Proline racemase

proline racemase
Identifiers
EC no.	5.1.1.4
CAS no.	9024-09-3
Databases
IntEnz	IntEnz view
BRENDA	BRENDA entry
ExPASy	NiceZyme view
KEGG	KEGG entry
MetaCyc	metabolic pathway
PRIAM	profile
PDB structures	RCSB PDB PDBe PDBsum
Gene Ontology	AmiGO / QuickGO
Search PMC articles PubMed articles NCBI proteins

Proline racemase
Identifiers
Symbol	Pro_racemase
Pfam	PF05544
InterPro	IPR008794
Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary PDB 2azp A:6-313 1tm0 B:9-333

In enzymology, a proline racemase (EC 5.1.1.4) is an enzyme that catalyzes the chemical reaction

L-proline ${\displaystyle \rightleftharpoons }$ D-proline

Hence, this enzyme has two substrates, L- and D-proline, and two products, D- and L- proline.

This enzyme belongs to the family of proline racemases acting on free amino acids. The systematic name of this enzyme class is proline racemase. This enzyme participates in arginine and proline metabolism. These enzymes catalyse the interconversion of L- and D-proline in bacteria. ^[1]

Species distribution

This first eukaryotic proline racemase was identified in Trypanosoma cruzi and fully characterized Q9NCP4 . The parasite enzyme, TcPRAC, is as a co-factor-independent proline racemase and displays B-cell mitogenic properties when released by T. cruzi upon infection, contributing to parasite escape. ^{[2] [3]}

Novel proline racemases of medical and veterinary importance were described respectively in Clostridioides difficile (bacteria) ( Q17ZY4 ) ^[4] and Trypanosoma vivax ( B8LFE4 ). ^[5] These studies showed that a peptide motif used as a minimal pattern signature to identify putative proline racemases (motif III*) is insufficient stringent per se to discriminate proline racemases from 4-hydroxyproline epimerases (HyPRE). Also, additional, non-dissociated elements that account for the discrimination of these enzymes were identified, based for instance on polarity constraints imposed by specific residues of the catalytic pockets. Based on those elements, enzymes incorrectly described as proline racemases were biochemically proved to be hydroxyproline epimerases (i.e. HyPREs from Pseudomonas aeruginosa (Q9I476), Burkholderia pseudomallei ( Q63NG7 ), Brucella abortus ( Q57B94 ), Brucella suis ( Q8FYS0 ) and Brucella melitensis ( Q8YJ29 ). ^[4]

Structural studies

The biochemical mechanism of proline racemase was first put forward in the late sixties by Cardinale and Abeles ^[6] using the Clostridium sticklandii enzyme, CsPRAC. The catalytic mechanism of proline racemase was late revisited by Buschiazzo, Goytia and collaborators that, in 2006, resolved the structure of the parasite TcPRAC co-crystallyzed with its known competitive inhibitor - pyrrole carboxylic acid (PYC). ^[7] Those studies showed that each active enzyme contains two catalytic pockets. Isothermal titration calorimetry then showed that two molecules of PYC associate with TcPRAC in solution, and that this association is time-dependent and most probably based on mechanism of negative cooperativity. Complementary biochemical findings are consistent with the presence of two active catalytic sites per homodimer, each pertaining to one enzyme subunit, challenging the previously proposed mechanism of one catalytic site per homodimer previously proposed. ^[8]

Mechanism

The proline racemase active site contains two general bases, each of them a Cys, located on either side of the alpha-carbon of the substrate. In order to work properly, one Cys must be protonated (a thiol, RSH) and the other must be deprotonated (a thiolate, RS–).

Inhibition

P2C in Proline Racemase

Proline racemase is inhibited by pyrrole-2-carboxylic acid (P2C), a transition state analogue that is flat like the transition state. P2C acts as a competitive inhibitor, due to the chemical’s similarity to the transition state of the natural proline substrate. Inhibition of the active site Cys130 and Cys300 residues prevents the conversion of proline enantiomers. Pyrrole-2-carboxylic acid (P2C) reveals the presence of one catalytic center per monomer, with two Cys residues present to perform acid/base catalysis, utilizing a carbanion stabilization mechanism. The catalytic residues are 3.5 angstroms away from the molecule of P2C. ^[9]

References

1. Fisher LM, Albery WJ, Knowles JR (May 1986). "Energetics of proline racemase: racemization of unlabeled proline in the unsaturated, saturated, and oversaturated regimes". Biochemistry. 25 (9): 2529–37. doi:10.1021/bi00357a037. PMID   3755058.
2. Reina-San-Martín B, Degrave W, Rougeot C, Cosson A, Chamond N, Cordeiro-Da-Silva A, Arala-Chaves M, Coutinho A, Minoprio P (August 2000). "A B-cell mitogen from a pathogenic trypanosome is a eukaryotic proline racemase". Nature Medicine. 6 (8): 890–7. doi:10.1038/78651. PMID   10932226. S2CID   9163196.
3. Chamond N, Goytia M, Coatnoan N, Barale JC, Cosson A, Degrave WM, Minoprio P (October 2005). "Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity". Molecular Microbiology. 58 (1): 46–60. doi: 10.1111/j.1365-2958.2005.04808.x . PMID   16164548. S2CID   28436244.
4. Goytia M, Chamond N, Cosson A, Coatnoan N, Hermant D, Berneman A, Minoprio P (2007). "Molecular and structural discrimination of proline racemase and hydroxyproline-2-epimerase from nosocomial and bacterial pathogens". PLOS ONE. 2 (9): e885. Bibcode:2007PLoSO...2..885G. doi: 10.1371/journal.pone.0000885 . PMC   1964878 . PMID   17849014.
5. Chamond N, Cosson A, Coatnoan N, Minoprio P (June 2009). "Proline racemases are conserved mitogens: characterization of a Trypanosoma vivax proline racemase". Molecular and Biochemical Parasitology. 165 (2): 170–9. doi:10.1016/j.molbiopara.2009.02.002. PMID   19428664.
6. Cardinale GJ, Abeles RH (November 1968). "Purification and mechanism of action of proline racemase". Biochemistry. 7 (11): 3970–8. doi:10.1021/bi00851a026. PMID   5722267.
7. PDB: 1W61 ​ and PDB: 1W62 ​; Buschiazzo A, Goytia M, Schaeffer F, Degrave W, Shepard W, Grégoire C, Chamond N, Cosson A, Berneman A, Coatnoan N, Alzari PM, Minoprio P (February 2006). "Crystal structure, catalytic mechanism, and mitogenic properties of Trypanosoma cruzi proline racemase". Proceedings of the National Academy of Sciences. 103 (6): 1705–10. Bibcode:2006PNAS..103.1705B. doi: 10.1073/pnas.0509010103 . PMC   1413642 . PMID   16446443.
8. Albery WJ, Knowles JR (May 1986). "Energetics and mechanism of proline racemase". Biochemistry. 25 (9): 2572–7. doi:10.1021/bi00357a043. PMID   3718964.
9. Buschiazzo, Alejandro (February 2006). "Crystal structure, catalytic mechanism, and mitogenic properties of Trypanosoma cruzi proline racemase". Proc Natl Acad Sci U S A. 103 (6): 1705–10. doi:10.1073/pnas.0509010103. PMC   1413642 .

Further reading

• Stadtman TC, Elliott P (October 1957). "Studies on the enzymic reduction of amino acids. II. Purification and properties of D-proline reductase and a proline racemase from Clostridium sticklandii". The Journal of Biological Chemistry. 228 (2): 983–97. doi: 10.1016/S0021-9258(18)70675-3 . PMID   13475375.
• Stenta M, Calvaresi M, Altoè P, Spinelli D, Garavelli M, Bottoni A (January 2008). "The catalytic activity of proline racemase: a quantum mechanical/molecular mechanical study". The Journal of Physical Chemistry B. 112 (4): 1057–9. doi:10.1021/jp7104105. PMID   18044876.
• Chamond N, Goytia M, Coatnoan N, Barale JC, Cosson A, Degrave WM, Minoprio P (October 2005). "Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity". Molecular Microbiology. 58 (1): 46–60. doi: 10.1111/j.1365-2958.2005.04808.x . PMID   16164548. S2CID   28436244.
• Chamond N, Cosson A, Blom-Potar MC, Jouvion G, D'Archivio S, Medina M, Droin-Bergère S, Huerre M, Goyard S, Minoprio P (2010). "Trypanosoma vivax infections: pushing ahead with mouse models for the study of Nagana. I. Parasitological, hematological and pathological parameters". PLOS Neglected Tropical Diseases. 4 (8): e792. doi: 10.1371/journal.pntd.0000792 . PMC   2919405 . PMID   20706595.
• Blom-Potar MC, Chamond N, Cosson A, Jouvion G, Droin-Bergère S, Huerre M, Minoprio P (2010). "Trypanosoma vivax infections: pushing ahead with mouse models for the study of Nagana. II. Immunobiological dysfunctions". PLOS Neglected Tropical Diseases. 4 (8): e793. doi: 10.1371/journal.pntd.0000793 . PMC   2919407 . PMID   20711524.
• Conti P, Tamborini L, Pinto A, Blondel A, Minoprio P, Mozzarelli A, De Micheli C (September 2011). "Drug Discovery Targeting Amino Acid Racemases" (PDF). Chemical Reviews. 111 (11): 6919–46. doi:10.1021/cr2000702. PMID   21913633.