Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240.
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 Km value for 4-hydroxybenzoate was 596 microM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the Km 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 Km 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.
Matsui
Tsuyoshi
T
Department of Biomolecular Science, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan.
Yoshida
Toyokazu
T
Hayashi
Toshihisa
T
Nagasawa
Toru
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D016972:Enterobacter cloacae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data
D002262:Carboxy-Lyases; C065828:4-hydroxybenzoate decarboxylase