Enhancement of L-tryptophan 5-hydroxylation activity by structure-based modification of L-phenylalanine 4-hydroxylase from Chromobacterium violaceum(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
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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 k_<cat> values for L-tryptophan hydroxylation were increased from 0.40 (wild-type) to 1.02 (L101Y) and 0.51s^<-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 2.08s^<-1>, showing a 5.2-fold increase compared to wild-type enzyme levels.
- 社団法人日本生物工学会の論文
著者
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Kino Kuniki
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University
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Kino K
Department Of Applied Chemistry School Of Science And Engineering Waseda University
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Kino Kuniki
Department Of Applied Chemistry Faculty Of Science And Engineering Waseda University
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Kino Kuniki
Department Of Applied Chemistry School Of Science And Engineering Waseda University
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Kino Kuniki
Dep. Of Applied Chemistry Fac. Of Sci. And Engineering Waseda Univ. 3-4-1 Ohkubo Shinjuku-ku Tokyo 1
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HARA Ryotaro
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University
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NOZAWA Ai
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University
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Nozawa Ai
Department Of Applied Chemistry Faculty Of Science And Engineering Waseda University
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Hara Ryotaro
Department Of Applied Chemistry Faculty Of Science And Engineering Waseda University
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