Metal coordination by L-amino acid oxidase derived from flounder Platichthys stellatus is structurally essential and regulates antibacterial activity

Autor:	Tomisato Miura, Kentaro Ariyoshi, Akihide Nitta, Toshiya Nakamura, Kosuke Kasai, Yudai Ito
Rok vydání:	2020
Předmět:	chemistry.chemical_element Flounder L-Amino Acid Oxidase L-amino-acid oxidase Applied Microbiology and Biotechnology Pichia pastoris 03 medical and health sciences Metalloprotein Animals Chelation Magnesium ion Histidine 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology 030306 microbiology Chemistry Magnesium General Medicine biology.organism_classification Recombinant Proteins Anti-Bacterial Agents Biochemistry Saccharomycetales Antibacterial activity Biotechnology
Zdroj:	Applied Microbiology and Biotechnology. 104:9645-9654
ISSN:	1432-0614 0175-7598
DOI:	10.1007/s00253-020-10914-3
Popis:	L-amino acid oxidases (LAAOs) have antibacterial activity and play important roles in innate immunity. We have previously identified a LAAO of ~52 kDa in size from the mucus layer of the flounder Platichthys stellate (psLAAO1) and have successfully produced psLAAO1 as a secreted bioactive recombinant protein by using Pichia pastoris (P. pastoris). The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in the mucus layer. In this study, homology modeling of psLAAO1 predicted metal coordination by residues Y241, H348, and D406. We show that the Michaelis constant (Km) of psLAAO1 decreased and the catalytic constant (Kcat/Km) value increased following pre-treatment of the protein with a chelating agent. In contrast to the non-chelated protein sample, enzymatic activity of EDTA-treated psLAAO1 gradually decreased or was absent after one or two freeze-thaw cycles. The H348A psLAAO1 mutant generated by site-directed mutagenesis and recombinantly produced by P. pastoris did not display antibacterial activity. The results of the metal detection assay revealed that for the non-metal coordinating histidine mutant (H209A, control), the levels of iron, zinc, and magnesium were similar to those of wild-type psLAAO1, whereas magnesium was not detected in the H348A mutant sample. A wild-type psLAAO1 sample treated with chelating agent did not contain zinc and magnesium ions. In conclusion, metal coordination by psLAAO1 affects enzymatic activity, and H348 is involved in the coordination of magnesium, and metal coordination by psLAAO1 provides essential structural stability. KEY POINTS: • Homology modeling of psLAAO1 predicted metal coordination by residue H348 • The H348A psLAAO1 mutant showed no antibacterial activity or magnesium coordination • Metal coordination by H348 affects enzyme activity and structural stability.
Databáze:	OpenAIRE
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