A single Phe54Tyr substitution improves the catalytic activity and thermostability of Trigonopsis variabilis D-amino acid oxidase.
The industrial importance of Trigonopsis variabilisd-amino acid oxidase (TvDAAO) is represented by its biocatalytic oxidative deamination of cephalosporin C (CPC) to yield glutaryl-7-aminocephalosporanic acid (GL-7-ACA). The process has been incorporated into a two-step bioconversion to produce 7-aminocephalosporanic acid, the crucial synthetic nucleus for several semi-synthetic cephalosporin antibiotics. A homology model of TvDAAO indicated that residue F54 is in a close proximity to the in silico docked CPC. Substitution of this F54 to Tyr (F54Y) resulted in 6-fold improvement in k(cat,app) and approximately 2.5-fold increase in K(i) of GL-7-ACA. Heat treatment (55 degrees C, 60 min) did not decrease the activity of F54Y. It is suggested that the Tyr substitution might initiate hydrogen bond formation with the amino group of CPC and facilitate deamination. Faster substrate turnover, reduced GL-7-ACA inhibition and improved thermostability of the F54Y substitution render it a useful candidate in industrial production of semi-synthetic cephems.
Wong
Kin-Sing
KS
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
Fong
Wing-Ping
WP
Tsang
Paul Wai-Kei
PW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D002511:Cephalosporins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D004718:Saccharomycetales / Q000201:enzymology*; D016415:Sequence Alignment; D013696:Temperature; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D002511:Cephalosporins; D005656:Fungal Proteins; C081063:glutaryl-7-aminocephalosporanic acid; C025163:cephalosporin C; D014443:Tyrosine; D010649:Phenylalanine; C030735:7-aminocephalosporanic acid; D003605:D-Amino-Acid Oxidase