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Properties of the reversible nonoxidative vanillate / 4-hydroxybenzoate decarboxylase from Bacillus subtilis

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Abstract:

Bacillus subtilis (ATCC 6051) reversibly decarboxylates vanillate and 4-hydroxybenzoate under both aerobic and anoxic conditions. Thus, we have identified on the basis of gene sequence homology with Sedimentibacter hydroxybenzoicus and Streptomyces sp. strain D7, a putative B. subtilis hydroxybenzoate decarboxylase. The native form of this enzyme is encoded by 3 genes yclBCD (GI Sequence Identification Nos.: 2632649, 2632650, 2632651) that we have renamed during this research as bsdBCD to align with existing nomenclature. The bsdD gene is reported in the database to be 690 bp; however, our sequence analysis revealed that the size of this gene is in fact 228 bp, an observation that results in a shortening of YclD (i.e., BsdD) from 229 to 75 aa. The corresponding bsdBCD genes were cloned into Escherichia coli, and the heterologously expressed enzyme was assayed for activity. The decarboxylase exhibited a narrow substrate range, with only 2 of the tested substrates, vanillate (Kmapp = 4 mmol·L-1) and 4-hydroxybenzoate (Kmapp = ~1 mmol·L-1), being decarboxylated. The recombinant enzyme had properties similar to that of the native enzyme in respect to specific activity, kinetic properties, bidirectional decarboxylase-carboxylase activity, oxygen insensitivity, and substrate specificity.

Le Bacillus subtilis (ATCC 6051) décarboxyle de façon réversible le vanillate et le 4-hydroxybenzoate sous conditions aérobies et anaérobies. Nous avons donc identifié une hydroxybenzoate décarboxylase présumée chez le B. subtilis, sur la base de l’homologie de séquence avec le gène du Sedimentibacter hydroxybenzoicus et de Streptomyces sp. souche D7. La forme native de cette enzyme est codée par 3 gènes yclBCD (nos d'identification de séquence GI : 2632649, 2632650, 2632651), qui ont été renommés bsdBCD au cours de cette recherche, en accord avec la nomenclature existante. On rapporte dans la base de données que le gène bsdD a une longueur de 690 pb; cependant, notre analyse de séquence a révélé que la taille du gène est en fait de 228 pb, une observation qui résulte en un raccourcissement de YclD (BsdD) de 229 à 75 aa. Les gènes bsdBCD correspondant ont été clonés dans l'Escherichia coli et l’activité de l’enzyme exprimée de façon hétérologue a été testée. La décarboxylase présentait une gamme restreinte de substrats parmi ceux testés, le vanillate (Kmapp = 4 mmol·L-1) et le 4-hydroxybenzoate (Kmapp = ~1 mmol·L-1) étant les 2 seuls substrats décarboxylés. L’enzyme recombinante avait des propriétés similaires à celles de l’enzyme native quant à l’activité spécifique, les propriétés cinétiques, l’activité bidirectionnelle décarboxylase-carboxylase, l’insensibilité à l’oxygène et la spécificité de substrat.

Document Type: Research Article

Publication date: January 1, 2008

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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