Purification of Leuconostoc mesenteroides citrate lyase and cloning and characterization of the citCDEFG gene cluster

• Published in: Journal of bacteriology Vol. 180; no. 3; pp. 647 - 654
• Main Authors: Bekal, S, Beeumen, J. van, Samyn, B, Garmyn, D, Henini, S, Divies, C, Prevost, H
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.02.1998
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ACYL CARRIER PROTEIN; Acyl Carrier Protein - genetics; Acyl Carrier Protein - isolation & purification; Amino Acid Sequence; AMINO ACID SEQUENCES; Amino acids; BACTERIAL PROTEINS; Bacteriology; Base Sequence; Biochemistry; Carbon-Sulfur Ligases - genetics; Carbon-Sulfur Ligases - isolation & purification; CHEMICAL COMPOSITION; CITRATE LYASE LIGASE; CITRATE:ACETYL-ACP TRANSFERASE; CITRYL-S-ACP LYASE; CLONACION; CLONAGE; CLONING; Cloning, Molecular; Coenzyme A-Transferases - genetics; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Deoxyribonucleic acid; DNA; DNA, Bacterial; Enzymes and Proteins; ENZYMIC ACTIVITY; Escherichia coli - metabolism; GENBANK/U10621; GENE; Gene Expression; GENES; Genes, Bacterial; GENETICA; GENETICS; GENETIQUE; Leuconostoc - enzymology; Leuconostoc - genetics; LEUCONOSTOC MESENTEROIDES; LEUCONOSTOC MESENTEROIDES SUBSP. CREMORIS; LIASAS; LIGASAS; LIGASE; LIGASES; LYASE; LYASES; MICROBIAL PROTEINS; Microbiology; MOLECULAR SEQUENCE DATA; Multienzyme Complexes - genetics; Multienzyme Complexes - isolation & purification; Multigene Family; NUCLEOTIDE SEQUENCE; OXO-ACID-LYASES; Oxo-Acid-Lyases - genetics; Oxo-Acid-Lyases - isolation & purification; Peptides; PROTEINAS MICROBIANAS; PROTEINE MICROBIENNE; PURIFICACION; PURIFICATION; RESTRICTION MAPPING; SECUENCIA NUCLEOTIDICA; Sequence Analysis, DNA; Sequence Homology, Amino Acid; SEQUENCE NUCLEOTIDIQUE; STRUCTURAL GENES; TRANSFERASAS; TRANSFERASE; TRANSFERASES
• ISSN: 0021-9193; 1098-5530
• DOI: 10.1128/JB.180.3.647-654.1998

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 beta subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the beta 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 gamma (acyl carrier protein [ACP]), beta (citryl-S-ACP lyase; EC 4.1.3.34), and alpha (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