AN OPERON FROM LACTOBACILLUS-HELVETICUS COMPOSED OF A PROLINE IMINOPEPTIDASE GENE (PEPL) AND 2 GENES-CODING FOR PUTATIVE MEMBERS OF THE ABCTRANSPORTER FAMILY OF PROTEINS

A proline iminopeptidase gene (pepl) of an industrial Lactobacillus he lveticus strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 wa s preceded by a typical prokaryotic promoter region, and a putative tr anscription terminator was found downstream of ORF3, identified as the pepl gene. Using primer-extension analyses, only one transcription st art site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis eit her with ORF1-, ORF2- or pepl-specific probes, reverse transcription-P CR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50.7, 24.5 and 33.8 kDa proteins, respectively. The ORF3-encoded Pepl protein showed 65% iden tity with the Pepl proteins from Lactobacillus delbrueckii subsp. bulg aricus and Lactobacillus delbrueckii subsp. lactis. The ORF1-encoded p rotein had significant homology with several members of the ABC transp orter family but, with two distinct putative ATP-binding sites, it wou ld represent an unusual type among the bacterial ABC transporters. ORF 2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The pepl gene was overexpressed in Escheri chia coli. Purified Pepl hydrolysed only di- and tripeptides with prol ine in the first position, Optimum Pepl activity was observed at ph 7. 5 and 40 degrees C. A gel filtration analysis indicated that Pepl is a dimer of M(r) 53000. Pepl was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic stud ies with proline-p-nitroanilide as substrate revealed K-m and V-max va lues of 0.8 mM and 350 mmol min(-1) mg(-1), respectively, and a very h igh turnover number of 135000 s(-1).