scholarly journals Riboflavin Synthase ofEscherichia coli

2001 ◽  
Vol 276 (15) ◽  
pp. 11524-11530 ◽  
Author(s):  
Boris Illarionov ◽  
Kristina Kemter ◽  
Sabine Eberhardt ◽  
Gerald Richter ◽  
Mark Cushman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Amino Acid ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Sequence Motif ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Substrate Binding Sites

Conserved amino acid residues of riboflavin synthase fromEscherichia coliwere modified by site-directed mutagenesis. Replacement or deletion of phenylalanine 2 afforded catalytically inactive proteins. S41A and H102Q mutants had substantially reduced reaction velocities. Replacements of various other conserved polar residues had little impact on catalytic activity.19F NMR protein perturbation experiments using a fluorinated intermediate analog suggest that the N-terminal sequence motif MFTG is part of one of the substrate-binding sites of the protein.

Analysis of essential carboxylic amino acid residues for catalytic activity of fungal chitosanases by site-directed mutagenesis

2005 ◽  
Vol 100 (5) ◽  
pp. 545-550 ◽  
Author(s):  
Makoto Shimosaka ◽  
Kazuaki Sato ◽  
Naohide Nishiwaki ◽  
Takashi Miyazawa ◽  
Mitsuo Okazaki
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues

scholarly journals Restoration of a Defective Lactococcus lactis Xylose Isomerase

2004 ◽  
Vol 70 (7) ◽  
pp. 4318-4325 ◽  
Author(s):  
Joo-Heon Park ◽  
Carl A. Batt
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Lactococcus Lactis ◽  
Xylose Isomerase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Triple Mutant ◽  
Sequence Homologies

ABSTRACT The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp. lactis strains, 210 (Xyl−) and IO-1 (Xyl+), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated. The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs. The purified IO-1 XI was soluble with K m and k cat being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive. Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble. The K m and k cat of this mutant were 4.15 mM and 141/s, respectively. One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI. The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility. The dissolution of XI activity in L. lactis subsp. lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme.

scholarly journals Two Amino Acid Residues of Transposase Contributing to Differential Transposability of IS1 Elements inEscherichia coli

1998 ◽  
Vol 180 (19) ◽  
pp. 5279-5283 ◽  
Author(s):  
Jiann-Hwa Chen ◽  
Wen-Ben Hsu ◽  
Jiing-Luen Hwang
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Inescherichia Coli ◽  
Amino Acid Residues ◽  
Insertion Element ◽  
Resistance Plasmid ◽  
Insertion Mutants

ABSTRACT Escherichia coli W3110 contains four types of IS1 elements in the chromosome. Using an insertion element entrapping system, we collected 116 IS1 plasmid insertion mutants, which resulted from a minimum of 26 independent IS1 insertion events. All of them had insertions of IS1 of the IS1A (IS1E and IS1G) type. Inspection of the transposase sequences of the four IS1 types and the IS1 of the resistance plasmid R100 showed that two amino acid residues, His-193 and Leu-217 of transposase, might contribute to differential transposability of IS1 elements in W3110. The two amino acid residues of the transposase in IS1A (IS1E and IS1G) were altered separately by site-directed mutagenesis, and each mutant was found to mediate transposition at a frequency about 30-fold lower than that of IS1A (IS1E and IS1G). Thus, the assumption that His-193 and Leu-217 of transposase contribute to differential transposability of IS1 elements in W3110 was confirmed.

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.

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