1887

Abstract

Summary: A proline iminopeptidase gene () of an industrial strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or -specific probes, reverse transcription-PCR 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% identity with the Pepl proteins from subsp. and subsp. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The gene was overexpressed in Purified Pepl hydrolysed only diand tripeptides with proline in the first position. Optimum Pepl activity was observed at pH 7.5 and 40 C. A gel filtration analysis indicated that Pepl is a dimer of 53000. Pepl was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline--nitroanilide as substrate revealed and values of 0.8 mM and 350 mmol min mg, respectively, and a very high turnover number of 135000 s.

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/content/journal/micro/10.1099/13500872-142-12-3459
1996-12-01
2019-10-19
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http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-142-12-3459
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