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.