Kurosawa Nami, Hirata Tomoko, Suzuki Haruo
Department of Biosciences, Kitasato University, Sagamihara-shi, Kanagawa-ken, Japan.
J Biochem. 2009 Jul;146(1):23-32. doi: 10.1093/jb/mvp040. Epub 2009 Mar 20.
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.
来自青枯雷尔氏菌的一种假定色氨酸单加氧酶（PTMO）的氨基酸序列与来自假单胞菌属P-501的L-苯丙氨酸氧化酶（脱氨基和脱羧）（PAO）的酶原（proPAO）在整体序列上具有同源性。PTMO在大肠杆菌中表达并纯化，但对氧化L-苯丙氨酸没有催化活性。通过用各种蛋白酶处理PTMO，经链霉蛋白酶处理的PTMO（PTMOp）对氧化L-苯丙氨酸、L-色氨酸、L-酪氨酸和L-蛋氨酸表现出相对较高的活性。对反应化学计量学的研究表明，L-苯丙氨酸和L-酪氨酸大多被氧化，L-蛋氨酸大多被氧化，并且观察到L-色氨酸的氧化和加氧反应。以L-苯丙氨酸和L-酪氨酸为底物时，初始速度模式为乒乓型，以L-色氨酸和L-蛋氨酸为底物时为顺序型。用足量这些底物还原酶后的酶光谱显示，以L-苯丙氨酸为底物时呈现长波长物质（紫色复合物），而以L-酪氨酸、L-色氨酸和L-蛋氨酸为底物时则没有。这些结果得出结论，PTMO和PTMOp分别属于proPAO和PAO。
