Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone
Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone
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通过活性位点工程改变甲苯4-单加氧酶以合成3-甲氧基儿茶酚、甲氧基氢醌和甲基氢醌
DOI：
10.1128/jb.186.14.4705-4713.2004
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发表时间：
2004
影响因子：
3.2
通讯作者：
T. Wood
中科院分区：
文献类型：
--
作者：
Y. Tao;A. Fishman;W. Bentley;T. Wood
ABSTRACT Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent V max values of 6.6 ± 0.9 to 10.7 ± 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 ± 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184 :344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41 :3176-3188, 2002). By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 ± 0.2 versus 0.21 ± 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol. Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.
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羟化酶活性位点残基和效应蛋白结合联合参与甲苯 4-单加氧酶区域特异性的对位至邻位调节。
影响因子：
2.9
作者：
Mitchell,KevinH;Studts,JoeyM;Fox,BrianG
通讯作者：
Fox,BrianG
影响因子：
2.9
作者：
J. Pikus;J. Studts;K. McClay;R. Steffan;B. Fox
通讯作者：
J. Pikus;J. Studts;K. McClay;R. Steffan;B. Fox
DOI：
10.1021/jo010958s
发表时间：
2002-04
期刊：
The Journal of organic chemistry
影响因子：
--
作者：
D. Hua;M. Tamura;X. Huang;H. Stephany;B. Helfrich;E. Perchellet;B. J. Sperfslage;J. Perchellet;Suping Jiang;D. Kyle;P. Chiang
通讯作者：
D. Hua;M. Tamura;X. Huang;H. Stephany;B. Helfrich;E. Perchellet;B. J. Sperfslage;J. Perchellet;Suping Jiang;D. Kyle;P. Chiang
酚羟化酶机制的研究：突变体 Tyr289Phe、Asp54Asn 和 Arg281Met。
影响因子：
2.9
作者：
Xu,D;Ballou,DP;Massey,V
通讯作者：
Massey,V
甲苯4-单加氧酶羟化酶的优化表达和纯化。
DOI：
10.1006/prep.2000.1281
发表时间：
2000
期刊：
Protein expression and purification.
影响因子：
--
作者：
Studts,JM;Mitchell,KH;Pikus,JD;McClay,K;Steffan,RJ;Fox,BG
通讯作者：
Fox,BG