Single amino acid substitution in Bacillus sphaericus phenylalanine dehydrogenase dramatically increases its discrimination between phenylalanine and tyrosine substrates.
Homology-based modeling of phenylalanine dehydrogenases (PheDHs) from various sources, using the structures of homologous enzymes Clostridium symbiosum glutamate dehydrogenase and Bacillus sphaericus leucine dehydrogenase as a guide, revealed that an asparagine residue at position 145 of B. sphaericus PheDH was replaced by valine or alanine in PheDHs from other sources. This difference was proposed to be the basis for the poor discrimination by the B. sphaericus enzyme between the substrates L-phenylalanine and L-tyrosine. Residue 145 of this enzyme was altered, by site-specific mutagenesis, to hydrophobic residues alanine, valine, leucine, and isoleucine, respectively. The resultant mutants showed a high discrimination, above 50-fold, between L-phenylalanine and L-tyrosine. This higher specificity toward L-phenylalanine was due to K(m) values for L-phenylalanine lowered more than 20-fold compared to the values for L-tyrosine. The greater specificity for L-phenylalanine in the wild-type Bacillus badius enzyme, which has a valine residue in the corresponding position, was also found to be largely due to a lower K(m) for this substrate. Activities were also measured with a range of six amino acids with aliphatic, nonpolar side chains, and with the corresponding oxoacids, and in all cases the specificity constants for these substrates were increased in the mutant enzymes. As with phenylalanine, these increases are mainly attributable to large decreases in K(m) values.
Seah
Stephen Y K
SY
Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
Britton
K Linda
KL
Rice
David W
DW
Asano
Yasuhisa
Y
Engel
Paul C
PC
D000409:Alanine; D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010649:Phenylalanine / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase; D000409:Alanine