Purification of Leuconostoc mesenteroides Citrate Lyase and Cloning and Characterization of the citCDEFG Gene Cluster

Abstract

ABSTRACT A citrate lyase (EC 4.1.3.6 ) was purified 25-fold from Leuconostoc mesenteroides and was shown to contain three subunits. The first 42 amino acids of the β subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the α subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from Leuconostoc mesenteroides subsp. cremoris . This fragment was used as a probe for screening a Leuconostoc genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of L. mesenteroides is organized in three open reading frames, citD , citE , and citF , encoding, respectively, the three citrate lyase subunits γ (acyl carrier protein [ACP]), β (citryl-S-ACP lyase; EC 4.1.3.34 ), and α (citrate:acetyl-ACP transferase; EC 2.8.3.10 ). The gene ( citC ) encoding the citrate lyase ligase (EC 6.2.1.22 ) was localized in the region upstream of citD . Protein comparisons show similarities with the citrate lyase ligase and citrate lyase of Klebsiella pneumoniae and Haemophilus influenzae . Downstream of the citrate lyase cluster, a 1.4-kb open reading frame encoding a 52-kDa protein was found. The deduced protein is similar to CitG of the other bacteria, and its function remains unknown. Expression of the citCDEFG gene cluster in Escherichia coli led to the detection of a citrate lyase activity only in the presence of acetyl coenzyme A, which is a structural analog of the prosthetic group. This shows that the acetyl-ACP group of the citrate lyase form in E. coli is not complete or not linked to the protein.