scholarly journals Contribution of interactions with the core domain of hirudin to the stability of its complex with thrombin

1994 ◽  
Vol 298 (2) ◽  
pp. 507-510 ◽  
Author(s):  
A Betz ◽  
P C R Hopkins ◽  
B F Le Bonniec ◽  
S R Stone
Keyword(s):  
Crystal Structure ◽  
Binding Energy ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Ionic Interactions ◽  
Directed Mutagenesis ◽  
Side Chains ◽  
Core Domain ◽  
The Core ◽  
The Stability

The importance of interactions with residues 15-21 in the core domain of hirudin for the formation of the complex with thrombin has been investigated by site-directed mutagenesis. Contacts made by Leu-15 were found to be particularly important; replacement of this residue by alanine led to a decrease in the binding energy (delta delta Gbo) of 15 kJ.mol-1. Comparison with effects obtained in previous mutagenesis studies indicate that interactions with the side chain of Leu-15 contribute more to the stability of the complex than those of any other hirudin residues. Interactions with the side chains of Glu-17, Asn-20 and Val-21 also contributed significantly to binding energy; the delta delta Gbo value for these mutations was between 3 and 6 kJ.mol-1. Examination of the crystal structure of the thrombin-hirudin complex suggested the possibility that ionic interactions that would increase binding energy could be engineered by mutating Ser-19, Asn-20 and Gln-49 to acidic residues. The stability of the thrombin-hirudin complex was not, however, increased by these substitutions. The results obtained are discussed in terms of the crystal structure of the thrombin-hirudin complex.

scholarly journals Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic efficiency

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Canfang Niu ◽  
Peilong Yang ◽  
Huiying Luo ◽  
Huoqing Huang ◽  
Yaru Wang ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Side Chains ◽  
Cleavage Sites ◽  
Corn Meal ◽  
Feed Enzymes ◽  
The Stability ◽  
Phytate Content

Abstract Strong resistance to proteolytic attack is important for feed enzymes. Here, we selected three predicted pepsin cleavage sites, L99, L162, and E230 (numbering from the initiator M of premature proteins), in pepsin-sensitive HAP phytases YkAPPA from Yersinia kristensenii and YeAPPA from Y. enterocolitica, which corresponded to L99, V162, and D230 in pepsin-resistant YrAPPA from Y. rohdei. We constructed mutants with different side chain structures at these sites using site-directed mutagenesis and produced all enzymes in Escherichia coli for catalytic and biochemical characterization. The substitutions E230G/A/P/R/S/T/D, L162G/A/V, L99A, L99A/L162G, and L99A/L162G/E230G improved the pepsin resistance. Moreover, E230G/A and L162G/V conferred enhanced pepsin resistance on YkAPPA and YeAPPA, increased their catalytic efficiency 1.3–2.4-fold, improved their stability at 60 °C and pH 1.0–2.0 and alleviated inhibition by metal ions. In addition, E230G increased the ability of YkAPPA and YeAPPA to hydrolyze phytate from corn meal at a high pepsin concentration and low pH, which indicated that optimization of the pepsin cleavage site side chains may enhance the pepsin resistance, improve the stability at acidic pH, and increase the catalytic activity. This study proposes an efficient approach to improve enzyme performance in monogastric animals fed feed with a high phytate content.

scholarly journals Ionic interactions in the formation of the thrombin-hirudin complex

1991 ◽  
Vol 275 (3) ◽  
pp. 801-803 ◽  
Author(s):  
A Betz ◽  
J Hofsteenge ◽  
S R Stone
Keyword(s):  
Amino Acids ◽  
Binding Energy ◽  
Glutamic Acid ◽  
Crystal Structures ◽  
Site Directed Mutagenesis ◽  
Ionic Interactions ◽  
Directed Mutagenesis ◽  
Salt Bridges ◽  
Acidic Residues ◽  
Terminal Amino

Site-directed mutagenesis has been used to examine the importance of each of the acidic C-terminal residues of hirudin in the formation of its complex with alpha-thrombin. The contribution to binding energy of acidic residues in the 11 C-terminal amino acids varied from 2.3 to 5.9 kJ.mol-1. The differences between the contributions of individual residues were smaller than would be expected from the crystal structures of the thrombin-hirudin complex. In particular, the small effect (2.4 kJ.mol-1) for the replacement of Asp-55 was surprising in view of the two salt bridges made by this residue. The results of studies involving multiple mutations indicated that the additivity of the effects varied with the position of the mutation. Whereas the effect of mutations involving the glutamic acid residues at positions 61 and 62 were additive, non-additivity was observed with the glutamic acid residues at positions 57 and 58.

Engineering Enzymes for Enhanced Thermostability

1990 ◽  
Vol 218 ◽  
Author(s):  
Phoebe Shih ◽  
Bruce A. Malcolm ◽  
Jack F. Kirsch
Keyword(s):  
Amino Acid ◽  
Linear Correlation ◽  
Egg White ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Individual Amino Acid ◽  
Amino Acid Residues ◽  
The Core ◽  
Egg White Lysozyme

AbstractChicken egg-white lysozyme (CEWL) is used as a model to attempt to engineer proteins for enhanced thermostability. Site-directed mutagenesis is employed for selective amino acid substitution to probe the contribution of an individual amino acid in a given sequence to thermostability. A linear correlation is found between the side-chain volume of a triplet of amino acid residues located at the interior core of the protein and its thermostability. Additional mutant constructs at the core position reveal that hyperpacking can disrupt other intramolecular contacts and offset the hydrophobic stabilization due to denser packing. Multiple substitutions at different loci of the protein are combined to analyze the additivity of thermostability mutations.

scholarly journals Threonine 57 is required for the post-translational activation ofEscherichia coliaspartate α-decarboxylase

2014 ◽  
Vol 70 (4) ◽  
pp. 1166-1172 ◽  
Author(s):  
Michael E. Webb ◽  
Briony A. Yorke ◽  
Tom Kershaw ◽  
Sarah Lovelock ◽  
Carina M. C. Lobley ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Intramolecular Rearrangement ◽  
Directed Mutagenesis ◽  
Vitamin B ◽  
Biosynthesis Pathway ◽  
Serine Residue ◽  
Translational Activation

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formedviathe intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

scholarly journals Crystal Structure and Site-directed Mutagenesis ofBacillus maceransEndo-1,31,4--glucanase

1995 ◽  
Vol 270 (7) ◽  
pp. 3081-3088 ◽  
Author(s):  
Michael Hahn ◽  
Ole Olsen ◽  
Oliver Politz ◽  
Rainer Borriss ◽  
Udo Heinemann
Keyword(s):  
Crystal Structure ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Crystal Structure of African Swine Fever Virus pS273R Protease and Implications for Inhibitor Design

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Guobang Li ◽  
Xiaoxia Liu ◽  
Mengyuan Yang ◽  
Guangshun Zhang ◽  
Zhengyang Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Information ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Hydrolytic Activity ◽  
Viral Disease ◽  
Fever Virus ◽  
Dna Virus ◽  
Core Domain ◽  
The Core

ABSTRACT African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs that is responsible for serious economic and production losses. It is caused by the African swine fever virus (ASFV), a large and complex icosahedral DNA virus of the Asfarviridae family. Currently, there is no effective treatment or approved vaccine against the ASFV. pS273R, a specific SUMO-1 cysteine protease, catalyzes the maturation of the pp220 and pp62 polyprotein precursors into core-shell proteins. Here, we present the crystal structure of the ASFV pS273R protease at a resolution of 2.3 Å. The overall structure of the pS273R protease is represented by two domains named the “core domain” and the N-terminal “arm domain.” The “arm domain” contains the residues from M1 to N83, and the “core domain” contains the residues from N84 to A273. A structure analysis reveals that the “core domain” shares a high degree of structural similarity with chlamydial deubiquitinating enzyme, sentrin-specific protease, and adenovirus protease, while the “arm domain” is unique to ASFV. Further, experiments indicated that the “arm domain” plays an important role in maintaining the enzyme activity of ASFV pS273R. Moreover, based on the structural information of pS273R, we designed and synthesized several peptidomimetic aldehyde compounds at a submolar 50% inhibitory concentration, which paves the way for the design of inhibitors to target this severe pathogen. IMPORTANCE African swine fever virus, a large and complex icosahedral DNA virus, causes a deadly infection in domestic pigs. In addition to Africa and Europe, countries in Asia, including China, Vietnam, and Mongolia, were negatively affected by the hazards posed by ASFV outbreaks in 2018 and 2019, at which time more than 30 million pigs were culled. Until now, there has been no vaccine for protection against ASFV infection or effective treatments to cure ASF. Here, we solved the high-resolution crystal structure of the ASFV pS273R protease. The pS273R protease has a two-domain structure that distinguishes it from other members of the SUMO protease family, while the unique “arm domain” has been proven to be essential for its hydrolytic activity. Moreover, the peptidomimetic aldehyde compounds designed to target the substrate binding pocket exert prominent inhibitory effects and can thus be used in a potential lead for anti-ASFV drug development.

Site-directed mutagenesis and homology modeling indicate an important role of cysteine 439 in the stability of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

Biochimie ◽  
2005 ◽  
Vol 87 (12) ◽  
pp. 1056-1064 ◽  
Author(s):  
Lilian González-Segura ◽  
Roberto Velasco-García ◽  
Enrique Rudiño-Piñera ◽  
Carlos Mújica-Jiménez ◽  
Rosario A. Muñoz-Clares
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Homology Modeling ◽  
Aldehyde Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
The Stability
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