Identification of a novel beta-N-acetylhexosaminidase (Pcb-NAHA1) from marine Zoanthid Palythoa caribaeorum (Cnidaria, Anthozoa, Zoanthidea).
beta-N-Acetylhexosaminidases (EC 3.2.1.52) belong to an enzyme family that hydrolyzes terminal beta-d-N-glucosamine and beta-d-N-galactosamine residues from oligosaccharides. In this report, we purified a novel beta-N-acetylhexosaminidase (Pcb-NAHA1) from the marine zoanthid Palythoa caribaeorum by applying ammonium sulfate fractionation, affinity chromatography on a chitin column, followed by two rounds of size exclusion chromatography. SDS-PAGE analysis indicated a single band protein of apparent homogeneity with a molecular mass of 25kDa. The purified enzyme preferentially hydrolyzed p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylglucosamide (pNP-GlcNAc) and to a lesser extent p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylgalactosamide (pNP-GalNAc). Detailed kinetic analysis using pNP-GlcNAc resulted in a specific activity of 57.9 U/mg, a K(m) value of 0.53 mM and a V(max) value of 88.1 micromol/h/mg and k(cat) value of 0.61s(-1). Furthermore, purified Pcb-NAHA1 enzyme activity was decreased by Hg Cl(2) or maltose and stimulated in the presence of Na(2)SeO(4,) BaCl(2), MgCl(2,) chondroitin 6-sulfate, and phenylmethylsulfonylfluoride. The optimum activity of Pcb-NAHA1 was observed at pH 5.0 and elevated temperatures (45-60 degrees C). Direct sequencing of proteolytic fragments generated from Pcb-NAHA1 revealed remarkable similarities to plant chitinases, which belong to family 18, although no chitinase activity was detected with Pcb-NAHA1. We conclude that beta-N-acetylhexosaminidases, representing a type of exochitinolytic activity, and endo-chitinases share common functional domains and/or may have evolved from a common ancestor.
Souza
Djair S L
DS
Embrapa Recursos Genéticos e Biotecnologia, PqEB-Final W5 Norte-Cp02372, Brasilia, DF, Brazil.
Grossi-de-Sa
Maria F
MF
Silva
Luciano P
LP
Franco
Octavio L
OL
Gomes-Junior
José E
JE
Oliveira
Gustavo R
GR
Rocha
Thales L
TL
Magalhães
Cláudio P
CP
Marra
Brener M
BM
Grossi-de-Sa
Maíra
M
Romano
Eduardo
E
de Sá
César Martins
CM
Kombrink
Erich
E
Jiménez
Arnubio V
AV
Abreu
Luiz R D
LR
D000595:Amino Acid Sequence; D000818:Animals; D037421:Anthozoa / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D001619:beta-N-Acetylhexosaminidases