kinetic characterization of active-site histidine as a proton shuttle in catalysis by human carbonic anhydrase II. Biochemistry 44(4):1097–1105. https://doi.org/10.1021/bi0480279 CrossRefPubMedGoogle Scholar
- Fusetti F, Schroter KH, Steiner RA, van Noort PI, Pijning T, Rozeboom HJ, Kalk KH, Egmond MR, Dijkstra BW (2002) Crystal structure of the copper-containing quercetin 2,3-dioxygenase from Aspergillus japonicus. Structure 10(2):259–268. https://doi.org/10.1016/s0969-2126(02)00704-9 CrossRefPubMedGoogle Scholar
- Liu Z, Qin J, Gao C, Hua D, Ma C, Li L, Wang Y, Xu P (2011) Production of (2S,3S)-2,3-butanediol and (3S)-acetoin from glucose using resting cells of Klebsiella pneumonia and Bacillus subtilis. Bioresour Technol 102(22):10741–10744. https://doi.org/10.1016/j.biortech.2011.08.110 CrossRefPubMedGoogle Scholar
- Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ (1998) Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem 19(14):1639–1662. https://doi.org/10.1002/(SICI)1096-987X(19981115)19:14<1639::AID-JCC10>3.0.CO;2-B CrossRefGoogle Scholar
- Najmudin S, Andersen JT, Patkar SA, Borchert TV, Crout DHG, Fulop V (2003) Purification, crystallization and preliminary X-ray crystallographic studies on acetolactate decarboxylase. Acta Crystallogr D Biol Crystallogr 59:1073-1075. https://doi.org/10.1107/s0907444903006978 CrossRefPubMedGoogle Scholar
- Schrodinger, LLC (2015) The PyMOL molecular graphics system, version 1.8. Schrodinger, LLC, New YorkGoogle Scholar
- Vinogradov M, Kaplun A, Vyazmensky M, Engel S, Golbik R, Tittmann K, Uhlemann K, Meshalkina L, Barak Z, Hübner G, Chipman DM (2005) Monitoring the acetohydroxy acid synthase reaction and related carboligations by circular dichroism spectroscopy. Anal Biochem 342(1):126–133. https://doi.org/10.1016/j.ab.2005.03.049 CrossRefPubMedGoogle Scholar
- Wang T (2015) Recombinant expression and enzymatic characterization of acetolactate decarboxylase in vitro. Dalian University of Technology, DalianGoogle Scholar
- Wechsler C, Meyer D, Loschonsky S, Funk LM, Neumann P, Ficner R, Brodhun F, Müller M, Tittmann K (2015) Tuning and Switching Enantioselectivity of Asymmetric Carboligation in an Enzyme through Mutational Analysis of a Single Hot Spot. ChemBioChem 16(18):2580–2584. https://doi.org/10.1002/cbic.201500529 CrossRefPubMedGoogle Scholar
- Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AGW, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr, Sect D: Biol Crystallogr 67(4):235–242. https://doi.org/10.1107/s0907444910045749 CrossRefGoogle Scholar
- Xiao Z, Qiao S, Ma C, Xu P (2010) Acetoin production associated with the increase of cell biomass in Bacillus pumilus ATCC 14884. Afr J Microbiol Res 4(19):1997–2003Google Scholar
- Zhang X, Yang T, Lin Q, Xu M, Xia H, Xu Z, Li H, Rao Z (2011) Isolation and identification of an acetoin high production bacterium that can reverse transform 2,3-butanediol to acetoin at the decline phase of fermentation. World J Microbiol Biotechnol 27(12):2785–2790. https://doi.org/10.1007/s11274-011-0754-y CrossRefGoogle Scholar