Expression, Purification, 3-D Homology Modelling and Immunoanalysis of Glucosyltransferases in Streptococcus mutans

• Main Authors: Yau-Wei Tsai, 蔡耀緯
• Other Authors: Kuo-Long Lou
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/02164008115640759582

碩士 === 國立臺灣大學 === 口腔生物科學研究所 === 88 === Glucosyltransferases (GTFs) of the Streptococcus mutans are responsible for the dental plaque formation in human oral cariogenesis by synthesizing glucans through hydrolysis of sucrose. There are three different kinds of GTFs in S. mutans: GtfB and GtfC are of membrane-associated form and responsible for synthesizing water-insoluble glucan, whereas GtfD is secretion form and responsible for water-soluble glucan. Much experimental work has been carried out to indicate the functional roles of these enzymes. All the three GTFs show very high homology in amino acid sequence while revealing great difference in the aspect of functional analysis. However, in present stage, no direct structural information is available to explain such functional discrimination. Therefore we have proceeded this work to approach such problem by comparing various GTFs in the aspect of three-dimensional structural characterization. (A) Based on a combined sequence and secondary structure alignment against known crystal structures of segments from closely related proteins, molecular modelling has been carried out to build up the 3-D models of GTFs and correlate the structure-functional interpretation in order to understand the catalytic behavior of GTFs. (B) Chia and coworkers have results suggested that monoclonal antibody recognizing a 19-residue fragment, Gtf-P1, can reduce the sucrase activity of GTFs to a large extent. We have accomplished the functional analyses in order to supplement structure-functional correlation on such inhibition mechanism. (C) With a longer viewing, only crystal structure can provide the complete direct structural evidence for molecular mechanism in atomic level. Therefore, the third part of our project is to proceed the crystallization of GTFs in order to obtain crystals prepared for the 3-D structural determination. 3-D models of all three GTFs in Streptococcus mutans have been accomplished. The results suggested that these enzymes retain a Tim-barrel structure constructing the active sites inside the barrels, which may provide the specificity pocket for the entrance of substrate sucrose, as well as the binding to enzymes and the catalysis. Gtf-P1 is located on the surface of the molecule and protrudes the N-terminal residues of its long a-helix into the vicinity of active site. This may allow the antibody binding resulting conformational change to affect the active site, and furthermore, reduce the activity. Our results have provided sufficient interpretation for such structure-functional correlation. However, more direct evidence to describe the reaction mechanisms is anticipated through the forthcoming crystal structural determination.