TY - JOUR
T1 - Molecular cloning and heterologous expression in Pichia pastoris of X-prolyl-dipeptidyl aminopeptidase from basidiomycete Ustilago maydis
AU - Juárez-Montiel, Margarita
AU - Ibarra, J. Antonio
AU - Chávez-Camarillo, Griselda
AU - Hernández-Rodríguez, César
AU - Villa-Tanaca, Lourdes
N1 - Funding Information:
Acknowledgments Authors would like to thank Martha Thayer for reviewing this manuscript thoroughly. MJM was a recipient of fellowships from CONACyT, ICyT-DF, and PIFI-IPN; LVT and CHHR received support from COFAA-IPN and EDI-IPN, as well as grants from ICyT-DF (PICSO10-95, Convenio 254/10) and CONACyT (208247, SIP-IPN 20120750, and 20131171). JAI was hired through the “Programa Institucional de Contratación de Personal Académico de Excelencia IPN.”
PY - 2014/3
Y1 - 2014/3
N2 - Dipeptidyl aminopeptidases are enzymes involved in the posttranslational control of bioactive peptides. Here we identified the gene dapUm in Ustilago maydis by homology with other fungal dipeptidyl aminopeptidases. Analysis of the dapUm-deduced amino acid sequence indicated that it encodes for membrane-type serine protease with a characteristic prolyl oligopeptidase catalytic motif triad: Ser, Asp, His. In order to overexpress the DapUm, the gene encoding for it was cloned and transformed into Pichia. Using this system, we observed a ∼125-kDa recombinant protein with an optimal enzymatic activity at pH 6.0 and at 40 °C for the Ala-Pro-p-nitroanilide substrate and an experimental pH of 6.9. U. maydis DapUm was specifically inhibited by phenylmethylsulfonyl fluoride and Pefabloc, confirming the presence of a serine residue in the active site. To our knowledge, this study is the first report on the cloning and expression of a DPP IV dipeptidyl aminopeptidase from a basidiomycete organism. Moreover, the use of recombinant DapUm will allow us to further study and characterize this enzyme, in addition to testing chemical compounds for pharmaceutical purposes.
AB - Dipeptidyl aminopeptidases are enzymes involved in the posttranslational control of bioactive peptides. Here we identified the gene dapUm in Ustilago maydis by homology with other fungal dipeptidyl aminopeptidases. Analysis of the dapUm-deduced amino acid sequence indicated that it encodes for membrane-type serine protease with a characteristic prolyl oligopeptidase catalytic motif triad: Ser, Asp, His. In order to overexpress the DapUm, the gene encoding for it was cloned and transformed into Pichia. Using this system, we observed a ∼125-kDa recombinant protein with an optimal enzymatic activity at pH 6.0 and at 40 °C for the Ala-Pro-p-nitroanilide substrate and an experimental pH of 6.9. U. maydis DapUm was specifically inhibited by phenylmethylsulfonyl fluoride and Pefabloc, confirming the presence of a serine residue in the active site. To our knowledge, this study is the first report on the cloning and expression of a DPP IV dipeptidyl aminopeptidase from a basidiomycete organism. Moreover, the use of recombinant DapUm will allow us to further study and characterize this enzyme, in addition to testing chemical compounds for pharmaceutical purposes.
KW - DPP IV
KW - EC 3.4.14
KW - U. maydis protease
UR - http://www.scopus.com/inward/record.url?scp=84899460731&partnerID=8YFLogxK
U2 - 10.1007/s12010-013-0682-4
DO - 10.1007/s12010-013-0682-4
M3 - Artículo
C2 - 24402567
AN - SCOPUS:84899460731
SN - 0273-2289
VL - 172
SP - 2530
EP - 2539
JO - Applied Biochemistry and Biotechnology
JF - Applied Biochemistry and Biotechnology
IS - 5
ER -