Enhancement of l-tryptophan 5-hydroxylation activity by structure-based modification of l-phenylalanine 4-hydroxylase from Chromobacterium violaceum

Kuniki Kino, Ryotaro Hara, Ai Nozawa

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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 L101 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 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 kcat 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 kcat value of L101Y-W180F increased to 2.08 s- 1, showing a 5.2-fold increase compared to wild-type enzyme levels.

Original languageEnglish
Pages (from-to)184-189
Number of pages6
JournalJournal of Bioscience and Bioengineering
Volume108
Issue number3
DOIs
Publication statusPublished - 2009 Sep 1

Keywords

  • 5-Hydroxy-l-tryptophan
  • Aromatic amino acid hydroxylase
  • Chromobacterium violaceum
  • Saturation mutagenesis
  • l-Phenylalanine 4-hydroxylase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Fingerprint Dive into the research topics of 'Enhancement of l-tryptophan 5-hydroxylation activity by structure-based modification of l-phenylalanine 4-hydroxylase from Chromobacterium violaceum'. Together they form a unique fingerprint.

  • Cite this