Sequence of a Gene Cluster from Klebsiella Pneumoniae Encoding Malonate Decarboxylase and Expression of the Enzyme in Escherichia Coli

Abstract

Malonate decarboxylase of Klebsiella pneumoniae consists of four different subunits and catalyzes the conversion of malonate plus H' to acetate and CO,. The catalysis proceeds via acetyl and malonyl thioester residues with the phosphribosyl-dephospho-CoA prosthetic group of the acyl carrier protein (ACP) subunit. From a cosmid library of K. pneumoniae, a gene cluster of 9 kb has been isolated and sequenced that included the structural genes for the malonate decarboxylase. The cluster consisted of the eight consecutive genes mdcABCDEFCH and the divergently oriented mdcR gene. The intergenic regions were short (usually < 17 bp, 136 bp between mdcE and mdcF) and ribosome binding sites were found 4-10 bp before each gene. According to N-terminal protein sequencing, the mdcA, C, D and E genes encoded subunits a, 6, / 3 and y of malonate decarboxylase. Data bank searches for related proteins with known function revealed that MdcA represents the ACP-transferase and that MdcD and E together probably function as malonyl-S-ACP decarboxylase. MdcC is the (apo) ACP subunit. MdcB and MdcG could be involved in the synthesis and attachment of the prosthetic group. MdcH is similar to various malonylCoA:ACP-SH transacylases and therefore probably 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 plasmids harbouring the entire gene cluster including mdcR. As partial deletion of the mdcR gene impaired growth of the transformants on malonate, MdcR is probably a transcriptional regulator of the mdc genes.

Formation of catalytically active S‐acetyl(malonate decarboxylase) requires malonyl‐coenzyme A

“…Furthermore, this compound has to be linked to the apoprotein and its SH-group has to be acetylated to gain catalytic power. These features are in accord with the presence of more genes than required to synthesize the individual polypeptides of the catalysts in the mdc [12] and mad [13] gene clusters of K. pneumoniae and M. rubra, respectively.…”

“…Malonate decarboxylase from K. pneumoniae was also activated with acetate and ATP, but activation was more efficient with low concentrations (mm) of malonyl-CoA [4]. In correlation to this observation is the presence of the mdcH gene in the mdc gene cluster of K. pneumoniae conferring malonate utilization [12]. MdcH is clearly Correspondence to P. Dimroth, Mikrobiologisches…”

“…In order to construct the MdcH deletion plasmid pKS-DH, the malonate decarboxylase genes containing plasmid pHS15 [12] was digested with BamHI, the resulting 9.6-kb fragment was gel purified using the QIAEX kit (Quiagen) and religated. This procedure removed a 1379-bp DNA fragment coding for amino acids 76±300 of MdcH.…”

“…The coding region of the mdcH gene was amplified from plasmid pSH41 [12] using primers that introduced a NdeI restriction site at the initiation codon (primer mh-a: 5 H -TTTCTTCCATATGAAGATTTTATTTACCTTTCC-3 H ) and an EcoRI site after the stop codon (mh-z: 5 H -TGCG-GGGAATTCTTAATCGCTAAGCCGCTGC-3 H ). The polymerase chain reaction (PCR) was performed with Pfu polymerase (Stratagene) in glass capillaries on an Air Thermocycler (model 1605, Idaho Technology).…”

EIR1, a root-specific protein involved in auxin transport, is required for gravitropism inArabidopsis thaliana

“…4C). The mdcE (U95087) protein is a potential malonate transporter from Klebsiella pneumoniae (Hoenke et al 1997), whereas livM (P22729) is involved in high affinity uptake of leucine into Escherichia coli (Adams et al 1990). …”