The Experts below are selected from a list of 21 Experts worldwide ranked by ideXlab platform
William H. Simmons - One of the best experts on this subject based on the ideXlab platform.
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Xaa-Pro Aminopeptidase (Prokaryote)
Handbook of Proteolytic Enzymes, 2012Co-Authors: William H. SimmonsAbstract:Publisher Summary This chapter elaborates the structural chemistry and the biological aspects of X-Pro Aminopeptidase (XProAP). E. coli XProAP removes the N-terminal residue from peptides that have a proline residue in the penultimate position. The PI' proline can be substituted with 3,4-dehydroproline, but not with trans-4-hydroxyproline. The enzyme can accommodate a variety of amino acids including proline in both the PI and P2 positions. It is stereospecific and requires L-amino acids in PI and L-proline in PI. Xaaj-Pro dipeptides are cleaved more slowly than larger peptides. Only the trans form of the Xaa–Pro bond is hydrolyzed. The first nucleotide and deduced amino acid sequences for XProAP were determined for E. coli type II enzyme. E. coli enzyme is 440 amino acids long, with an Mr of 49,684. Monomers form very stable dimers and dimers form tetramers at biologically significant concentrations. XProAP is possibly involved in intracellular protein turnover by hydrolyzing Xaa-Pro-Y peptides resistant to other peptidases. Mutants of Salmonella typhimurium lacking this enzyme have a much reduced ability to produce free proline during starvation-induced protein breakdown. The structurally related X-Pro dipeptidase has been found in bacteria and is specific for Xaa-Pro dipeptides.
Véronique Monnet - One of the best experts on this subject based on the ideXlab platform.
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Xaa-Pro Aminopeptidase ( Lactococcus )
Handbook of Proteolytic Enzymes, 2012Co-Authors: Véronique MonnetAbstract:Publisher Summary This chapter elaborates the structural chemistry and the biological aspects of X-Pro Aminopeptidase. The presence of an X-Pro Aminopeptidase was reported both in lactococci and in lactobacilli. The enzyme was purified from Lactococcus lactis and characterized. The corresponding gene, named pepP, was cloned and sequenced. The enzyme is recommended to be called XPro Aminopeptidase and the present form may be referred to as X-Pro Aminopeptidase. pepP is described as a monomeric enzyme with a molecular mass of 43,000 Da estimated by gel filtration, which fits well with the size of the corresponding gene. The sequences surrounding the cobalt ligands and the catalytic residues of the methionyl Aminopeptidase family are well conserved also for the lactococcal enzyme. pepP is found in the cytoplasm of Lactococcus lactis, which is widely used in cheese-making technology. However, a significant role of PepP in bacterial nutrition and during cheese ripening remains unlikely. On the opposite, pepP is the only peptidase gene the transcription of which is enhanced in presence of galactose. The presence of four potential catabolite responsive element boxes in its promoter region suggests that expression of pepP is directly controlled by catabolic repression.
Anthony J Turner - One of the best experts on this subject based on the ideXlab platform.
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structural studies of Aminopeptidase p a novel cellular peptidase
Advances in Experimental Medicine and Biology, 1997Co-Authors: Anthony J Turner, Ralph J Hyde, Nigel M. HooperAbstract:The plasma membrane of many cell types contains a cohort of peptidases that serve to modulate the activity of circulating regulatory peptides. Many of these are zinc metallopeptidases and these enzymes are particularly abundant in the brush border membranes of renal and intestinal microvilli. Some of them such as neprilysin (NEP; EC 3.4.24.11) and Aminopeptidase N (AP-N; EC 3.4.11.2) also exist as cluster differentiation antigens (CD10 and CD 13 respectively) on the surface of leukocytes and may play a role in regulation of the immune system1. Both NEP and AP-N also have roles in the metabolism of certain cardiovascular and neuropeptides, e.g. natriuretic peptides2 and enkephalins3. NEP and AP-N are type ll integral membrane proteins and possess the typical HExxH zincin motif4 characteristic of many zinc peptidases. Recently, we have focused on another brush border zinc peptidase, Aminopeptidase P (AP-P; X-Pro Aminopeptidase; EC 3.4.11.9) which we showed to be unusual among the membrane peptidases in being anchored to the membrane by a glycosylphosphatidylinositol (GPI) moiety5. AP-P also shows a number of other atypical features which led us to attempt the molecular cloning of the pig kidney enzyme.
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molecular cloning and expression in cos 1 cells of pig kidney Aminopeptidase p
Biochemical Journal, 1996Co-Authors: Ralph J Hyde, Nigel M. Hooper, Anthony J TurnerAbstract:Aminopeptidase P (AP-P; X-Pro Aminopeptidase; EC 3.4.11.9), a key enzyme in the metabolism of the vasodilator bradykinin, has been cloned from a pig kidney cortex cDNA library following the use of the PCR to identify sub-libraries enriched in AP-P clones. The complete primary sequence of the enzyme has been deduced from a full-length cDNA clone. This predicts a protein of 673 amino acids with a cleavable N-terminal signal sequence and six potential N-linked glycosylation sites. A stretch of mainly hydrophobic amino acids at the C-terminus is predicted to co-ordinate the attachment of a glycosyl-phosphatidylinositol (GPI) membrane anchor. Although AP-P is a zinc metallopeptidase, the predicted primary sequence does not contain any recognizable zinc-binding motif. Transient expression of AP-P cDNA in COS-1 cells resulted in enzymic activity characteristic of AP-P, namely apstatin- and EDTA-sensitive hydrolysis of bradykinin and Gly-Pro-Hyp. The expressed protein was recognized as a polypeptide of M(r)91,000 under reducing conditions, following immunoblotting of COS-1 membranes with a polyclonal antibody raised against purified pig kidney AP-P. The presence of a GPI anchor on expressed AP-P was established by demonstrating release of the enzyme from a membrane fraction following treatment with bacterial phosphatidylinositol-specific phospholipase C and its corresponding conversion from an amphipathic to a hydrophilic form, as assessed by phase separation in Triton X-114. Sequence comparisons confirm that AP-P is a member of the proline peptidase family of hydrolytic enzymes and is unrelated in sequence to other brush-border membrane peptidases.
Sung Gyun Kang - One of the best experts on this subject based on the ideXlab platform.
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cloning expression and characterization of Aminopeptidase p from the hyperthermophilic archaeon thermococcus sp strain na1
Applied and Environmental Microbiology, 2006Co-Authors: Jeong Ho Jeon, Byeong Chul Jeong, Sung Gyun KangAbstract:Aminopeptidase P (APP, or X-Pro Aminopeptidase; EC 3.4.11.9) is a peptidase that specifically removes the N-terminal amino acids from peptides in which the penultimate residue is proline (5). Since the time an enzyme with the specificity of APP was first purified from Escherichia coli (24), APPs have been characterized from diverse sources, including bacteria (16), nematodes (15), insects (14), plants (10), and tissues from several mammalian species (9, 11). While the physiological role of APP in bacteria is unclear, mammalian APP is involved in the protein turnover of collagen and the regulation of biologically active peptides, such as substance P and bradykinin (5, 23, 26). It has been shown that APPs from a number of lactococcal strains may contribute to the abolition of bitterness during the ripening of cheese by participating in peptide degradation following release into the cheese matrix (17). To date, however, there have been no reports on the properties of an APP from either an archaeon or a hyperthermophile. With the availability of a generally applicable combination of conventional genetic engineering and genomic research techniques, the genome sequences of some hyperthermophilic microorganisms are of considerable biotechnological interest because of their heat-stable enzymes, and many extremely thermostable enzymes are being developed for biotechnological purposes (22). Furthermore, recent advances in the application of molecular biological tools to hyperthermophilic archaea, such as gene knockout techniques and efficient transformation systems, could facilitate the study of hyperthermophilic archaeal gene function and contribute to an understanding of the physiology of hyperthermophilic archaea. To facilitate the search for valuable and extremely thermostable enzymes and to help answer questions concerning the physiology of hyperthermophilic archaea grown at extremely high temperatures, we recently isolated a hyperthermophilic archaeon, Thermococcus sp. strain NA1 (S. S. Bae et al., unpublished data), and its whole genome sequence was determined (J.-H. Lee et al., unpublished data). Analysis of the genome information of Thermococcus sp. strain NA1 revealed an APP gene that is similar to those for other APPs. In the present study, the gene corresponding to an APP was cloned and expressed in E. coli. The recombinant enzyme was purified, and its characteristics were examined.
Elwyn R Isaac - One of the best experts on this subject based on the ideXlab platform.
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functional expression and characterization of the cytoplasmic Aminopeptidase p of caenorhabditis elegans
FEBS Journal, 2001Co-Authors: Virginie Laurent, Darren R Brooks, David Coates, Elwyn R IsaacAbstract:Aminopeptidase P (AP-P; X-Pro Aminopeptidase; EC 3.4.11.9) cleaves the N-terminal X-Pro bond of peptides and occurs in mammals as both cytosolic and plasma membrane forms, encoded by separate genes. In mammals, the plasma membrane AP-P can function as a kininase, but little is known about the physiological role of the cytosolic enzyme. The C. elegans genome contains a single gene encoding AP-P (W03G9.4), analysis of which predicts regions displaying high levels of amino-acid sequence homology between the predicted gene product and mammalian cytoplasmic AP-P, with the absolute conservation of key catalytic residues. The sequence of an EST (yk91g4), comprising the open reading frame of W03G9.4, confirmed the predicted genomic structure of the gene and the prediction that W03G9.4 codes for a nonsecreted protein with a molecular mass of 68 kDa. Nematodes transformed with a promoter reporter construct, W03G9.4::GFP, showed high levels of fluorescence in the intestine of larvae and adult hermaphrodites, indicating that the intestine is a major site of W03G9.4 expression. yk91g4 tagged with a hexahistidine and DLYDDDDK peptide epitope was expressed in Escherichia coli to yield, after affinity purification, a recombinant protein with a molecular mass of 71 kDa. The recombinant W03G9.4 removed the N-terminal amino acid from bradykinin (RPPGFSPFR), a Caenorhabditis elegans neuropeptide (KPSFVRFamide) and Lem Trp 1 (APSGFLGVRamide), but did not display activity towards angiotensin I (NRVYIHPFHL), des-Arg bradykinin and AF1 (KNEFIRFamide). The activity towards bradykinin was inhibited by EDTA and 1, 10 phenanthroline, as expected for a metalloenzyme, and also by apstatin (IC50, 1 µm), a selective inhibitor of mammalian AP-P. A Km of 45 µm and an optimum pH of 7–8 was observed with bradykinin as the substrate. The activity of the nematode AP-P, like its mammalian counterparts, was strongly influenced by metal ions, with Co2+, Mn2+ and Zn2+ all inhibiting the hydrolysis of bradykinin. We conclude that W03G9.4 codes for a cytoplasmic AP-P with very similar enzymatic properties to those of mammalian AP-P, and we suggest that the enzyme has a physiological role in the intracellular hydrolysis of proline-containing peptides absorbed from the lumen of the intestine.
