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

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.