Lactobacillus delbrueckii subsp. lactis DSM7290 pepG gene encodes a novel cysteine aminopeptidase.
A number of Escherichia coli clones were isolated from a Lactobacillus delbrueckii subsp. lactis gene library capable of hydrolysing the chromogenic substrate Gly-Ala-beta-naphthylamide (Gly-Ala-beta NA). Some of the recombinant plasmids carried by these clones have been shown to encode the cysteine aminopeptidase gene pepC. Nucleotide sequence analyses of the plasmid inserts of the remaining clones resulted in the identification of two adjacent ORFs encoding proteins exhibiting a high degree of similarity between themselves (72.6%) and with PepC. One gene, designated pepG, was overexpressed in E. coli and the crude extracts obtained were shown to be peptidolytically active both against chromogenic substrates and peptides, and in a Salmonella typhimurium growth test. PepC and PepG activities were compared using chromogenic beta NA and p-nitroanilide substrates and leucine or proline-containing peptides were applied in growth experiments of recombinant Sal. typhimurium. The results indicate that the enzymes, although structurally related, have different substrate preferences. No enzyme activity could be ascribed to the second ORF (orfW), despite the production of a visible protein using a T7 RNA polymerase system. Primer extension analysis, using mRNA isolated from Lb. delbrueckii subsp. lactis DSM7290 did establish that orfW was transcribed.
Klein
Jurgen R
JR
Abteilung Mikrobiologie der Universität,Postfach 3049, 67653 Kaiserslautern,Germany.
Schick
Joachim
J
Abteilung Mikrobiologie der Universität,Postfach 3049, 67653 Kaiserslautern,Germany.
Henrich
Bemhard
B
Abteilung Mikrobiologie der Universität,Postfach 3049, 67653 Kaiserslautern,Germany.
Plapp
Roland
R
Abteilung Mikrobiologie der Universität,Postfach 3049, 67653 Kaiserslautern,Germany.
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000626:Aminopeptidases; C086319:PepC thiol aminopeptidase; C075408:cysteine aminopeptidase