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J. Biol. Chem., Vol. 282, Issue 27, 19969-19978, July 6, 2007

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A Systematic and Comprehensive Combinatorial Approach to Simultaneously Improve the Activity, Reaction Specificity, and Thermal Stability of p-Hydroxybenzoate Hydroxylase^*

From the National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

We have simultaneously improved the activity, reaction specificity, and thermal stability of p-hydroxybenzoate hydroxylase by means of systematic and comprehensive combinatorial mutagenesis starting from available single mutations. Introduction of random mutations at the positions of four cysteine and eight methionine residues provided 216 single mutants as stably expressed forms in Escherichia coli host cells. Four characteristics, hydroxylase activity toward p-hydroxybenzoate (main activity), protocatechuate-dependent NADPH oxidase activity (sub-activity), ratio of sub-activity to main activity (reaction specificity), and thermal stability, of the purified mutants were determined. To improve the above characteristics for diagnostic use of the enzyme, 11 single mutations (C152V, C211I, C332A, M52V, M52Q, M110L, M110I, M213G, M213L, M276Q, and M349A) were selected for further combinatorial mutagenesis. All possible combinations of the mutations provided 18 variants with double mutations and further combinatorial mutagenesis provided 6 variants with triple mutations and 9 variants with quadruple mutations with the simultaneously improved four properties.

Received for publication, November 6, 2006 , and in revised form, February 27, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1-3 and Figs. 7-9.

¹ To whom correspondence should be addressed. Tel.: 81-29-861-6179; Fax: 81-29-856-4055; E-mail: masa-iwakura{at}aist.go.jp.

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