Structural insights into the enhanced thermostability of cysteine substitution mutants of L-histidine decarboxylase from Photobacterium phosphoreum

Abstract

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.

Keywords: Abbreviations: AADCs, amino acid decarboxylase; ASA, accessible surface area; CL, catalytic loop; HDC, L-histidine decarboxylase; HDH, histamine dehydrogenase; HsHDC, HDC from humans; LL, long loop; 1-methoxy PMS, 1-methoxy-5-methylphenazinium methylsulfate; MjTDC, tyrosine decarboxylase from Methanocaldococcus jannaschii; MR, molecular replacement; PLP, pyridoxal 5′-phosphate; PpHDC, L-histidine decarboxylase from Photobacterium phosphoreum; PpHDC-SM, PpHDC C57S mutant; PpHDC-TM, PpHDC C57S/C101V/C282V mutant; (WST)-8, water-soluble tetrazolium salt; WT, wild-type; crystal structure; histidine decarboxylase; protein engineering; thermostability; thermostable mutant.