scholarly journals Evolution of tunnels in alpha/beta-hydrolase fold proteins - what can we learn from studying epoxide hydrolases?

2021 ◽  
Author(s):  
Maria Bzowka ◽  
Karolina Mitusinska ◽  
Agata Raczynska ◽  
Tomasz Skalski ◽  
Aleksandra Samol ◽  
...  
Keyword(s):  
Protein Function ◽  
Active Sites ◽  
Tunnel Lining ◽  
Fine Tuning ◽  
Evolutionary Analysis ◽  
Active Site Residues ◽  
Epoxide Hydrolases ◽  
Fold Family ◽  
Hydrolase Fold ◽  
Tunnel Inspection

The evolutionary variability of a protein's residues is highly dependent on protein region and protein function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues. Active site residues are considered to be conserved as they ensure an enzyme's activity and selectivity. The abovementioned rules apply also to α/β-hydrolase fold proteins - an example of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We hypothesised two scenarios: (1) tunnels are lined by mostly variable residues, allowing adaptation to the evolutionary pressures of a changeable environment; or (2) tunnels are lined by mostly conserved amino acids, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. We also wanted to check if evolutionary analysis can help distinguish functional and non-functional tunnels. Soluble epoxide hydrolases (sEHs) represent a good case study for the analysis of the evolution of tunnels in an α/β-hydrolase fold family due to their size and architecture. Here, we propose methods for the comparison of tunnels detected in both crystal structures and molecular dynamics simulations, as well as the assignment of tunnel functionality, and we identify critical steps for careful tunnel inspection. We also compare the entropy values of the tunnel-lining residues and system-specific compartments in seven selected sEHs from different clades. We present three different cases of entropy distribution among tunnel-lining residues. As a result, we propose a 'perforation' model for tunnel evolution via the merging of internal cavities or surface perforations. We also report an approach for the identification of highly variable tunnel-lining residues as potential targets to be used for the fine-tuning of selected enzymes.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

The prolyl aminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus belongs to the α/β hydrolase fold family

1999 ◽  
Vol 1429 (2) ◽  
pp. 501-505 ◽  
Author(s):  
Fabienne Morel ◽  
Christophe Gilbert ◽  
Christophe Geourjon ◽  
Jacques Frot-Coutaz ◽  
Raymond Portalier ◽  
...  
Keyword(s):  
Lactobacillus Delbrueckii ◽  
Prolyl Aminopeptidase ◽  
Fold Family ◽  
Hydrolase Fold ◽  
Lactobacillus Delbrueckii Subsp

Enhanced photocatalytic H2 evolution over micro-SiC by coupling with CdS under visible light irradiation

2014 ◽  
Vol 2 (18) ◽  
pp. 6296-6300 ◽  
Author(s):  
Yuan Peng ◽  
Zhongnan Guo ◽  
Jingjing Yang ◽  
Da Wang ◽  
Wenxia Yuan
Keyword(s):  
Visible Light ◽  
Visible Light Irradiation ◽  
Active Sites ◽  
Fine Tuning ◽  
Surface Activation ◽  
H2 Evolution ◽  
Effective Surface ◽  
Suitable Amount ◽  
Surface Active ◽  
Photocatalytic H2 Evolution

Fine-tuning surface activation of the micro-SiC catalyst via hybridizing a suitable amount of CdS to obtain effective surface active sites.

scholarly journals Gene Expression Profile of RNA N1-methyladenosine methyltransferases

2020 ◽  
Vol 218 ◽  
pp. 03052
Author(s):  
Mingzhu Rao
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Active Sites ◽  
Evolutionary Analysis ◽  
Base Modification ◽  
Binding Domains ◽  
Non Coding Rna ◽  
Beta Sheet Structure ◽  
The Family

N1-methyladenosine (m1A) is a kind of common and abundant methylation modification in eukaryotic mRNA and long-chain non-coding RNA. Nucleoside methyltransferase (MTase) of m1A is a diverse protein family, which is characterized by the presence of methyltransferases like domains and conserved S-adenosylmethionine (SAM) binding domains formed by the central sevenstranded beta-sheet structure. However, comprehensive analysis of the gene expression profile of such enzymes has not been performed to classify them according to evolutionary criteria and to guide the functional prediction. Here, we conducted extensive searches of databases to collect all members of previously identified m1A RNA methyltransferases. And we report bioinformatics studies on gene expression profile based on evolutionary analysis, sequence alignment, expression in tissues and cells within the family of RNA methyltransferases. Our analysis showed that the base modification behavior mediated by m1A RNA methyltransferases evolved from invertebrate, and the active sites of m1A RNA methyltransferases were highly conserved during the evolution from invertebrates to human. And m1A RNA methyltransferases have low tissue and cell specificity.

Methylation of the nuclear poly(A)-binding protein by type I protein arginine methyltransferases – how and why

2013 ◽  
Vol 394 (8) ◽  
pp. 1029-1043 ◽  
Author(s):  
Elmar Wahle ◽  
Bodo Moritz
Keyword(s):  
Active Sites ◽  
Binding Protein ◽  
Arginine Methylation ◽  
Fine Tuning ◽  
General Mechanism ◽  
Type I ◽  
Protein Arginine Methyltransferases ◽  
Low Protein ◽  
Backbone Conformation ◽  
Arginine Residues

Abstract Asymmetric dimethylation of arginine side chains in proteins is a frequent posttranslational modification, catalyzed by type I protein arginine methyltransferases (PRMTs). This article summarizes what is known about this modification in the nuclear poly(A)-binding protein (PABPN1). PABPN1 contains 13 dimethylated arginine residues in its C-terminal domain. Three enzymes, PRMT1, 3, and 6, can methylate PABPN1. Although 26 methyl groups are transferred to one PABPN1 molecule, the PRMTs do so in a distributive reaction, i.e., only a single methyl group is transferred per binding event. As PRMTs form dimers, with the active sites accessible from a small central cavity, backbone conformation around the methyl-accepting arginine is an important determinant of substrate specificity. Neither the association of PABPN1 with poly(A) nor its role in poly(A) tail synthesis is affected by arginine methylation. At least at low protein concentration, methylation does not affect the protein’s tendency to oligomerize. The dimethylarginine residues of PABPN1 are located in the binding site for its nuclear import receptor, transportin. Arginine methylation weakens this interaction about 10-fold. Very recent evidence suggests that arginine methylation as a way of fine-tuning the interactions between transportin and its cargo may be a general mechanism.

scholarly journals AidH, an Alpha/Beta-Hydrolase Fold Family Member from an Ochrobactrum sp. Strain, Is a Novel N-Acylhomoserine Lactonase

2010 ◽  
Vol 76 (15) ◽  
pp. 4933-4942 ◽  
Author(s):  
Gui-Ying Mei ◽  
Xiao-Xue Yan ◽  
Ali Turak ◽  
Zhao-Qing Luo ◽  
Li-Qun Zhang
Keyword(s):  
Pathogenic Bacteria ◽  
Divalent Cations ◽  
Hydrolytic Enzyme ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Great Promise ◽  
Fold Family ◽  
Hydrolase Fold ◽  
Enzymatic Inactivation ◽  
Alpha Beta

ABSTRACT N-Acylhomoserine lactones (AHLs) are signaling molecules in many quorum-sensing (QS) systems that regulate interactions between various pathogenic bacteria and their hosts. Quorum quenching by the enzymatic inactivation of AHLs holds great promise in preventing and treating infections, and several such enzymes have been reported. In this study, we report the characterization of a novel AHL-degrading protein from the soil bacterium Ochrobactrum sp. strain T63. This protein, termed AidH, shares no similarity with any of the known AHL degradases but is highly homologous with a hydrolytic enzyme from Ochrobactrum anthropi ATCC 49188 that contains the alpha/beta-hydrolase fold. By liquid chromatography-mass spectrometry (MS) analysis, we demonstrate that AidH functions as an AHL-lactonase that hydrolyzes the ester bond of the homoserine lactone ring of AHLs. Mutational analyses indicate that the G-X-Nuc-X-G motif or the histidine residue conserved among alpha/beta-hydrolases is critical for the activity of AidH. Furthermore, the AHL-inactivating activity of AidH requires Mn2+ but not several other tested divalent cations. We also showed that AidH significantly reduces biofilm formation by Pseudomonas fluorescens 2P24 and the pathogenicity of Pectobacterium carotovorum, indicating that this enzyme is able to effectively quench QS-dependent functions in these bacteria by degrading AHLs.

scholarly journals The Convergence of the Hedgehog/Intein Fold in Different Protein Splicing Mechanisms

2020 ◽  
Vol 21 (21) ◽  
pp. 8367
Author(s):  
Hannes M. Beyer ◽  
Salla I. Virtanen ◽  
A. Sesilja Aranko ◽  
Kornelia M. Mikula ◽  
George T. Lountos ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Sites ◽  
Bond Cleavage ◽  
Structural Basis ◽  
Protein Splicing ◽  
Active Site Residues ◽  
Class 1 ◽  
Characteristic Sequences ◽  
Dynamics Simulations ◽  
Esterification Reactions

Protein splicing catalyzed by inteins utilizes many different combinations of amino-acid types at active sites. Inteins have been classified into three classes based on their characteristic sequences. We investigated the structural basis of the protein splicing mechanism of class 3 inteins by determining crystal structures of variants of a class 3 intein from Mycobacterium chimaera and molecular dynamics simulations, which suggested that the class 3 intein utilizes a different splicing mechanism from that of class 1 and 2 inteins. The class 3 intein uses a bond cleavage strategy reminiscent of proteases but share the same Hedgehog/INTein (HINT) fold of other intein classes. Engineering of class 3 inteins from a class 1 intein indicated that a class 3 intein would unlikely evolve directly from a class 1 or 2 intein. The HINT fold appears as structural and functional solution for trans-peptidyl and trans-esterification reactions commonly exploited by diverse mechanisms using different combinations of amino-acid types for the active-site residues.

scholarly journals Roles of active-site residues in catalysis, substrate binding, cooperativity, and the reaction mechanism of the quinoprotein glycine oxidase

2020 ◽  
Vol 295 (19) ◽  
pp. 6472-6481
Author(s):  
Kyle J. Mamounis ◽  
Erik T. Yukl ◽  
Victor L. Davidson
Keyword(s):  
Active Site ◽  
Protein Function ◽  
Marine Bacterium ◽  
Substrate Binding ◽  
Active Site Residues ◽  
X Ray Crystallography ◽  
Steady State Kinetics ◽  
Hill Coefficients ◽  
The Hill ◽  
Glycine Oxidase

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

scholarly journals Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with anIn VivoCompetition Assay

2015 ◽  
Vol 82 (4) ◽  
pp. 1050-1059 ◽  
Author(s):  
Zhao Jin ◽  
Sara C. Di Rienzi ◽  
Anders Janzon ◽  
Jeff J. Werner ◽  
Largus T. Angenent ◽  
...  
Keyword(s):  
Protein Function ◽  
Gene Diversity ◽  
Functional Characterization ◽  
Acc Deaminase ◽  
Active Site Residues ◽  
Protein Coding ◽  
Content Type ◽  
Soil Microbial ◽  
Starting Point

ABSTRACTMetagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen toEscherichia coliin vitro. The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate.

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