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Glycoside Hydrolase Family 12
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|Glycoside Hydrolase Family GH12|
|Active site residues||known|
|CAZy DB link|
The substrate specificities found among the glycoside hydrolases of family 12 are: endo-β-1,4-glucanase (EC 22.214.171.124), xyloglucan endo-hydrolase (EC 126.96.36.199), endo-β-1,3-1,4-glucanase (EC 188.8.131.52). Xyloglucan endo-transglycosylase (XET, EC 184.108.40.206) activity has been observed in a single fungal GH12 member (GenBank AAN89225.1) using a XET-specific screen , although this may represent a side activity of a predominant xyloglucan endo-hydrolase [2, 3].
Kinetics and Mechanism
GH12 enzymes are retaining enzymes, as first shown by NMR studies  on endoglucanase 3 from Humicola insolens, and is believed to follow a classical Koshland double-displacement mechanism in which a glycosyl-enzyme intermediate is formed and subsequently this intermediate is hydrolysed via oxocarbenium-ion transition states. No detailed studies involving both steady state and pre-steady state kinetic have yet been reported for GH12.
The catalytic nucleophile and the general acid/base catalyst of GH12 enzymes was initialy predicted by sequence homology to the xylanase members of GH11, a glycoside hydrolase family where the catalytic nucleophile was first identified in the Bacillus circulans endo-xylanase through trapping of the 2-deoxy-2-fluoroxylobiosyl-enzyme intermediate and subsequent peptide mapping via LC-MS/MS technologies . GH11 and GH12 together form clan GH-C. The prediction of the catalytic nucleophile and the general acid/base of GH family 12 enzymes was later confirmed to be correct when the first three dimensional structure of a GH family 12 enzyme was determined, that of Streptomyces Lividans CelB . The catalytic nucleophile was subsequentialy confirmed in Streptomyces Lividans CelB to be Glu 120 by using the same labeling strategy used for detecting the catalytic nucleophile of GH family 11 .
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- First sterochemistry determination
- Humicola insolens endoglucanase 3 by NMR .
- First catalytic nucleophile identification
- First general acid/base residue identification
- First 3-D structure
- Nielsen, RI. (2002) Microbial xyloglucan endotransglycosylase (XET) Patent US6448056.
- Gilbert HJ, Stålbrand H, and Brumer H. How the walls come crumbling down: recent structural biochemistry of plant polysaccharide degradation. Curr Opin Plant Biol. 2008 Jun;11(3):338-48. DOI:10.1016/j.pbi.2008.03.004 |
- Eklöf JM and Brumer H. The XTH gene family: an update on enzyme structure, function, and phylogeny in xyloglucan remodeling. Plant Physiol. 2010 Jun;153(2):456-66. DOI:10.1104/pp.110.156844 |
- Schou C, Rasmussen G, Kaltoft MB, Henrissat B, and Schülein M. Stereochemistry, specificity and kinetics of the hydrolysis of reduced cellodextrins by nine cellulases. Eur J Biochem. 1993 Nov 1;217(3):947-53.
- Sulzenbacher G, Shareck F, Morosoli R, Dupont C, and Davies GJ. The Streptomyces lividans family 12 endoglucanase: construction of the catalytic cre, expression, and X-ray structure at 1.75 A resolution. Biochemistry. 1997 Dec 23;36(51):16032-9. DOI:10.1021/bi972407v |
- Miao S, Ziser L, Aebersold R, and Withers SG. Identification of glutamic acid 78 as the active site nucleophile in Bacillus subtilis xylanase using electrospray tandem mass spectrometry. Biochemistry. 1994 Jun 14;33(23):7027-32.
- Zechel DL, He S, Dupont C, and Withers SG. Identification of Glu-120 as the catalytic nucleophile in Streptomyces lividans endoglucanase celB. Biochem J. 1998 Nov 15;336 ( Pt 1):139-45.