Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin

Biochemistry ◽  
1987 ◽  
Vol 26 (9) ◽  
pp. 2616-2623 ◽  
Author(s):  
Laszlo Graf ◽  
Charles S. Craik ◽  
Andras Patthy ◽  
Steven Roczniak ◽  
Robert J. Fletterick ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Aspartic Acid ◽  
Binding Pocket

scholarly journals Altering the Substrate Specificity of Acetyl-CoA Synthetase by Rational Mutagenesis of the Carboxylate Binding Pocket

2019 ◽  
Vol 8 (6) ◽  
pp. 1325-1336 ◽  
Author(s):  
Naazneen Sofeo ◽  
Jason H. Hart ◽  
Brandon Butler ◽  
David J. Oliver ◽  
Marna D. Yandeau-Nelson ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Binding Pocket ◽  
Acetyl Coa ◽  
Rational Mutagenesis

scholarly journals Main Structural Targets for Engineering Lipase Substrate Specificity

Catalysts ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 747
Author(s):  
Samah Hashim Albayati ◽  
Malihe Masomian ◽  
Siti Nor Hasmah Ishak ◽  
Mohd Shukuri bin Mohamad Ali ◽  
Adam Leow Thean ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Experimental Studies ◽  
Random Mutagenesis ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Process Conditions

Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipases.

scholarly journals CFTR: the nucleotide binding folds regulate the accessibility and stability of the activated state.

1996 ◽  
Vol 107 (1) ◽  
pp. 103-119 ◽  
Author(s):  
D J Wilkinson ◽  
M K Mansoura ◽  
P Y Watson ◽  
L S Smit ◽  
F S Collins ◽  
...  
Keyword(s):  
Aspartic Acid ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Active State ◽  
Slow Phase ◽  
Atp Binding ◽  
Wild Type ◽  
Rate Analysis ◽  
Activated State

The functional roles of the two nucleotide binding folds, NBF1 and NBF2, in the activation of the cystic fibrosis transmembrane conductance regulator (CFTR) were investigated by measuring the rates of activation and deactivation of CFTR Cl- conductance in Xenopus oocytes. Activation of wild-type CFTR in response to application of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was described by a single exponential. Deactivation after washout of the cocktail consisted of two phases: an initial slow phase, described by a latency, and an exponential decline. Rate analysis of CFTR variants bearing analogous mutations in NBF1 and NBF2 permitted us to characterize amino acid substitutions according to their effects on the accessibility and stability of the active state. Access to the active state was very sensitive to substitutions for the invariant glycine (G551) in NBF1, where mutations to alanine (A), serine (S), or aspartic acid (D) reduced the apparent on rate by more than tenfold. The analogous substitutions in NBF2 (G1349) also reduced the on rate, by twofold to 10-fold, but substantially destabilized the active state as well, as judged by increased deactivation rates. In the putative ATP-binding pocket of either NBF, substitution of alanine, glutamine (Q), or arginine (R) for the invariant lysine (K464 or K1250) reduced the on rate similarly, by two- to fourfold. In contrast, these analogous substitutions produced opposite effects on the deactivation rate. NBF1 mutations destabilized the active state, whereas the analogous substitutions in NBF2 stabilized the active state such that activation was prolonged compared with that seen with wild-type CFTR. Substitution of asparagine (N) for a highly conserved aspartic acid (D572) in the ATP-binding pocket of NBF1 dramatically slowed the on rate and destabilized the active state. In contrast, the analogous substitution in NBF2 (D1370N) did not appreciably affect the on rate and markedly stabilized the active state. These results are consistent with a hypothesis for CFTR activation that invokes the binding and hydrolysis of ATP at NBF1 as a crucial step in activation, while at NBF2, ATP binding enhances access to the active state, but the rate of ATP hydrolysis controls the duration of the active state. The relatively slow time courses for activation and deactivation suggest that slow processes modulate ATP-dependent gating.

scholarly journals The Metalloid Reductase of Pseudomonas Moravenis Stanleyae Conveys Nanoparticle Mediated Metalloid Tolerance

2018 ◽  
Author(s):  
Richard Nemeth ◽  
Mackenzie Neubert ◽  
Thomas Ni ◽  
Christopher J. Ackerson
Keyword(s):  
Structural Analysis ◽  
Substrate Specificity ◽  
Glutathione Reductase ◽  
Cell Lines ◽  
Bacterial Cell ◽  
Binding Pocket ◽  
Oxidized Glutathione ◽  
Se Nanoparticles ◽  
In Cells

In the present work we have identified a glutathione reductase like metalloid reductase (GRLMR) responsible for mediating selenite tolerance in <i>Pseudomonas moravenis</i> stanleyae through the enzymatic generation of Se(0) nanoparticles. This enzyme has an unprecedented substrate specificity for selenodiglutathione (K<sub>m</sub>= 336 μM) over oxidized glutathione (K<sub>m</sub>=8.22 mM). This enzyme was able to induce selenite tolerance in foreign bacterial cell lines by increasing the IC<sub>90</sub> for selenite from 1.9 mM in cell lacking the GRLMR gene to 21.3 mM for cells containing the GRLMR gene. It was later confirmed by STEM and EDS that Se nanoparticles were absent in control cells and present in cells expressing GRLMR. Structural analysis suggests the lack of a sulfur residue in the substrate/product binding pocket may be responsible for this unique substrate specificity.

scholarly journals Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

2019 ◽  
Vol 476 (22) ◽  
pp. 3475-3492 ◽  
Author(s):  
Robert D. Grinshpon ◽  
Suman Shrestha ◽  
James Titus-McQuillan ◽  
Paul T. Hamilton ◽  
Paul D. Swartz ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Binding Pocket ◽  
Gene Duplications ◽  
Substrate Selection ◽  
Charged Amino Acids ◽  
Effector Caspase ◽  
Effector Caspases ◽  
Hydrophobic Binding ◽  
Caspase 6 ◽  
Aspartate Residue

Apoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases prefer an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared with caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates a preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspase-hemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

scholarly journals Characterization and Molecular Determinants for β-Lactam Specificity of the Multidrug Efflux Pump AcrD from Salmonella typhimurium

Antibiotics ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1494
Author(s):  
Jenifer Cuesta Bernal ◽  
Jasmin El-Delik ◽  
Stephan Göttig ◽  
Klaas M. Pos
Keyword(s):  
Substrate Specificity ◽  
Salmonella Typhimurium ◽  
Efflux Pump ◽  
Binding Pocket ◽  
Salt Resistance ◽  
Drug Binding ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Molecular Determinants ◽  
Groove Region

Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.

scholarly journals Reprogramming protein kinase substrate specificity through synthetic mutations

2016 ◽  
Author(s):  
Joshua M. Lubner ◽  
George M. Church ◽  
Michael F. Chou ◽  
Daniel Schwartz
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Structure Function ◽  
Binding Pocket ◽  
Missense Mutations ◽  
New Paradigm ◽  
Substrate Binding Pocket ◽  
Kinase Substrate ◽  
Substrate Preferences ◽  
Kinase Specificity

Protein kinase specificity is largely imparted through substrate binding pocket motifs. Missense mutations in these regions are frequently associated with human disease, and in some cases can alter substrate specificity. However, current efforts at decoding the influence of mutations on substrate specificity have been focused on disease-associated mutations. Here, we adapted the Proteomic Peptide Library (ProPeL) approach for determining kinase specificity to the task of exploring structure-function relationships in kinase specificity by interrogating the effects of synthetic mutation. We established a specificity model for the wild-type DYRK1A kinase with unprecedented resolution. Using existing crystallographic and sequence homology data, we rationally designed mutations that precisely reprogrammed the DYRK1A kinase at the P+1 position to mimic the substrate preferences of a related kinase, CK II. This study illustrates a new synthetic biological approach to reprogram kinase specificity by design, and a powerful new paradigm to investigate structure-function relationships underpinning kinase substrate specificity.

Expanding Substrate Specificity of ω-Transaminase by Rational Remodeling of a Large Substrate-Binding Pocket

2015 ◽  
Vol 357 (12) ◽  
pp. 2712-2720 ◽  
Author(s):  
Sang-Woo Han ◽  
Eul-Soo Park ◽  
Joo-Young Dong ◽  
Jong-Shik Shin
Keyword(s):  
Substrate Specificity ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket

Aspartic Acid 405 Contributes to the Substrate Specificity of Aminopeptidase B

Biochemistry ◽  
2006 ◽  
Vol 45 (38) ◽  
pp. 11425-11431 ◽  
Author(s):  
Kayoko M. Fukasawa ◽  
Junzo Hirose ◽  
Toshiyuki Hata ◽  
Yukio Ono
Keyword(s):  
Substrate Specificity ◽  
Aspartic Acid
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