scholarly journals Identification of the Substrate Specificity-conferring Amino Acid Residues of 4-Coumarate:Coenzyme A Ligase Allows the Rational Design of Mutant Enzymes with New Catalytic Properties

2001 ◽  
Vol 276 (29) ◽  
pp. 26893-26897 ◽  
Author(s):  
Hans-Peter Stuible ◽  
Erich Kombrink
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Rational Design ◽  
Catalytic Properties ◽  
Amino Acid Residues

scholarly journals Rational design of novel amphipathic antimicrobial peptides focused on the distribution of cationic amino acid residues

MedChemComm ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 896-900 ◽  
Author(s):  
Takashi Misawa ◽  
Chihiro Goto ◽  
Norihito Shibata ◽  
Motoharu Hirano ◽  
Yutaka Kikuchi ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Amino Acid ◽  
Human Cell ◽  
Hemolytic Activity ◽  
Rational Design ◽  
Cell Cytotoxicity ◽  
Amino Acid Residues ◽  
High Antimicrobial Activity ◽  
Cationic Amino Acid ◽  
Helical Peptide

Amphipathic helical peptideStripeshowed high antimicrobial activity, low hemolytic activity, and low human cell cytotoxicity.

scholarly journals Efficient Synthesis of Rare Disaccharides by Engineered β-Glucosidase Based on Hydropathy Index

2020 ◽  
Author(s):  
Kangle Niu ◽  
Zhengyao Liu ◽  
Yuhui Feng ◽  
Tianlong Gao ◽  
Zhenzhen Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rational Design ◽  
Large Scale ◽  
Catalytic Site ◽  
Scale Production ◽  
Total Production ◽  
Amino Acid Residues ◽  
Hydropathy Index ◽  
Reverse Hydrolysis ◽  
Hydrolysis Activity

<p>Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis, especially rare disaccharides, is reverse hydrolysis by <i>β</i>-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants<i> Tr</i>Cel1b<sup>I177S</sup>, <i>Tr</i>Cel1b<sup>I177S/I174S</sup>, and <i>Tr</i>Cel1b<sup>I177S/I174S/W173H</sup>, and one negative variant <i>Tr</i>Cel1b<sup>N240I</sup> were designed according to the <u>H</u>ydropathy <u>I</u>ndex <u>F</u>or <u>E</u>nzyme <u>A</u>ctivity (HIFEA) strategy. The reverse hydrolysis with <i>Tr</i>Cel1b<sup>I177S/I174S/W173H </sup>was accelerated and then the maximum total production (<a>195.8 mg/ml/mg enzyme</a>) of the synthesized disaccharides was increased 3.5-fold compared to that of wildtype. On the contrary, <a><i>Tr</i>Cel1b</a><sup>N240I</sup> lost reverse hydrolysis activity. The results demonstrate that<a> </a><a>the average hydropathy index</a> of <a>the key amino acid residues </a>in the catalytic site of<i> Tr</i>Cel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of <i>β</i>-glucosidases used for the synthesis of oligosaccharides.</p>

Prediction of amino acid residues participated in substrate recognition by cytochrome P450 subfamilies with broad substrate specificity

2013 ◽  
Vol 26 (2) ◽  
pp. 86-91 ◽  
Author(s):  
Maria S. Zharkova ◽  
Boris N. Sobolev ◽  
Nina Yu. Oparina ◽  
Alexander V. Veselovsky ◽  
Alexander I. Archakov
Keyword(s):  
Cytochrome P450 ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Substrate Recognition ◽  
Amino Acid Residues ◽  
Broad Substrate Specificity

scholarly journals Gene Cloning and Molecular Characterization of Lysine Decarboxylase from Selenomonas ruminantiumDelineate Its Evolutionary Relationship to Ornithine Decarboxylases from Eukaryotes

2000 ◽  
Vol 182 (23) ◽  
pp. 6732-6741 ◽  
Author(s):  
Yumiko Takatsuka ◽  
Yoshihiro Yamaguchi ◽  
Minenobu Ono ◽  
Yoshiyuki Kamio
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Pyridoxal Phosphate ◽  
Evolutionary Relationship ◽  
Binding Domain ◽  
Sequencing Analysis ◽  
Lysine Decarboxylase ◽  
Amino Acid Residues ◽  
Phosphate Binding ◽  
Link Type

ABSTRACT Lysine decarboxylase (LDC; EC 4.1.1.18 ) from Selenomonas ruminantium comprises two identical monomeric subunits of 43 kDa and has decarboxylating activities toward both l-lysine andl-ornithine with similar Km andVmax values (Y. Takatsuka, M. Onoda, T. Sugiyama, K. Muramoto, T. Tomita, and Y. Kamio, Biosci. Biotechnol. Biochem. 62:1063–1069, 1999). Here, the LDC-encoding gene (ldc) of this bacterium was cloned and characterized. DNA sequencing analysis revealed that the amino acid sequence of S. ruminantium LDC is 35% identical to those of eukaryotic ornithine decarboxylases (ODCs; EC 4.1.1.17 ), including the mouse,Saccharomyces cerevisiae, Neurospora crassa,Trypanosoma brucei, and Caenorhabditis elegansenzymes. In addition, 26 amino acid residues, K69, D88, E94, D134, R154, K169, H197, D233, G235, G236, G237, F238, E274, G276, R277, Y278, K294, Y323, Y331, D332, C360, D361, D364, G387, Y389, and F397 (mouse ODC numbering), all of which are implicated in the formation of the pyridoxal phosphate-binding domain and the substrate-binding domain and in dimer stabilization with the eukaryotic ODCs, were also conserved inS. ruminantium LDC. Computer analysis of the putative secondary structure of S. ruminantium LDC showed that it is approximately 70% identical to that of mouse ODC. We identified five amino acid residues, A44, G45, V46, P54, and S322, within the LDC catalytic domain that confer decarboxylase activities toward bothl-lysine and l-ornithine with a substrate specificity ratio of 0.83 (defined as thek cat/Km ratio obtained with l-ornithine relative to that obtained withl-lysine). We have succeeded in converting S. ruminantium LDC to form with a substrate specificity ratio of 58 (70 times that of wild-type LDC) by constructing a mutant protein, A44V/G45T/V46P/P54D/S322A. In this study, we also showed that G350 is a crucial residue for stabilization of the dimer in S. ruminantium LDC.

scholarly journals Amino Acid Residues Involved in the Substrate Specificity of TauT/SLC6A6 for Taurine and γ-Aminobutyric Acid

2014 ◽  
Vol 37 (5) ◽  
pp. 817-825 ◽  
Author(s):  
Tohru Yahara ◽  
Masanori Tachikawa ◽  
Shin-ichi Akanuma ◽  
Yoshiyuki Kubo ◽  
Ken-ichi Hosoya
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Aminobutyric Acid ◽  
Amino Acid Residues

scholarly journals Protein engineering of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4

1993 ◽  
Vol 292 (1) ◽  
pp. 69-74 ◽  
Author(s):  
W Asmara ◽  
U Murdiyatmo ◽  
A J Baines ◽  
A T Bull ◽  
D J Hardman
Keyword(s):  
Amino Acid ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Protonated Form ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Pseudomonas Cepacia ◽  
Amino Acid Residues ◽  
Substrate Complex ◽  
Key Residues

The chemical modification of L-2-haloacid halidohydrolase IVa (Hdl IVa), originally identified in Pseudomonas cepacia MBA4, produced as a recombinant protein in Escherichia coli DH5 alpha, led to the identification of histidine and arginine as amino acid residues likely to play a part in the catalytic mechanism of the enzyme. These results, together with DNA sequence and analyses [Murdiyatmo, Asmara, Baines, Bull and Hardman (1992) Biochem. J. 284, 87-93] provided the basis for the rational design of a series of random- and site-directed-mutagenesis experiments of the Hdl IVa structural gene (hdl IVa). Subsequent apparent kinetic analyses of purified mutant enzymes identified His-20 and Arg-42 as the key residues in the activity of this halidohydrolase. It is also proposed that Asp-18 is implicated in the functioning of the enzyme, possibly by positioning the correct tautomer of His-20 for catalysis in the enzyme-substrate complex and stabilizing the protonated form of His-20 in the transition-state complex. Comparison of conserved amino acid sequences between the Hdl IVa and other halidohydrolases suggests that L-2-haloacid halidohydrolases contain conserved amino acid sequences that are not found in halidohydrolases active towards both D- and L-2-monochloropropionate.

scholarly journals Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases

2015 ◽  
Vol 57 (1) ◽  
pp. 89-99 ◽  
Author(s):  
Kenshi Watanabe ◽  
Makoto Ohno ◽  
Masahiro Taguchi ◽  
Seiji Kawamoto ◽  
Kazuhisa Ono ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Residues ◽  
Fatty Acid Desaturases ◽  
Front End ◽  
Membrane Bound

scholarly journals A novel strategy for efficient disaccharides synthesis from glucose by β-glucosidase

2020 ◽  
Author(s):  
Kangle Niu ◽  
Zhengyao Liu ◽  
Yuhui Feng ◽  
Tianlong Gao ◽  
Zhenzhen Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rational Design ◽  
Large Scale ◽  
Catalytic Site ◽  
Scale Production ◽  
Total Production ◽  
Amino Acid Residues ◽  
Hydropathy Index ◽  
Reverse Hydrolysis ◽  
Hydrolysis Activity

Abstract Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis is reverse hydrolysis by β-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants TrCel1bI177S, TrCel1bI177S/I174S, and TrCel1bI177S/I174S/W173H, and one negative variant TrCel1bN240I were designed according to the Hydropathy Index For Enzyme Activity (HIFEA) strategy. The reverse hydrolysis with TrCel1bI177S/I174S/W173H was accelerated and then the maximum total production (195.8 mg/ml/mg enzyme) of the synthesized disaccharides was increased 3.5-fold compared to that of wildtype. On the contrary, TrCel1bN240I lost reverse hydrolysis activity. The results demonstrate that the average hydropathy index of the key amino acid residues in the catalytic site of TrCel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of β-glucosidases used for the synthesis of oligosaccharides.

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