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Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240

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Abstract

We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its K m value for 4-hydroxybenzoate was 596 μM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the K m value was 6.80 mM. In the presence of 3 M KHCO3 and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The K m value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96–97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.

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Acknowledgments

This study was carried out as a part of The Project for Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of New Industrial Science and Technology Frontiers by Ministry of Economy, Trade & Industry (METI), and entrusted by the New Energy and Industrial Technology Development Organization (NEDO).

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  1. Department of Biomolecular Science, Gifu University, Yanagido 1-1, Gifu, 501-1193, Japan

    Tsuyoshi Matsui, Toyokazu Yoshida, Toshihisa Hayashi & Toru Nagasawa

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  1. Tsuyoshi Matsui
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  2. Toyokazu Yoshida
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  3. Toshihisa Hayashi
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  4. Toru Nagasawa
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Correspondence to Toru Nagasawa.

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Matsui, T., Yoshida, T., Hayashi, T. et al. Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240. Arch Microbiol 186, 21–29 (2006). https://doi.org/10.1007/s00203-006-0117-5

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  • DOI: https://doi.org/10.1007/s00203-006-0117-5

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