Studies on the Substrate Binding Segments and Catalytic Action of Lanosterol Synthase. Affinity Labeling with Carbocations Derived from Mechanism-Based Analogs of 2,3-Oxidosqualene and Site-Directed Mutagenesis Probes

1997 ◽  
Vol 119 (6) ◽  
pp. 1289-1296 ◽  
Author(s):  
E. J. Corey ◽  
Hengmiao Cheng ◽  
C. Hunter Baker ◽  
Seiichi P. T. Matsuda ◽  
Ding Li ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Catalytic Action ◽  
Site Directed Mutagenesis ◽  
Affinity Labeling ◽  
Directed Mutagenesis ◽  
Lanosterol Synthase

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

scholarly journals Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Sara Pintar ◽  
Jure Borišek ◽  
Aleksandra Usenik ◽  
Andrej Perdih ◽  
Dušan Turk
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Drug Targets ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Pathogen ◽  
Active Site Cleft ◽  
Novel Drug ◽  
Novel Drug Targets

AbstractTo achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.

Investigation of the ATP Binding Site ofEscherichia coliAminoimidazole Ribonucleotide Synthetase Using Affinity Labeling and Site-Directed Mutagenesis†

Biochemistry ◽  
1999 ◽  
Vol 38 (31) ◽  
pp. 9831-9839 ◽  
Author(s):  
E. J. Mueller ◽  
S. Oh ◽  
E. Kavalerchik ◽  
T. J. Kappock ◽  
E. Meyer ◽  
...  
Keyword(s):  
Binding Site ◽  
Site Directed Mutagenesis ◽  
Affinity Labeling ◽  
Directed Mutagenesis ◽  
Atp Binding ◽  
Atp Binding Site

Lys631 residue in the active site of the bacteriophage T7 RNA polymerase. Affinity labeling and site-directed mutagenesis

1991 ◽  
Vol 195 (3) ◽  
pp. 841-847 ◽  
Author(s):  
Tatyana G. MAKSIMOVA ◽  
Arkady A. MUSTAYEV ◽  
Evgeny F. ZAYCHIKOV ◽  
Dmitry L. LYAKHOV ◽  
Vera L. TUNITSKAYA ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
Affinity Labeling ◽  
Directed Mutagenesis
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