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

Akio Suemori,Masahiro Iwakura

doi:10.1074/jbc.m610320200

Akio Suemori, Masahiro Iwakura

Open Access

https://doi.org/10.1074/jbc.m610320200

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Abstract

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.

Highlights

Among the 20 naturally occurring amino acids, only Cys and Met residues contain a sulfur atom in their side chains
Protein Design by Means of an Adaptive Walk Search in Sequence Space—To demonstrate the applicability of our QAW strategy, an evolutional design approach, for simultaneous improvement of particular properties, p-hydroxybenzoate hydroxylase (PHBH) was used as a model protein in this study to improve the characteristics in terms of main activity, sub-activity, reaction specificity, and thermal stability, using a diminished amino acid component such as Cys- and Met-reduced [41,42,43]
The total number of variants tested was only 249, which resulted in eight quadruple variants with improved characteristics, where the size of the sequence space to be considered is 1812 ϭ 1.2 ϫ 1015 for constructing a complete library with mutations at four Cys and eight Met residues [44], i.e. an ϳ5 ϫ 1012-fold reduction in the number to be tested was attained in this study

Summary

EXPERIMENTAL PROCEDURES

Materials—The PCR, DNA extraction, DNA purification, DNA sequencing, and nickel-nitrilotriacetic acid protein purification kits were purchased from Qiagen; the protein extraction kit was from Novagen; and S-Gal/LB Agar Blend was purchased from Sigma.

Simultaneous Improvement of Properties of PHBH

RESULTS

DISCUSSION