Enhancement of L-tryptophan 5-hydroxylation activity by structure-based modification of L-phenylalanine 4-hydroxylase from Chromobacterium violaceum.
The objective of this study was to enhance l-tryptophan hydroxylation activity of l-phenylalanine 4-hydroxylase. It had been known that l-phenylalanine 4-hydroxylase from Chromobacterium violaceum could convert l-tryptophan to 5-hydroxy-l-tryptophan and l-phenylalanine to l-tyrosine; however, the activity for l-tryptophan was extremely low compared to l-phenylalanine activity levels. We used the information on the crystal structures of aromatic amino acid hydroxylases to generate C. violaceuml-phenylalanine 4-hydroxylase with high l-tryptophan hydroxylating activity. In silico structural modeling analysis suggested that hydrophobic and/or stacking interactions with the substrate and cofactor at L101 and W180 in C. violaceuml-phenylalanine 4-hydroxylase would increase hydroxylation activity. Based on this hypothesis, we introduced a saturation mutagenesis towards these sites followed by the evaluation of 5-hydroxy-l-tryptophan productivity using a modified Gibbs assay. Three and nine positive mutants were obtained from the L101 and W180 mutant libraries, respectively. Among the mutants, L101Y and W180F showed the highest l-tryptophan hydroxylation activity at the respective residues. Steady-state kinetic analysis revealed that k(cat) values for l-tryptophan hydroxylation were increased from 0.40 (wild-type) to 1.02 (L101Y) and 0.51 s(-1) (W180F). In addition, the double mutant (L101Y-W180F) displayed higher l-tryptophan hydroxylation activity than the wild-type and the W180F and L101Y mutants. The k(cat) value of L101Y-W180F increased to 2.08 s(-1), showing a 5.2-fold increase compared to wild-type enzyme levels.
Kino
Kuniki
K
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan. kkino@waseda.jp
Hara
Ryotaro
R
Nozawa
Ai
A
D000595:Amino Acid Sequence; D000818:Animals; D002374:Catalase / Q000737:chemistry; D002417:Cattle; D002861:Chromobacterium / Q000201:enzymology*; D006900:Hydroxylation; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D002374:Catalase; D010651:Phenylalanine Hydroxylase