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Journal of Biochemistry Advance Access originally published online on March 20, 2009
Journal of Biochemistry 2009 146(1):23-32; doi:10.1093/jb/mvp040
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Characterization of putative tryptophan monooxygenase from Ralstonia solanasearum

Nami Kurosawa1, Tomoko Hirata2 and Haruo Suzuki1,2,*

1Department of Biosciences, School of Science; and 2Division of Biosciences, The Graduate School of Fundamental Life Science, Kitasato University, Kitasato 1-15-1, Sagamihara-shi, Kanagawa-ken 228-8555, Japan

*To whom correspondence should be addressed. Tel: +81-42-741-5526; E-mail: suzukih{at}kitasato-u.ac.jp

Received January 9, 2009; Accepted February 25, 2009


  Abstract

The amino-acid sequence of a putative tryptophan monooxygenase (PTMO) from Ralstonia solanacearum is homologous with that of proenzyme (proPAO) of L-Phe oxidase (deaminating and decarboxylating) (PAO) from Pseudomonas sp. P-501 in their overall sequences. PTMO was expressed in E. coli and purified, but had no catalytic activity to oxidize L-Phe. By treating PTMO with various proteases, the Pronase-treated PTMO (PTMOp) showed a relatively high activity to oxidize L-Phe, L-Trp, L-Tyr and L-Met. Studies on the stoichiometry of the reaction showed that L-Phe and L-Tyr were mostly oxygenated, that L-Met was mostly oxidized, and both oxygenation and oxidation of L-Trp was observed. Initial velocity patterns were a ping-pong type with L-Phe and L-Tyr, and a sequential type with L-Trp and L-Met as substrate. The spectrum of enzymes with sufficient amounts of these substrates to reduce the enzyme showed a long wavelength species (purple complex) with L-Phe, but not with L-Tyr, L-Trp and L-Met. These results lead to the conclusion that PTMO and PTMOp belong to proPAO and PAO, respectively.

Key Words: flavoproteins, proteolytic activation, tryptophan monooxygenase, steady-state kinetics, stoichiometry of reaction

Abbreviations: IAA, indole-3-acetic acid; IPTG, isopropyl-β-D-thiogalactopyranoside; PAO, L-phenylalanine oxidase; PAOpt, PAO activated by Pronase and trypsin; proPAO, noncatalytic proenzyme of PAO; PTMO, putative tryptophan monooxygenase; PTMOp, PTMO activated by Pronase; X-gal, 5-bromo-4-chloro-3-indoltl-β-D-galactoside


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