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Expression, purification, and characterization of a membrane-bound D-amino acid dehydrogenase from Proteus mirabilis
Jinjin Xu1, Yajun Bai2, Taiping Fan2,3
1The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
Objectives:
To characterize a novel membrane-bound D -amino acid dehydrogenase from Proteus mirabilis JN458 (PmDAD).
Results:
The recombinant PmDAD protein, encoding a peptide of 434 amino acids with a MW of 47.7 kDa, exhibited broad substrate specificity with D -alanine the most preferred substrate. The K m and V max values for D -alanine were 9 mM and 20 μmol min-1 mg-1, respectively. Optimal activity was at pH 8 and 45 °C. Additionally, this PmDAD generated H2O2 and exhibited 68 and 60% similarity with E. coli K12 DAD and Pseudomonas aeruginosa DAD, respectively, with low degrees of sequence similarity with other bacterial DADs.
Conclusions:
D-Amino acid dehydrogenase from Proteus mirabilis JN458 was expressed and characterized for the first time, DAD was confirmed to be an alanine dehydrogenase.
Insights
Researchers characterized a novel membrane-bound D-amino acid dehydrogenase (DAD) from Proteus mirabilis JN458. This enzyme, PmDAD, functions as an alanine dehydrogenase, with optimal activity at pH 8 and 45°C.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- D-amino acid dehydrogenases (DADs) are enzymes involved in amino acid metabolism.
- Understanding bacterial DADs is crucial for various biotechnological applications.
Purpose of the Study:
- To express and characterize a novel membrane-bound D-amino acid dehydrogenase from Proteus mirabilis JN458 (PmDAD).
Main Methods:
- Recombinant expression of PmDAD.
- Enzyme activity assays to determine substrate specificity, kinetics, and optimal conditions.
- Sequence homology analysis.
Main Results:
- The recombinant PmDAD protein (434 amino acids, 47.7 kDa) showed broad substrate specificity, with D-alanine as the preferred substrate.
- Kinetic parameters for D-alanine: Km = 9 mM, Vmax = 20 μmol min-1 mg-1.
- Optimal activity at pH 8 and 45°C; enzyme generates H2O2; 68% and 60% similarity to E. coli and P. aeruginosa DADs, respectively.
Conclusions:
- D-amino acid dehydrogenase from Proteus mirabilis JN458 was successfully expressed and characterized for the first time.
- The enzyme was confirmed to be an alanine dehydrogenase.
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