Reaction pathway of tryptophanase-catalyzed l-tryptophan synthesis from d-serine

Shimada, Akihiko; Ozaki, Haruka; Saito, Takeshi; Fujii, Nobutaka

doi:10.1016/j.jchromb.2011.04.028

Cited by 5 publications

(2 citation statements)

References 28 publications

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“…Tryptophanase enantioselectivity is very flexible in the presence of DAP, contrary to conventional knowledge about enantioselectivity. D-tryptophan or D-serine, respectively, was degraded or synthesized through β-elimination or β-replacement reactions after D-tryptophan or D-serine formed an aldimine bond with pyridoxal 5'-phosphate [ 12 ]. The formation of the external aldimine bond between the D-enantiomers and pyridoxal 5'-phosphate was the first step for D-enantiomers to function as active substrates.…”

Section: Introductionmentioning

confidence: 99%

Flexible Enantioselectivity of Tryptophanase Attributable to Benzene Ring in Heterocyclic Moiety of D-Tryptophan

Shimada

Ozaki

2012

Life

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The invariance principle of enzyme enantioselectivity must be absolute because it is absolutely essential to the homochiral biological world. Most enzymes are strictly enantioselective, and tryptophanase is one of the enzymes with extreme absolute enantioselectivity for L-tryptophan. Contrary to conventional knowledge about the principle, tryptophanase becomes flexible to catalyze D-tryptophan in the presence of diammonium hydrogenphosphate. Since D-amino acids are ordinarily inert or function as inhibitors even though they are bound to the active site, the inhibition behavior of D-tryptophan and several inhibitors involved in this process was examined in terms of kinetics to explain the reason for this flexible enantioselectivity in the presence of diammonium hydrogenphosphate. Diammonium hydrogenphosphate gave tryptophanase a small conformational change so that D-tryptophan could work as a substrate. As opposed to other D-amino acids, D-tryptophan is a very bulky amino acid with a benzene ring in its heterocyclic moiety, and so we suggest that this structural feature makes the catalysis of D-tryptophan degradation possible, consequently leading to the flexible enantioselectivity. The present results not only help to understand the mechanism of enzyme enantioselectivity, but also shed light on the origin of homochirality.

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Section: Introductionmentioning

confidence: 99%

Flexible Enantioselectivity of Tryptophanase Attributable to Benzene Ring in Heterocyclic Moiety of D-Tryptophan

Shimada

Ozaki

2012

Life

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“…This enzyme has been of increasing interest in biotechnology owing to its reverse α,β-elimination and β-substitution reactions. Thus, it has been utilized as a biocatalyst in the synthesis of L-tryptophan and for the production of amino acid analogs [4][5][6][7][8].…”

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Characterization of Tryptophanase from Vibrio cholerae

Nuidate

Tansila

Chomchuen

et al. 2014

Appl Biochem Biotechnol

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Tryptophanase (Trpase) is a pyridoxal phosphate (PLP)-dependent enzyme responsible for the production of indole, an important intra- and interspecies signaling molecule in bacteria. In this study, the tnaA gene of Vibrio cholerae coding for VcTrpase was cloned into the pET-20b(+) vector and expressed in Escherichia coli BL21(DE3) tn5:tnaA. Using Ni(2+)-nitrilotriacetic acid (NTA) chromatography, VcTrpase was purified, and it possessed a molecular mass of ∼49 kDa with specific absorption peaks at 330 and 435 nm and a specific activity of 3 U/mg protein. The VcTrpase had an 80 % homology to the Trpase of Haemophilus influenzae and E. coli, but only around 50 % identity to the Trpase of Proteus vulgaris and Porphyromonas gingivalis. The optimum conditions for the enzyme were at pH 9.0 and 45 °C. Recombinant VcTrpase exhibited analogous kinetic reactivity to the EcTrpase with K m and k cat values of 0.612 × 10(-3) M and 5.252 s(-1), respectively. The enzyme catalyzed S-methyl-L-cysteine and S-benzyl-L-cysteine degradation, but not L-phenylalanine and L-serine. Using a site-directed mutagenesis technique, eight residues (Thr52, Tyr74, Arg103, Asp137, Arg230, Lys269, Lys270, and His463) were conserved for maintaining enzyme catalysis. All amino acid substitutions at these sites either eliminated or remarkably diminished Trpase activity. These sites are thus potential targets for the design of drugs to control the V. cholerae Trpase and to further investigate its functions.

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d -Amino Acids and Cross-Linked Amino Acids in Food

Cartus

2017

Chemical Contaminants and Residues in Food

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Reaction pathway of tryptophanase-catalyzed l-tryptophan synthesis from d-serine

Cited by 5 publications

References 28 publications

Flexible Enantioselectivity of Tryptophanase Attributable to Benzene Ring in Heterocyclic Moiety of D-Tryptophan

Flexible Enantioselectivity of Tryptophanase Attributable to Benzene Ring in Heterocyclic Moiety of D-Tryptophan

Characterization of Tryptophanase from Vibrio cholerae

d -Amino Acids and Cross-Linked Amino Acids in Food

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