Elucidation of exo-beta-D-glucosaminidase activity of a family 9 glycoside hydrolase (PBPRA0520) from Photobacterium profundum SS9

Glycobiology. 2011 Apr;21(4):503-11. doi: 10.1093/glycob/cwq191. Epub 2010 Nov 22.

Abstract

A glycoside hydrolase (GH) gene from Photobacterium profundum SS9 (PBPRA0520) belonging to GH family 9 was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it is an intracellular enzyme. The recombinant protein showed hydrolytic activity toward chitobiose [(GlcN)(2)] and cellobiose (CG(2)) in various disaccharides. This protein also released 4-nitrophenol (PNP) from both 4-nitrophenyl-β-D-glucosaminide (GlcN-PNP) and 4-nitrophenyl-β-D-glucoside (Glc-PNP). The hydrolytic pattern observed in chitooligosaccharides and cellooligosaccharides suggested that the reaction proceeded from the nonreducing end in an exo-type manner. Time-dependent (1)H-nuclear magnetic resonance (NMR) analysis of the anomeric form of the enzymatic reaction products indicated that the protein is an inverting enzyme. k(cat)/K(m) of (GlcN)(2) hydrolysis was 14 times greater than that of CG(2) hydrolysis. These results suggested that the protein is an exo-β-D-glucosaminidase (EC 3.2.1.165) rather than a glucan 1,4-β-D-glucosidase (EC 3.2.1.74). Based on the results, we suggest that the function of conserved GH9 proteins in the chitin catabolic operon is to cleave a (GlcN)(2)-phosphate derivative by hydrolysis during intracellular chitooligosaccharide catabolism in Vibrionaceae.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, High Pressure Liquid
  • Chromatography, Thin Layer
  • Disaccharides / metabolism
  • Enzyme Assays
  • Enzyme Stability
  • Hexosaminidases / biosynthesis*
  • Hexosaminidases / chemistry
  • Hexosaminidases / isolation & purification
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Molecular Weight
  • Photobacterium / enzymology*
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Temperature

Substances

  • Disaccharides
  • Recombinant Proteins
  • Hexosaminidases