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. violaceum L-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 L701 and W180 in C. violaceum L-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 L701 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 hydroxlylation 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. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.