Elucidation of the Role of Functional Amino Acid Residues of the Small Sialidase from Clostridium perfringens by Site-Directed Mutagenesis

2001 ◽  
Vol 382 (2) ◽  
Author(s):  
Reinhard G. Kleineidam ◽  
Susanne Kruse ◽  
Peter Roggentin ◽  
Roland Schauer
Keyword(s):  
Amino Acid ◽  
Clostridium Perfringens ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues

scholarly journals Identification of amino acid residues responsible for differences in substrate specificity and inhibitor sensitivity between two human liver dihydrodiol dehydrogenase isoenzymes by site-directed mutagenesis

1997 ◽  
Vol 323 (1) ◽  
pp. 61-64 ◽  
Author(s):  
Kazuya MATSUURA ◽  
Yoshihiro DEYASHIKI ◽  
Kumiko SATO ◽  
Naoko ISHIDA ◽  
Gunpei MIWA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Human Liver ◽  
Hydroxysteroid Dehydrogenase ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Dihydrodiol Dehydrogenase ◽  
Inactive Protein

Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20α-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3α-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors.

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.

Identification of Amino Acid Residues Underlying the Antiport Mechanism of the Mitochondrial Carnitine/Acylcarnitine Carrier by Site-Directed Mutagenesis and Chemical Labeling

Biochemistry ◽  
2014 ◽  
Vol 53 (44) ◽  
pp. 6924-6933 ◽  
Author(s):  
Nicola Giangregorio ◽  
Lara Console ◽  
Annamaria Tonazzi ◽  
Ferdinando Palmieri ◽  
Cesare Indiveri
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Chemical Labeling
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