Cloning and nucleotide sequence analysis of pepV, a carnosinase gene from Lactobacillus delbrueckii subsp. lactis DSM 7290, and partial characterization of the enzyme

Vongerichten, Klaus F.; Klein, Jürgen; Matern, Hugo; Plapp, Roland

doi:10.1099/00221287-140-10-2591

Cited by 53 publications

(32 citation statements)

References 40 publications

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“…lactis (42) (20). Two regions of both PepVs which conform to the two signature sequences of the ArgE/DapE/ACY1/CPG2/YscS family of proteins were identified (Fig.…”

Section: Discussionmentioning

confidence: 97%

“…(iii) The deduced amino acid sequence of the dipeptidase showed a high degree of similarity to PepV of L. delbrueckii subsp. lactis (42). (iv) The multiple-peptidase-negative E. coli CM89L carrying pepV on a plasmid displayed high dipeptidase activity.…”

Section: Discussionmentioning

confidence: 99%

“…From Lactobacillus delbrueckii subsp. lactis, the gene of a dipeptidase (pepV) has been cloned and sequenced (42). Dipeptidase genes pepD and pepDA have been cloned and sequenced from Lactobacillus helveticus 53/7 (41) and from Lactobacillus helveticus CNRZ32 (6), respectively.…”

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confidence: 99%

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Cloning and analysis of the pepV dipeptidase gene of Lactococcus lactis MG1363

et al. 1997

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The gene pepV, encoding a dipeptidase from Lactococcus lactis subsp. cremoris MG1363, was identified in a genomic library in pUC19 in a peptidase-deficient Escherichia coli strain and subsequently sequenced. PepV of L. lactis is enzymatically active in E. coli and hydrolyzes a broad range of dipeptides but no tri-, tetra-, or larger oligopeptides. Northern (RNA) and primer extension analyses indicate that pepV is a monocistronic transcriptional unit starting 24 bases upstream of the AUG translational start codon. The dipeptidase of L. lactis was shown to be similar to the dipeptidase encoded by pepV of L. delbrueckii subsp. lactis, with 46% identity in the deduced amino acid sequences. A PepV-negative mutant of L. lactis was constructed by single-crossover recombination. Growth of the mutant strain in milk was significantly slower than that of the wild type, but the strains ultimately reached the same final cell densities.The starter bacterium Lactococcus lactis subsp. cremoris is widely used in cheese making to provide optimal conditions for curd formation and for the development of texture and flavor. Lactococci are organisms with multiple amino acid auxotrophies, and as a consequence, their growth to high cell densities in milk depends on their ability to efficiently degrade the milk protein casein (11). The peptides which are initially released from casein by the cell envelope-associated proteinase and translocated into the cell by the oligopeptide transport system Opp are broken down into amino acids by various intracellular peptidases. In recent years, many lactococcal peptidases have been isolated and characterized both biochemically and genetically (11,12,23,25). Mutants lacking either PepX, PepO, PepT, PepN, PepC, PepF, or PepA have been constructed by gene disruption methods and analyzed for their ability to grow in milk. With the possible exception of PepA-and PepNdeficient mutants, no differences could be detected in growth rates and final cell densities between the various mutants and the wild-type strain (15,(22)(23)(24)(25)27). These observations indicate that none of these peptidases individually is essential for growth in milk. However, analysis of strains carrying multiple peptidase mutations showed that inactivation of several peptidases can lead to lower growth rates in milk, the general trend being that growth rates decrease when more peptidases are inactivated. A strain with mutations in five genes (pepX, pepO, pepT, pepC, and pepN) grew more than 10 times slower in milk than the wild-type strain did (24).A dipeptidase from L. lactis subsp. cremoris Wg2 has been purified to homogeneity by van Boven et al. (39). The enzyme was shown to be a metallopeptidase which hydrolyzes a wide range of dipeptides but not tripeptides or tetrapeptides. Also, dipeptides with proline, histidine, glycine, or glutamate as the N-terminal amino acid or with proline in the second position are not degraded (39). Similar enzymes have been purified from Lactobacillus delbrueckii subsp. bulgaricus (43), from Lactobac...

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“…lactis (42) (20). Two regions of both PepVs which conform to the two signature sequences of the ArgE/DapE/ACY1/CPG2/YscS family of proteins were identified (Fig.…”

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

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Cloning and analysis of the pepV dipeptidase gene of Lactococcus lactis MG1363

et al. 1997

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“…Several enzymes of the Acy1/M20 family are known as exopeptidases (Table I). They include CPG2 (9 -11), Saccharomyces cerevisiae carboxypeptidase Y (12), PepT and the dipeptidase enzyme PepV from various bacterial sources (13)(14)(15), Escherichia coli X-His dipeptidase (16), and human nonspecific dipeptidase and a brain-specific carnosinase that possibly plays a role in aging and neurodegenerative or psychiatric diseases (17). The enzymatic function of a related drought-induced polypeptide-1 from wild watermelon (Citrullus lanatus) remains unknown (18).…”

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confidence: 99%

Essential Roles of Zinc Ligation and Enzyme Dimerization for Catalysis in the Aminoacylase-1/M20 Family

Lindner

Лунин

Alary

et al. 2003

Journal of Biological Chemistry

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Members of the aminoacylase-1 (Acy1)/M20 family of aminoacylases and exopeptidases exist as either monomers or homodimers. They contain a zinc-binding domain and a second domain mediating dimerization in the latter case. The roles that both domains play in catalysis have been investigated for human Acy1 (hAcy1) by x-ray crystallography and by site-directed mutagenesis. Structure comparison of the dinuclear zinc center in a mutant of hAcy1 reported here with dizinc centers in related enzymes points to a difference in zinc ligation in the Acy1/M20 family. Mutational analysis supports catalytic roles of zinc ions, a vicinal glutamate, and a histidine from the dimerization domain. By complementing different active site mutants of hAcy1, we show that catalysis occurs at the dimer interface. Reinterpretation of the structure of a monomeric homolog, peptidase V, reveals that a domain insertion mimics dimerization. We conclude that monomeric and dimeric Acy1/M20 family members share a unique active site architecture involving both enzyme domains. The study may provide means to improve homologous carboxypeptidase G2 toward application in antibody-directed enzyme prodrug therapy.Zinc peptidases play roles in metabolic and signaling pathways throughout all kingdoms of life. A growing number of these enzymes have been found to contain two zinc ions at their active sites. Some are regarded as potential pharmaceutical targets (1). Recently, Wouters and Husain (2) pointed out that members of the MH and MF clans 1 of dizinc peptidases, together with the MC clan of monozinc peptidases display three different catalytic zinc centers that have evolved in a similar structural scaffold, which is exemplified by carboxypeptidase A of clan MC. Although they all appear to employ the same general base-like catalytic mechanism, neither all catalytic residues nor the substrate-binding sites are conserved among the three clans. A glutamate residue representing a putative catalytic base, for instance, resides in different regions of the polypeptide chain in the MH and MC clans. In the MF clan enzyme leucine aminopeptidase, a bicarbonate ion replaces the glutamate residue. Moreover, whereas the monozinc center in the MC clan is structurally equivalent to one of the two zincbinding sites in the dinuclear zinc center in the MH clan, it does not share any homology with the dinuclear zinc center in the MF clan anymore. As a consequence, members of families from each clan must be examined individually in order to gain a detailed understanding of their catalytic machineries.Aminoacylase-1 (Acy1 2 ; EC 3.5.1.14) was discovered in 1881 by virtue of its ability to hydrolyze hippuric acid in crude kidney homogenates (4) and is now classified in the M20 family of clan MH, also referred to as the Acy1 family (5). Acy1 plays a general role in the cytosolic breakdown of N ␣ -acetylated amino acids (6) generated during protein degradation (7). Other functional aminoacylase enzymes from the Acy1/M20 family (Table I) are implicated in the bacterial biosynthesi...

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“…11) Among the families in the clan MH, metal ligands were demonstrated to be well conserved. 12) The amino acid sequences were quite similar among the PepTs from bacteria from L. lactis subsp.…”

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confidence: 99%