title: P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: insights into substrate selectivity and kinetic behavior.
abstract: Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil.
authors.forename: Débora N
authors.initials: DN
authors.lastname: Okamoto
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil.
authors.forename: Marcia Y
authors.initials: MY
authors.lastname: Kondo
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil.
authors.forename: Lilian C G
authors.initials: LC
authors.lastname: Oliveira
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisas em Energia e Materiais, 13083-100 Campinas, SP, Brazil.
authors.forename: Rodrigo V
authors.initials: RV
authors.lastname: Honorato
authors.affiliation: Departamento de Orgânica, Instituto de Química, UNICAMP, 13083-970 Campinas, Brazil.
authors.forename: Leticia M
authors.initials: LM
authors.lastname: Zanphorlin
authors.affiliation: Departamento de Física, IBILCE, UNESP, 15054-000 São José do Rio Preto, Brazil.
authors.forename: Monika A
authors.initials: MA
authors.lastname: Coronado
authors.affiliation: Departamento de Bioquímica, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil.
authors.forename: Mariana S
authors.initials: MS
authors.lastname: Araújo
authors.affiliation: Departamento de Bioquímica, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil.
authors.forename: Guacyara
authors.initials: G
authors.lastname: da Motta
authors.affiliation: Departamento de Bioquímica, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil.
authors.forename: Camila L
authors.initials: CL
authors.lastname: Veronez
authors.affiliation: Departamento de Ginecologia, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil.
authors.forename: Sheila S
authors.initials: SS
authors.lastname: Andrade
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisas em Energia e Materiais, 13083-100 Campinas, SP, Brazil.
authors.forename: Paulo S L
authors.initials: PS
authors.lastname: Oliveira
authors.affiliation: Departamento de Física, IBILCE, UNESP, 15054-000 São José do Rio Preto, Brazil.
authors.forename: Raghuvir K
authors.initials: RK
authors.lastname: Arni
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, 14040-903 Ribeirão Preto, SP, Brazil.
authors.forename: Adelia C O
authors.initials: AC
authors.lastname: Cintra
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, 14040-903 Ribeirão Preto, SP, Brazil.
authors.forename: Suely V
authors.initials: SV
authors.lastname: Sampaio
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil.
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisas em Energia e Materiais, 13083-100 Campinas, SP, Brazil. Electronic address: mario.murakami@lnbio.cnpem.br.
authors.forename: Mário T
authors.initials: MT
authors.lastname: Murakami
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil. Electronic address: iurig@yahoo.com.
authors.forename: Iuri E
authors.initials: IE
authors.lastname: Gouvea
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops; D003435:Crotalid Venoms / Q000201:enzymology*; D006868:Hydrolysis; D007610:Kallikreins / Q000378:metabolism*; D007700:Kinetics; D045726:Metalloproteases / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D000085822:Prolyl Oligopeptidases; D011863:Radioimmunoassay; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D003435:Crotalid Venoms; D010455:Peptides; D045726:Metalloproteases; D007610:Kallikreins; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases
keywords: FRET peptides; Kininogenase activity; Molecular dynamics simulations; Snake venom metalloproteinase; Substrate specificity