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

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.

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Enzymic synthesis of N-acetyl-l-phenylalanine in Escherichia coli K12

Biochemical Journal ◽

10.1042/bj1240905 ◽

1971 ◽

Vol 124 (5) ◽

pp. 905-913 ◽

Cited By ~ 12

Author(s):

R. V. Krishna ◽

P. R. Krishnaswamy ◽

D. Rajagopal Rao

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Substrate Specificity ◽

Acetyl Coa ◽

Ph Optimum ◽

E Coli ◽

Enzymic Synthesis ◽

Escherichia Coli K12

1. Cell-free extracts of Escherichia coli K12 catalyse the synthesis of N-acetyl-l-phenylalanine from acetyl-CoA and l-phenylalanine. 2. The acetyl-CoA–l-phenylalanine α-N-acetyltransferase was purified 160-fold from cell-free extracts. 3. The enzyme has a pH optimum of 8 and catalyses the acetylation of l-phenylalanine. Other l-amino acids such as histidine and alanine are acetylated at slower rates. 4. A transacylase was also purified from E. coli extracts and its substrate specificity studied. 5. The properties of both these enzymes were compared with those of other known amino acid acetyltransferases and transacylases.

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

10.26434/chemrxiv.6267383 ◽

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 Pseudomonas moravenis stanleyae through the enzymatic generation of Se(0) nanoparticles. This enzyme has an unprecedented substrate specificity for selenodiglutathione (Km= 336 μM) over oxidized glutathione (Km=8.22 mM). This enzyme was able to induce selenite tolerance in foreign bacterial cell lines by increasing the IC90 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.

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

Biochemical Journal ◽

10.1042/bcj20190625 ◽

2019 ◽

Vol 476 (22) ◽

pp. 3475-3492 ◽

Cited By ~ 6

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.

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

Antibiotics ◽

10.3390/antibiotics10121494 ◽

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.

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Kinetic, Structural, and Mutational Analysis of Acyl-CoA Carboxylase From Thermobifida fusca YX

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.615614 ◽

2021 ◽

Vol 7 ◽

Author(s):

Kiran-Kumar Shivaiah ◽

Bryon Upton ◽

Basil J. Nikolau

Keyword(s):

Substrate Specificity ◽

Quaternary Structure ◽

Mutational Analysis ◽

Streptomyces Coelicolor ◽

Thermobifida Fusca ◽

Substrate Selectivity ◽

Acetyl Coa ◽

Catalytic Subunits ◽

Chain Acyl

Acyl-CoA carboxylases (AcCCase) are biotin-dependent enzymes that are capable of carboxylating more than one short chain acyl-CoA substrate. We have conducted structural and kinetic analyses of such an AcCCase from Thermobifida fusca YX, which exhibits promiscuity in carboxylating acetyl-CoA, propionyl-CoA, and butyryl-CoA. The enzyme consists of two catalytic subunits (TfAcCCA and TfAcCCB) and a non-catalytic subunit, TfAcCCE, and is organized in quaternary structure with a A6B6E6 stoichiometry. Moreover, this holoenzyme structure appears to be primarily assembled from two A3 and a B6E6 subcomplexes. The role of the TfAcCCE subunit is to facilitate the assembly of the holoenzyme complex, and thereby activate catalysis. Based on prior studies of an AcCCase from Streptomyces coelicolor, we explored whether a conserved Asp residue in the TfAcCCB subunit may have a role in determining the substrate selectivity of these types of enzymes. Mutating this D427 residue resulted in alterations in the substrate specificity of the TfAcCCase, increasing proficiency for carboxylating acetyl-CoA, while decreasing carboxylation proficiency with propionyl-CoA and butyryl-CoA. Collectively these results suggest that residue D427 of AcCCB subunits is an important, but not sole determinant of the substrate specificity of AcCCase enzymes.

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Reprogramming protein kinase substrate specificity through synthetic mutations

10.1101/091892 ◽

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.

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

Advanced Synthesis & Catalysis ◽

10.1002/adsc.201500239 ◽

2015 ◽

Vol 357 (12) ◽

pp. 2712-2720 ◽

Cited By ~ 20

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

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Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP

Biochemical Journal ◽

10.1042/bj20050263 ◽

2005 ◽

Vol 389 (1) ◽

pp. 173-180 ◽

Cited By ~ 19

Author(s):

Melinda DEMENDI ◽

Noboru ISHIYAMA ◽

Joseph S. LAM ◽

Albert M. BERGHUIS ◽

Carole CREUZENET

Keyword(s):

Enzyme Activity ◽

Substrate Specificity ◽

Molecular Basis ◽

Substrate Binding ◽

Structural Data ◽

Binding Pocket ◽

Substrate Binding Pocket ◽

Expected Effect ◽

Biochemical Assays ◽

Golden Opportunity

WbpP is the only genuine UDP-GlcNAc (UDP-N-acetylglucosamine) C4 epimerase for which both biochemical and structural data are available. This represents a golden opportunity to elucidate the molecular basis for its specificity for N-acetylated substrates. Based on the comparison of the substrate binding site of WbpP with that of other C4 epimerases that convert preferentially non-acetylated substrates, or that are able to convert both acetylated and non-acetylated substrates equally well, specific residues of WbpP were mutated, and the substrate specificity of the mutants was determined by direct biochemical assays and kinetic analyses. Most of the mutations tested were anticipated to trigger a significant switch in substrate specificity, mostly towards a preference for non-acetylated substrates. However, only one of the mutations (A209H) had the expected effect, and most others resulted in enhanced specificity of WbpP for N-acetylated substrates (Q201E, G102K, Q201E/G102K, A209N and S143A). One mutation (S144K) totally abolished enzyme activity. These data indicate that, although all residues targeted in the present study turned out to be important for catalysis, determinants of substrate specificity are not confined to the substrate-binding pocket and that longer range interactions are essential in allowing proper positioning of various ligands in the binding pocket. Hence prediction or engineering of substrate specificity solely based on sequence analysis, or even on modelling of the binding pocket, might lead to incorrect functional assignments.

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