title: A naturally variable residue in the S1 subsite of M1 family aminopeptidases modulates catalytic properties and promotes functional specialization.
abstract: M1 family metallo-aminopeptidases fulfill a wide range of critical and in some cases medically relevant roles in humans and human pathogens. The specificity of M1-aminopeptidases is dominated by the interaction of the well defined S1 subsite with the side chain of the first (P1) residue of the substrate and can vary widely. Extensive natural variation occurs at one of the residues that contributes to formation of the cylindrical S1 subsite. We investigated whether this natural variation contributes to diversity in S1 subsite specificity. Effects of 11 substitutions of the S1 subsite residue valine 459 in the Plasmodium falciparum aminopeptidase PfA-M1 and of three substitutions of the homologous residue methionine 260 in Escherichia coli aminopeptidase N were characterized. Many of these substitutions altered steady-state kinetic parameters for dipeptide hydrolysis and remodeled S1 subsite specificity. The most dramatic change in specificity resulted from substitution with proline, which collapsed S1 subsite specificity such that only substrates with P1-Arg, -Lys, or -Met were appreciably hydrolyzed. The structure of PfA-M1 V459P revealed that the proline substitution induced a local conformational change in the polypeptide backbone that resulted in a narrowed S1 subsite. The restricted specificity and active site backbone conformation of PfA-M1 V459P mirrored those of endoplasmic reticulum aminopeptidase 2, a human enzyme with proline in the variable S1 subsite position. Our results provide compelling evidence that changes in the variable residue in the S1 subsite of M1-aminopeptidases have facilitated the evolution of new specificities and ultimately novel functions for this important class of enzymes.
authors.affiliation: From the Departments of Biochemistry and.
authors.forename: Seema
authors.initials: S
authors.lastname: Dalal
authors.affiliation: From the Departments of Biochemistry and.
authors.forename: Daniel R T
authors.initials: DRT
authors.lastname: Ragheb
authors.affiliation: Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061.
authors.forename: Florian D
authors.initials: FD
authors.lastname: Schubot
authors.affiliation: From the Departments of Biochemistry and. Electronic address: klemba@vt.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Klemba
mesh_terms: D019943:Amino Acid Substitution; D018826:CD13 Antigens / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D004151:Dipeptides / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D019143:Evolution, Molecular; D006801:Humans; D006868:Hydrolysis; D020125:Mutation, Missense; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D004151:Dipeptides; D029968:Escherichia coli Proteins; D015800:Protozoan Proteins; D018826:CD13 Antigens
keywords: Aminopeptidase; ERAP2; Enzyme Kinetics; Enzyme Mutation; Enzyme Structure; Malaria
references.citation: Biochem J. 2011 Apr 15;435(2):411-20
references.pmid: 21314638
references.citation: Chem Biol. 2008 Sep 22;15(9):920-9
references.pmid: 18804029
references.citation: J Biol Chem. 2008 Oct 31;283(44):30010-4
references.pmid: 18768474
references.citation: Acta Crystallogr D Biol Crystallogr. 2009 Aug;65(Pt 8):814-22
references.pmid: 19622865
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):E526-34
references.pmid: 21844374
references.citation: Curr Pharm Des. 2009;15(31):3656-70
references.pmid: 19925418
references.citation: Methods Mol Biol. 2008;426:419-35
references.pmid: 18542881
references.citation: Nature. 2002 Oct 3;419(6906):498-511
references.pmid: 12368864
references.citation: Biochemistry. 2008 May 13;47(19):5303-11
references.pmid: 18416562
references.citation: Appl Microbiol Biotechnol. 1995 Dec;44(1-2):100-5
references.pmid: 8579823
references.citation: Protein Pept Lett. 2009;16(10):1256-66
references.pmid: 19508204
references.citation: Nat Immunol. 2005 Jul;6(7):689-97
references.pmid: 15908954
references.citation: Biochem Biophys Res Commun. 1967 Apr 20;27(2):157-62
references.pmid: 6035483
references.citation: Mol Biochem Parasitol. 2012 May;183(1):70-7
references.pmid: 22348949
references.citation: Nat Immunol. 2002 Dec;3(12):1169-76
references.pmid: 12436109
references.citation: Proc Natl Acad Sci U S A. 2009 Feb 24;106(8):2537-42
references.pmid: 19196988
references.citation: J Biol Chem. 2004 Oct 8;279(41):43000-7
references.pmid: 15304495
references.citation: J Biol Chem. 2003 Aug 22;278(34):32275-83
references.pmid: 12799365
references.citation: Biochemistry. 2012 Jan 10;51(1):286-95
references.pmid: 22106953
references.citation: J Biol Chem. 2010 Jan 29;285(5):3310-8
references.pmid: 19948737
references.citation: Microbiol Res. 2013 Jan 15;168(1):56-64
references.pmid: 22766257
references.citation: Trends Mol Med. 2008 Aug;14(8):361-71
references.pmid: 18603472
references.citation: J Biol Chem. 2011 Aug 5;286(31):27255-65
references.pmid: 21659511
references.citation: Proc Natl Acad Sci U S A. 2005 Nov 22;102(47):17107-12
references.pmid: 16286653
references.citation: J Biol Chem. 2006 Nov 3;281(44):33664-76
references.pmid: 16885166
references.citation: Malar J. 2010 Jun 30;9:189
references.pmid: 20591164
references.citation: Genome Res. 2004 Jun;14(6):1188-90
references.pmid: 15173120
references.citation: Nat Rev Rheumatol. 2010 Aug;6(8):461-7
references.pmid: 20531381
references.citation: Nature. 2002 Oct 3;419(6906):480-3
references.pmid: 12368856
references.citation: J Med Chem. 2007 Mar 22;50(6):1322-34
references.pmid: 17326615
references.citation: J Biol Chem. 2007 Dec 7;282(49):35978-87
references.pmid: 17895246
references.citation: Protein Eng. 1995 Feb;8(2):127-34
references.pmid: 7630882
references.citation: Proc Natl Acad Sci U S A. 2006 Sep 5;103(36):13339-44
references.pmid: 16938892