Characterisation of a thiamine diphosphate-dependent alpha-keto acid decarboxylase from Proteus mirabilis JN458

Biying Wang; Yajun Bai; Taiping Fan; Xiaohui Zheng; Yujie Cai

doi:10.1016/j.foodchem.2017.03.164

Characterisation of a thiamine diphosphate-dependent alpha-keto acid decarboxylase from Proteus mirabilis JN458

Food Chem. 2017 Oct 1:232:19-24. doi: 10.1016/j.foodchem.2017.03.164. Epub 2017 Apr 1.

Authors

Biying Wang¹, Yajun Bai², Taiping Fan³, Xiaohui Zheng⁴, Yujie Cai⁵

Affiliations

¹ The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
² College of Life Sciences, Northwest University, Xi'an, Shanxi 710069, China.
³ College of Life Sciences, Northwest University, Xi'an, Shanxi 710069, China; Department of Pharmacology, University of Cambridge, Cambridge CB2 1T, UK.
⁴ College of Life Sciences, Northwest University, Xi'an, Shanxi 710069, China. Electronic address: zhengxh@nwu.edu.cn.
⁵ The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. Electronic address: yjcai@jiangnan.edu.cn.

PMID: 28490063
DOI: 10.1016/j.foodchem.2017.03.164

Abstract

Alpha-keto acid decarboxylases can convert keto acids to their corresponding aldehydes, which are often volatile aroma compounds. The gene encoding α-keto acid decarboxylase in Proteus mirabilis JN458 was cloned, and the enzyme overexpressed in Escherichia coli BL21 (DE3), purified in high yield, and characterised. The molecular weight is 62.291kDa by MALDI-TOF MS, and optimum activity at pH 6.0 and 40-50°C. The enzyme is a typical decarboxylase, dependent on thiamine diphosphate and Mg²⁺ as cofactors. For the decarboxylation reaction, the enzyme displayed a broad substrate range. Kinetic parameters were determined using 4-methyl-2-oxopentanoic acid, phenyl pyruvate and 3-methyl-2-oxopentanoic acid as substrates. K_m and k_cat values for phenyl pyruvate were 0.62mM and 77.38s^-1, respectively, and the k_cat/K_m value was 124.81mM^-1s^-1. The enzyme properties suggest it may act effectively under cheese ripening conditions.

Keywords: 2-oxobutyric acid (PubChem CID: 58); 3-methyl-2-oxobutanoic acid (PubChem CID: 49); 3-methyl-2-oxopentanoic acid (PubChem CID: 47); 4-Hydroxyphenylpyruvate (PubChem CID: 979); 4-methyl-2-oxopentanoic acid (PubChem CID: 70); Alpha-keto acid decarboxylase; Cheese ripening; Proteus mirabilis; Thiamine diphosphate; Volatile aroma compound; alpha-ketobutyrate (PubChem CID: 3593277); benzoylformic acid (PubChem CID: 11915); indole-3-pyruvate (PubChem CID: 803); phenyl pyruvate (PubChem CID: 997); pyruvate (PubChem CID: 107735).

MeSH terms

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) / chemistry
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) / metabolism*
Bacterial Proteins / chemistry
Bacterial Proteins / metabolism*
Enzyme Activation
Kinetics
Proteus mirabilis / enzymology*
Thiamine Pyrophosphate / metabolism*

Substances

Bacterial Proteins
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
Thiamine Pyrophosphate