PEPD

Xaa-Pro dipeptidase, also known as prolidase, is an is_associated_with::enzyme that in humans is encoded by the PEPD is_associated_with::gene.

Function
Xaa-Pro dipeptidase is a cytosolic is_associated_with::dipeptidase that hydrolyzes dipeptides with is_associated_with::proline or is_associated_with::hydroxyproline at the carboxy terminus (but not Pro-Pro). It is important in collagen metabolism because of the high levels of is_associated_with::imino acids. Mutations at the PEPD locus cause is_associated_with::prolidase deficiency. This is characterised by Iminodipeptidurea, is_associated_with::skin ulcers, is_associated_with::mental retardation and recurrent infections.

Structure
Prolidases fall under a subclass of is_associated_with::metallopeptidases that involve binuclear is_associated_with::active site is_associated_with::metal clusters. This metal cluster facilitates is_associated_with::catalysis by serving as a substrate is_associated_with::binding site, activating is_associated_with::nucleophiles, and stabilizing the is_associated_with::transition state. Furthermore, prolidases are classified under a smaller family called “pita-bread” enzymes, which cleave amido-, imido-, and amidino- containing bonds. The “pita-bread” fold, containing a metal center flanked by two well-defined substrate binding pockets enabled prolidase to specifically cleave between any non-proline is_associated_with::amino acid and proline.



The first ever solved structure of prolidase came from the is_associated_with::hyperthermophilic is_associated_with::archaeon is_associated_with::Pyrococcus furiosus (Pfprol). This dimer has a crystal structure shows two approximately symmetrical is_associated_with::monomers that both have an N-terminal domain, made up of a six-stranded mixed β-sheet flanked by five α-helices, a helical linker, and C-terminal domain, consisting of a mixed six-stranded β-sheet flanked by four α-helices. The curved β-sheet of Domain II has a “pita-bread” fold. The active site lies on the inner surface of the β-sheet of Domain II, with a notable dinuclear Co cluster anchored by the side chains of two is_associated_with::aspartate residues (Asp209 and Asp220), two is_associated_with::glutamate residues (Glu313 and Glu327), and a is_associated_with::histidine residue (His284). is_associated_with::Carboxylate groups of aspartate and is_associated_with::glutamine residues serve as bridges between the two Co atoms. In the is_associated_with::crystallization process, the Co atoms are replaced with Zn, which hinders enzymatic activity.

Unlike Pfprol, the structure of the human variant remains poorly understood. Sequence homology between human and Pfprol yield only 25% identity and 43% similarity. The two available structures of human prolidase available on the is_associated_with::Protein Data Bank are is_associated_with::homodimers contain either Na or Mn, which bind to similar amino acids as those in Pfprol: Glu412 (Glu313 in Pfprol), binds to the first ion, Asp276 (Asp209 in Pfprol) binds to the second ion, and Asp287 and Glu452 bind to both (Asp220 and Glu327 in Pfprol).



Function
Due to proline’s cyclic structure, only few is_associated_with::peptidases could cleave the bond between proline and other amino acids. Along with is_associated_with::prolinase, prolidase are the only known enzymes that can break down dipeptides to yield free proline. Prolidase serve to hydrolyze both dietary and is_associated_with::endogenous Xaa-Pro dipeptides. More specifically, it is essential in catalyzing the last step of the degradation of procollagen, is_associated_with::collagen, and other proline-containing peptides into free amino acids to be used for cellular growth. Additionally, it also participates in the process of recycling proline from Xaa-Pro dipeptides for collagen resynthesis. Proline and hydroyxyproline make up a quarter of the amino acid residues in collegen, which is the most abundant protein in the body by mass and plays an important role in maintaining is_associated_with::connective tissue in the body.

Mechanism
The mechanism for prolidase catalytic activity remains largely uncharacterized. However, biochemical and structural analyses of is_associated_with::aminopeptidase (APPro), is_associated_with::methionine aminopeptidase (MetAP), and prolidase, all members of the “pita-bread” is_associated_with::metalloenzymes, suggest that they share a common mechanism scheme. The main difference arises in the location of the is_associated_with::carbonyl oxygen atom of the scissile peptide bond.



The following mechanism shows a proposed scheme for a metal-dependent “pita-bread” enzyme with residue numbering corresponding to those found in methionine aminopeptidase from E. coli. As shown in Intermediate I of the figure, three potential is_associated_with::acidic amino acid residues interact with the N-terminus of the substrate in a fashion that is yet to be determined. The carbonyl and amide groups of the scissile peptide bond interact with the first metal ion, M1, in addition to His178 and His79, respectively. M1 and Glu204 activate a water molecule to prepare it is_associated_with::nucleophilic attack at the carbonyl carbon of the scissile peptide bond. Then, the tetrahedral intermediate (Intermediate II) becomes stabilized from interactions with M1 and His178. Lastly, Glu204 donates a is_associated_with::proton to the amine of the leaving is_associated_with::peptide (P1’). This leads to the breakdown of the intermediate (Intermediate III), which retains its interactions with M1 and His178.

Regulation
Post-translational modifications of prolidase regulate its enzymatic abilities. is_associated_with::Phosphorylation of prolidase has been shown to increase its activity while is_associated_with::dephosphorylation leads to a decrease in enzyme activity. Analysis of known is_associated_with::consensus sequence required for is_associated_with::serine/is_associated_with::threonine phosphorylation revealed that prolidase contains at least three potential sites for serine/threonine phosphorylation. Nitric oxide, both exogenously acquired and endogenously generated, was shown to increase prolidase activity in a time- and dose-dependent manner via phosphorylation at these serine and threonine sites. Additionally, prolidase may also be regulated at is_associated_with::tyrosine phosphorylation sites, which are mediated by FAK and is_associated_with::MAPK signaling pathways.

Disease relevance
Deficiency in prolidase leads to a rare, severe autosomal recessive disorder (is_associated_with::prolidase deficiency) that causes many chronic, debilitating health conditions in humans. These phenotypical symptoms vary and may include is_associated_with::skin ulcerations, is_associated_with::mental retardation, is_associated_with::splenomegaly, recurrent is_associated_with::infections, is_associated_with::photosensitivity, is_associated_with::hyperkeratosis, and unusual facial appearance. Furthermore, prolidase activity was found to be abnormal compared to healthy levels in various medical conditions including but limited to: is_associated_with::bipolar disorder, is_associated_with::breast cancer, is_associated_with::endometrial cancer, is_associated_with::keloid scar formation, is_associated_with::erectile dysfunction, is_associated_with::liver disease, is_associated_with::lung cancer, is_associated_with::hypertension, is_associated_with::melanoma, and is_associated_with::chronic pancreatitis. In some cancers with increased levels of prolidase activity, such as melanoma, the differential expression of prolidase and its substrate specificity for dipeptides with proline at the is_associated_with::carboxyl end suggests the potential of prolidase in becoming a viable, selective is_associated_with::endogenous enzyme target for proline is_associated_with::prodrugs. Serum prolidase enzyme activity is also currently being explored as a possible, reliable marker for diseases including is_associated_with::chronic hepatitis B and is_associated_with::liver fibrosis

Other applications
is_associated_with::Decontamination: Prolidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pfprol) shows potential for application in decontamination of organophosphorus is_associated_with::nerve agents in chemical warfare agents. Additionally, prolidase could also serve to detect is_associated_with::fluorine-containing organophosphorus is_associated_with::neurotoxins, like the G-type chemical warfare agents, and could antagonize organophosphorous intoxication and protect against the effects of is_associated_with::diisopropylfluorophosphate when encapsulated in is_associated_with::liposomes.

Model organisms
is_associated_with::Model organisms have been used in the study of PEPD function. A conditional is_associated_with::knockout mouse line called Pepdtm1a(KOMP)Wtsi was generated at the is_associated_with::Wellcome Trust Sanger Institute. Male and female animals underwent a standardized is_associated_with::phenotypic screen to determine the effects of deletion. Additional screens performed: - In-depth immunological phenotyping

''