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A Conserved Residue of L-Alanine Dehydrogenase From Bacillus Pseudofirmus, Lys-73, Participates in the Catalytic Reaction Through Hydrogen Bonding

A Conserved Residue of L-Alanine Dehydrogenase From Bacillus Pseudofirmus, Lys-73, Participates in the Catalytic Reaction Through Hydrogen Bonding

Guangzheng He, Shujing Xu, Shanshan Wang, Qing Zhang, Dong Liu, Yuling Chen, Jiansong Ju, Baohua Zhao

Enzyme Microb Technol. 2018 Mar;110:61-68.

PMID: 29310857

Abstract:

A multiple protein sequence alignment of l-alanine dehydrogenases from different bacterial species revealed that five highly conserved amino acid residues Arg-15, Lys-73, Lys-75, His-96 and Asp-269 are potential catalytic residues of l-alanine dehydrogenase from Bacillus pseudofirmus OF4. In this study, recombinant OF4Ald and its mutants of five conserved residues were constructed, expressed in Escherichia coli, purified by His6-tag affinity column and gel filtration chromatography, structure homology modeling, and characterized. The purified protein OF4Ald displayed high specificity to l-alanine (15Umg-1) with an optimal temperature and pH of 40°C and 10.5, respectively. Enzymatic assay and activity staining in native gels showed that mutations at four conserved residue Arg-15, Lys-75, His-96 and Asp-269 (except residue Lys-73) resulted in a complete loss in enzymatic activity, which signified that these predicted active sites are indispensable for OF4Ald activity. In contrast, the mutant K73A resulted in 6-fold improvement in kcat/Km towards l-alanine as compared to the wild type protein. Further research of the residue Lys-73 substituted by various amino acids and structural modeling revealed that residue Lys-73 might be involved in the catalytic reaction of the enzyme by influencing the enzyme-substrate binding through the hydrogen-bonding interaction with conserved residue Lys-75.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP9029065	Alanine Dehydrogenase, recombinant		9029-06-5	Price

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