热稳定性
组氨酸
化学
明亮发光杆菌
突变体
半胱氨酸
生物化学
氨基酸
脱氮酶
酶
立体化学
有机化学
泛素
毒性
基因
作者
Yuki Oda,Kengo Nakata,Hiroshi Miyano,Toshimi Mizukoshi,H Yamaguchi,Tatsuki Kashiwagi
摘要
Enzymatic amino acid assays are important in physiological research and clinical diagnostics because abnormal amino acid concentrations in biofluids are associated with various diseases. L-histidine decarboxylase from Photobacterium phosphoreum (PpHDC) is a pyridoxal 5'-phosphate-dependent enzyme and a candidate for use in an L-histidine quantitation assay. Previous cysteine substitution experiments demonstrated that the PpHDC C57S mutant displayed improved long-term storage stability and thermostability when compared with those of the wild-type enzyme. In this study, combinational mutation experiments of single cysteine substitution mutants of PpHDC were performed, revealing that the PpHDC C57S/C101V/C282V mutant possessed the highest thermostability. The stabilizing mechanism of these mutations was elucidated by solving the structures of PpHDC C57S and C57S/C101V/C282V mutants by X-ray crystallography. In the crystal structures, two symmetry-related PpHDC molecules form a domain-swapped homodimer. The side chain of S57 is solvent exposed in the structure, indicating that the C57S mutation eliminates chemical oxidation or disulfide bond formation with a free thiol group, thereby providing greater stability. Residues 101 and 282 form hydrophobic interactions with neighboring hydrophobic residues. Mutations C101V and C282V enhanced thermostability of PpHDC by filling a cavity present in the hydrophobic core (C101V) and increasing hydrophobic interactions.
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