A novel xyloglucan-specific endo-beta-1,4-glucanase: biochemical properties and inhibition studies.
A novel xyloglucan-specific endo-beta-1,4-glucanase gene (xeg5A) was isolated, cloned, and expressed in Esherichia coli. The enzyme XEG5A consisted of a C-terminal catalytic domain and N-terminal sequence of approximately 90 amino acid residues with unknown function. The catalytic domain assumed an (alpha/beta)(8)-fold typical of glycoside hydrolase (GH) family 5, with the two catalytic residues Glu240 and Glu362 located on opposite sides of the surface groove of the molecule. The recombinant enzyme showed high specificity towards tamarind xyloglucan and decreasing activity towards xyloglucan oligosaccharide (HDP-XGO), carboxymethyl cellulose, and lichenan. Tamarind xyloglucan was hydrolyzed to three major fragments, XXXG, XXLG/XLXG, and XLLG. The hydrolysis followed the Michaelis-Menten kinetics, yielding K (m) and V (max) of 3.61 +/- 0.23 mg/ml and 0.30 +/- 0.01 mg/ml/min, respectively. However, the hydrolysis of HDP-XGO showed a decrease in the rate at high concentrations suggesting appearance of excess substrate inhibition. The addition of XXXG resulted in linear noncompetitive inhibition on the hydrolysis of tamarind xyloglucan giving a K (i) of 1.46 +/- 0.13 mM. The enzyme was devoid of transglycosylase activities.
Wong
Dominic D W S
DD
Western Regional Research Center, USDA-ARS, 800 Buchanan Street, Albany, CA 94710, USA. dwsw@pw.usda.gov
Chan
Victor J
VJ
McCormack
Amanda A
AA
Batt
Sarah B
SB
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D005936:Glucans / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012417:Rumen / Q000382:microbiology; D016415:Sequence Alignment; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism*
D001426:Bacterial Proteins; D005936:Glucans; D014990:Xylans; C029353:xyloglucan; D002480:Cellulase