SEQUENCE OF A GENE-CLUSTER FROM KLEBSIELLA-PNEUMONIAE ENCODING MALONATE DECARBOXYLASE AND EXPRESSION OF THE ENZYME IN ESCHERICHIA-COLI

Malonate decarboxylase of Klebsiella pneumoniae consists of four diffe rent subunits and catalyzes the conversion of malonate plus H+ to acet ate and CO2. The catalysis proceeds via acetyl and malonyl thioester r esidues with the phosphribosyl-dephospho-CoA prosthetic group of the a cyl carrier protein (ACP) subunit. From a cosmid library of K. pneumon iae, a gene cluster of () kb has been isolated and sequenced that incl uded the structural genes for the malonate decarboxylase. The cluster consisted of the eight consecutive genes mdcABCDEFGH and the divergent ly oriented mdcR gene. The intergenic regions were short (usually <17 bp, 136 bp between mdcE and mdcF) and ribosome binding sites were foun d 4-10 bp before each gene. According to N-terminal protein sequencing , the mdcA, C, D and E genes encoded subunits alpha, delta, beta and g amma of malonate decarboxylase. Data bank searches for related protein s with known function revealed that MdcA represents the ACP-transferas e and that MdcD and E together probably function as malonyl-S-ACP deca rboxylase. MdcC is the (ape) ACP subunit. MdcB and MdcG could be invol ved in the synthesis and attachment of the prosthetic group. MdcH is s imilar to various malonyl-CoA:ACP-SH transacylases and therefore proba bly involved in the initial activation of the enzyme by malonylation. MdcF is a membrane protein that could function as a malonate carrier. The mdcR gene encodes a protein of the LysR regulator family. Malonate decarboxylase was functionally expressed in Escherichia coli from pla smids harbouring the entire gene cluster including mdcR. As partial de letion of the mdcR gene impaired growth of the transformants on malona te, MdcR is probably a transcriptional regulator of the mdc genes.