A GENE-CLUSTER ENCODING MALONYL-COA DECARBOXYLASE (MATA), MALONYL-COASYNTHETASE (MATB) AND A PUTATIVE DICARBOXYLATE CARRIER PROTEIN (MATC)IN RHIZOBIUM-TRIFOLII - CLONING, SEQUENCING, AND EXPRESSION OF THE ENZYMES IN ESCHERICHIA-COLI

A gene cluster consisting of three consecutive genes, matABC, was isol ated using a probe prepared from amino acid sequence information of Rh izobium trifolii malonyl-CoA synthetase, and was subsequently sequence d. The sequences of matA and matB were overlapped by four base pairs, whereas the intergenic region between matB and matC had 95 base pairs. The upstream region contained DNA sequences which are typical for an Escherichia coli sigma(70) promoter, and no other open reading frame w as found within 400 bp downstream of matC. The ribosome-binding sites were found 7 to 12 base pairs upstream of each gene. MatA gene encoded a polypeptide of 462 amino acid residues, with deduced molecular mass of 51414 Da. A glutathione-S-transferase-MatA fusion protein has been purified and MatA was shown to have an intrinsic malonyl-CoA decarbox ylase activity (K-m = 0.47 mM; V-max = 52 mu mol.min(-1).mg(-1)). MatB encoded a polypeptide of 504 amino acid residues with deduced molecul ar mass of 54612 Da. MatB was also purified from E. coli transformant carrying the gene cluster. The enzyme was essentially indistinguishabl e from the wild-type malonyl-CoA synthetase of R. trifolii by the crit eria of polyacrylamide gel electrophoresis and biochemical properties. MatC encoded a 46453-Da protein with a high content of hydrophobic re sidues. The deduced amino acid sequences of matC showed identity to so me extent with anaerobic C-4-dicarboxylate carrier proteins from E. co li (25 %) and Haemophilus influenzae (17%). MatC protein appears to be an integral membrane protein that could function as a malonate carrie r. The formation of acetyl-CoA and malonyl-CoA from malonate was confi rmed by thin-layer chromatographic analysis. These results strongly su ggest that the gene cluster encodes proteins involved in the malonate- metabolizing system, malonate-->malonyl-CoA-->acetyl-CoA, in R. trifol ii and that the metabolic pathway in the malonate-rich clover nodule m ight play an important role in symbiosis.