Production in Pichia pastoris, antifungal activity and crystal structure of a class I chitinase from cowpea (Vigna unguiculata): Insights into sugar binding mode and hydrolytic action.
A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu2+caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 Å by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and Rfreevalues were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.
Laboratório de Genética Molecular, Departamento de Biologia, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
Patrícia G Castro
PGC
Landim
Mcgill University, Biology Department, Stewart Biology Building 1205 Docteur Penfield, Montreal, Quebec, H3A 1B1, Canada.
Tuana O
TO
Correia
Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
Fredy D A
FDA
Silva
Laboratório de Genética Molecular, Departamento de Biologia, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
Denise R
DR
Nepomuceno
Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
Helen P S
HPS
Costa
Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, São Paulo, Brazil.
Humberto M
HM
Pereira
Núcleo de Biologia Experimental, Universidade de Fortaleza, Fortaleza, Ceará, Brazil.
Marina D P
MDP
Lobo
Núcleo de Biologia Experimental, Universidade de Fortaleza, Fortaleza, Ceará, Brazil.
Frederico B M B
FBMB
Moreno
Diamond Light Source, Harwell Science and Innovation Campus Didcot, Oxfordshire, OX11 0DE, United Kingdom.
José
J
Brandão-Neto
Laboratório de Genética Molecular, Departamento de Biologia, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
Suelen C
SC
Medeiros
Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
Ilka M
IM
Vasconcelos
Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
José T A
JTA
Oliveira
Departamento de Física, Universidade Federal do Ceará, 60440-760, Fortaleza, Ceará, Brazil.
Bruno L
BL
Sousa
Departamento de Química Analítica e Fisico-química, Universidade Federal do Ceará, 60455-760, Fortaleza, Ceará, Brazil.
Ito L
IL
Barroso-Neto
Departamento de Física, Universidade Federal do Ceará, 60440-760, Fortaleza, Ceará, Brazil.
Valder N
VN
Freire
Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.
Cristina P S
CPS
Carvalho
Núcleo de Biologia Experimental, Universidade de Fortaleza, Fortaleza, Ceará, Brazil.
Ana C O
ACO
Monteiro-Moreira
Laboratório de Genética Molecular, Departamento de Biologia, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil. Electronic address: thalles@ufc.br.
Thalles B
TB
Grangeiro
D000935:Antifungal Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002688:Chitinases / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006868:Hydrolysis; D010407:Penicillium / Q000187:drug effects; D010843:Pichia / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D000070658:Vigna / Q000201:enzymology*
D000935:Antifungal Agents; D010940:Plant Proteins; D002688:Chitinases; C119825:chitinase C
Chitin; Family 19 glycoside hydrolase; Hevein; Leguminosae