Molecular cloning, sequencing, and expression of the gene encoding alkaline ceramidase from Pseudomonas aeruginosa - Cloning of a ceramidase homologue from Mycobacterium tuberculosis

We previously reported the purification and characterization of a novel typ e of alkaline ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N. , Tani, M., Imayama, S., and Ito, IM. (1998) J. Biol. Chem. 273, 14368-1437 3). Here, we report the molecular cloning, sequencing, and expression of th e gene encoding the ceramidase of this strain. Specific oligonucleotide pri mers were synthesized using the peptide sequences of the purified ceramidas e obtained by digestion with lysylendopeptidase and used for polymerase cha in reaction. DNA fragments thus amplified were used as probes to clone the gene encoding the ceramidase from a genomic library of strain AN17. The ope n reading frame of 2,010 nucleotides encoded a polypeptide of 670 amino aci ds including a signal sequence of 24 residues, 64 residues of which matched the amino acid sequence determined for the purified enzyme. The molecular weight of the mature enzyme was estimated to be 70,767 from the deduced ami no acid sequence. Expression of the ceramidase gene in Escherichia coli, re sulted in production of a soluble enzyme with the identical N-terminal amin o acid sequence. Recombinant ceramidase was purified to homogeneity from th e lysate of E. coli cells and con firmed to be identical to the Pseudomonas enzyme in its specificity and other enzymatic properties. No significant s equence similarities were found in other known functional proteins includin g human acid ceramidase. However, we found a sequence homologous to the cer amidase in hypothetical proteins encoded in Mycobacterium tuberculosis, Dic tyostelium discoideum, and Arabidopsis thaliana. The homologue of the ceram idase gene was thus cloned from an M. tuberculosis cosmid and expressed in E. coli, and the gene was demonstrated to encode an alkaline ceramidase. Th is is the first report for the cloning of an alkaline ceramidase.