Abstract：
:Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k(cat) values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Collins T,De Vos D,Hoyoux A,Savvides SN,Gerday C,Van Beeumen J,Feller Gdoi
10.1016/j.jmb.2005.09.064keywords:
subject
Has Abstractpub_date
2005-11-25 00:00:00pages
425-35issue
2eissn
0022-2836issn
1089-8638pii
S0022-2836(05)01144-7journal_volume
354pub_type
杂志文章abstract：:The B-DNA decanucleotide C-G-A-T-C-G-6meA-T-C-G has been crystallized under the same conditions used earlier for C-G-A-T-C-G-A-T-C-G, but is found to adopt a new trigonal P3(2)21 packing mode instead of the expected orthorhombic P2(1)2(1)2(1) form. Unit cell dimensions a = b = 33.38 A, c = 98.30 A, gamma = 120 degrees...
journal_title：Journal of molecular biology
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journal_title：Journal of molecular biology
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doi：10.1016/j.jmb.2005.05.053
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doi：10.1006/jmbi.1998.1682
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