scholarly journals On the possible origin of protein homochirality, structure, and biochemical function

2019 ◽  
Vol 116 (52) ◽  
pp. 26571-26579 ◽  
Author(s):  
Jeffrey Skolnick ◽  
Hongyi Zhou ◽  
Mu Gao
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Ribosomal Proteins ◽  
Active Sites ◽  
Cell Formation ◽  
Binding Pocket ◽  
Nucleotide Biosynthesis ◽  
Native Proteins ◽  
Structural And Functional Properties ◽  
Biochemical Function

Living systems have chiral molecules, e.g., native proteins that almost entirely contain L-amino acids. How protein homochirality emerged from a background of equal numbers of L and D amino acids is among many questions about life’s origin. The origin of homochirality and its implications are explored in computer simulations examining the stability and structural and functional properties of an artificial library of compact proteins containing 1:1 (termed demi-chiral), 3:1, and 1:3 ratios of D:L and purely L or D amino acids generated without functional selection. Demi-chiral proteins have shorter secondary structures and fewer internal hydrogen bonds and are less stable than homochiral proteins. Selection for hydrogen bonding yields a preponderance of L or D amino acids. Demi-chiral proteins have native global folds, including similarity to early ribosomal proteins, similar small molecule ligand binding pocket geometries, and many constellations of L-chiral amino acids with a 1.0-Å RMSD to native enzyme active sites. For a representative subset containing 550 active site geometries matching 457 (2) 4-digit (3-digit) enzyme classification (E.C.) numbers, native active site amino acids were generated at random for 472 of 550 cases. This increases to 548 of 550 cases when similar residues are allowed. The most frequently generated sequences correspond to ancient enzymatic functions, e.g., glycolysis, replication, and nucleotide biosynthesis. Surprisingly, even without selection, demi-chiral proteins possess the requisite marginal biochemical function and structure of modern proteins, but were thermodynamically less stable. If demi-chiral proteins were present, they could engage in early metabolism, which created the feedback loop for transcription and cell formation.

scholarly journals Crystal structures of the editing domain of Escherichia coli leucyl-tRNA synthetase and its complexes with Met and Ile reveal a lock-and-key mechanism for amino acid discrimination

2006 ◽  
Vol 394 (2) ◽  
pp. 399-407 ◽  
Author(s):  
Yunqing Liu ◽  
Jing Liao ◽  
Bin Zhu ◽  
En-Duo Wang ◽  
Jianping Ding
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Crystal Structures ◽  
Active Site ◽  
Binding Pocket ◽  
Trna Synthetase ◽  
Vital Role ◽  
Common Mechanism ◽  
Trna Synthetases ◽  
Editing Domain

aaRSs (aminoacyl-tRNA synthetases) are responsible for the covalent linking of amino acids to their cognate tRNAs via the aminoacylation reaction and play a vital role in maintaining the fidelity of protein synthesis. LeuRS (leucyl-tRNA synthetase) can link not only the cognate leucine but also the nearly cognate residues Ile and Met to tRNALeu. The editing domain of LeuRS deacylates the mischarged Ile–tRNALeu and Met–tRNALeu. We report here the crystal structures of ecLeuRS-ED (the editing domain of Escherichia coli LeuRS) in both the apo form and in complexes with Met and Ile at 2.0 Å, 2.4 Å, and 3.2 Å resolution respectively. The editing active site consists of a number of conserved amino acids, which are involved in the precise recognition and binding of the noncognate amino acids. The substrate-binding pocket has a rigid structure which has an optimal stereochemical fit for Ile and Met, but has steric hindrance for leucine. Based on our structural results and previously available biochemical data, we propose that ecLeuRS-ED uses a lock-and-key mechanism to recognize and discriminate between the amino acids. Structural comparison also reveals that all subclass Ia aaRSs share a conserved structure core consisting of the editing domain and conserved residues at the editing active site, suggesting that these enzymes may use a common mechanism for the editing function.

scholarly journals An Overview of the Proofreading Functions in Bacteria and in Severe Acute Respiratory Syndrome-Coronaviruses

2021 ◽  
pp. 33-62
Author(s):  
Peramachi Palanivelu
Keyword(s):  
Amino Acids ◽  
Severe Acute Respiratory Syndrome ◽  
Structure Function ◽  
Active Site ◽  
Active Sites ◽  
Dna Polymerases ◽  
Proton Acceptor ◽  
Conserved Motifs ◽  
Bacterial Dna ◽  
X Ray

Aim: To understand the structure-function relationship of the proofreading (PR) functions in eubacteria and viruses with special reference to Severe Acute Respiratory Syndrome-Coronaviruses (SARS-CoVs) and propose a plausible mechanism of action for PR exonucleases of SARS-CoVs. Study Design: Bioinformatics, biochemical, site-directed mutagenesis (SDM), X-ray crystallographic data were used to study the structure-function relationships of the PR exonucleases from bacteria and CoVs. Methodology: The protein sequences of the PR exonucleases of various DNA polymerases, and RNA polymerases of SARS, SARS-related and human CoVs (HCoVs) were obtained from PUBMED and SWISS-PROT databases. The advanced version of Clustal Omega was used for protein sequence analysis. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical, SDM experiments and X-ray crystallographic analysis on these enzymes were used to arrive at the possible active amino acids in the PR exonucleases of these crucial enzymes. Results:  A complete analysis of the active sites of the PR exonucleases from various bacteria and CoVs were done. The multiple sequence alignment (MSA) analysis showed many conserved amino acids, small and large peptide regions among them. Based on the conserved motifs, the PR exonucleases are found to fit broadly into two superfamilies, viz. DEDD and polymerase-histidinol phosphatase (PHP) superfamilies. The bacterial DNA polymerases I and II, RNase D, RNase T and ε-subunit of DNA polymerases III belong to the DEDD superfamily. The PR enzymes from SARS, SARS-related CoVs and other HCoVs also essentially belong to the DEDD superfamily. The DEDD superfamily either uses an invariant Tyr or a His as proton acceptor during catalysis. Depending on the proton acceptor, they are further classified into DEDHD and DEDYD subfamilies. RNase T, ε-subunit of DNA polymerases III and the SARS, SARS-related CoVs and other HCoVs belong to DEDHD subfamily.  However, the SARS, SARS-related CoVs and other HCoVs showed additional zinc finger motifs (ZFMs) in their active sites. DNA polymerases I, II and RNase D belong to DEDYD subfamily. The bacterial DNA polymerases X, YcdX phosphoesterases and the co-editing exonuclease of DNA polymerases III belong to the PHP superfamily. Based on the MSA, X-ray crystallographic analyses and SDM experiments, the proposed active-site proton acceptor is Tyr/His in DEDDY/H subfamilies and His in PHP superfamily of PR exonucleases.  Conclusions:   Based on the similarities of active site amino acids/motifs, it may be concluded that the DEDD and PHP superfamilies of PR exonucleases should have evolved from a common ancestor but diverged very long ago. The biochemical properties of these enzymes, including the four conserved acidic amino acid residues in the catalytic core, suggest that the CoVs might have acquired the exonuclease function, possibly from a prokaryote. However, the presence of two zinc fingers in the PR active site of the SARS, SARS-related CoVs and other HCoVs sets their PR exonucleases apart from other homologues.

scholarly journals The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 5053
Author(s):  
Alina K. Bakunova ◽  
Alena Yu. Nikolaeva ◽  
Tatiana V. Rakitina ◽  
Tatiana Y. Isaikina ◽  
Maria G. Khrenova ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Structural Analysis ◽  
Active Site ◽  
Active Sites ◽  
Structural Basis ◽  
Active Site Residues ◽  
Type Iv ◽  
Fold Type ◽  
Arginine Residues

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.

scholarly journals High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides

2010 ◽  
Vol 54 (10) ◽  
pp. 4343-4351 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Manuela Benvenuti ◽  
Vito Calderone ◽  
Magdalena Stoczko ◽  
Nicola Menciassi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Bradyrhizobium Japonicum ◽  
Active Sites ◽  
Structural Diversity ◽  
Binding Pocket ◽  
Structural Basis ◽  
Side Chain ◽  
Functional Heterogeneity ◽  
Hydrophobic Binding

ABSTRACT Metallo-β-lactamases (MBLs) are important enzymatic factors in resistance to β-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes β-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for β-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.

scholarly journals Substituent Effects on Carbon Acidity in Aqueous Solution and at Enzyme Active Sites

Synlett ◽  
2017 ◽  
Vol 28 (12) ◽  
pp. 1407-1421 ◽  
Author(s):  
John Richard ◽  
Tina Amyes
Keyword(s):  
Aqueous Solution ◽  
Amino Acids ◽  
Active Site ◽  
Active Sites ◽  
Substituent Effects ◽  
Imidazolium Cations ◽  
Enzyme Active Sites ◽  
Iminium Cations ◽  
Carbon Acids

Methods are described for the determination of pK as for weak carbon acids in water. The application of these methods to the determination of the pK as for a variety of carbon acids including nitriles, imidazolium cations, amino acids, peptides and their derivatives and, α-iminium cations is presented. The substituent effects on the acidity of these different classes of carbon acids are discussed, and the relevance of these results to catalysis of the deprotonation of amino acids by enzymes and by pyridoxal 5′-phosphate is reviewed. The procedure for estimating the pK a of uridine 5′-phosphate for C-6 deprotonation at the active site of orotidine 5′-phosphate decarboxylase is described, and the effect of a 5-F substituent on carbon acidity of the enzyme-bound substrate is discussed.1 Introduction2 The Carbon Acidity of Ethyl Thioacetate3 The Carbon Acidity of Carboxylic Acid Derivatives4 The Carbon Acidity of Imidazolium Cations5 The α-Carbon Acidity of Amino Acids, Peptides and Their Derivatives6 Electrophilic Catalysis of Deprotonation of Amino Acids: The α-Carbon Acidity of Iminium Cations7 pK as for Carbon Acids at Enzyme Active Sites8 Concluding Remarks

scholarly journals A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase

2012 ◽  
Vol 448 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Hugh P. Morgan ◽  
Martin J. Walsh ◽  
Elizabeth A. Blackburn ◽  
Martin A. Wear ◽  
Matthew B. Boxer ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Active Site ◽  
High Throughput Screening ◽  
Active Sites ◽  
Covalent Bond ◽  
Cell Types ◽  
Binding Pocket ◽  
Lysine Residue ◽  
Dependent Manner ◽  
Inhibitor Molecule

PYK (pyruvate kinase) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small-molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit LmPYK (Leishmania mexicana PYK) activity in a time- (and dose-) dependent manner, a characteristic of irreversible inhibition. The crystal structure of DBS {4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid} complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS-binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.

scholarly journals Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis

2013 ◽  
Vol 69 (10) ◽  
pp. 1876-1888 ◽  
Author(s):  
Fabienne Furt ◽  
William J. Allen ◽  
Joshua R. Widhalm ◽  
Peter Madzelan ◽  
Robert C. Rizzo ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Active Site ◽  
Active Sites ◽  
Binding Pocket ◽  
Flowering Plant ◽  
Structural Basis ◽  
Photosynthetic Organisms ◽  
Functional Convergence ◽  
Hotdog Fold ◽  
Hydrolysis Of

The synthesis of phylloquinone (vitamin K1) in photosynthetic organisms requires a thioesterase that hydrolyzes 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA) to release 1,4-dihydroxy-2-naphthoate (DHNA). Cyanobacteria and plants contain distantly related hotdog-fold thioesterases that catalyze this reaction, although the structural basis of these convergent enzymatic activities is unknown. To investigate this, the crystal structures of hotdog-fold DHNA-CoA thioesterases from the cyanobacteriumSynechocystis(Slr0204) and the flowering plantArabidopsis thaliana(AtDHNAT1) were determined. These enzymes form distinct homotetramers and use different active sites to catalyze hydrolysis of DHNA-CoA, similar to the 4-hydroxybenzoyl-CoA (4-HBA-CoA) thioesterases fromPseudomonasandArthrobacter. Like the 4-HBA-CoA thioesterases, the DHNA-CoA thioesterases contain either an active-site aspartate (Slr0204) or glutamate (AtDHNAT1) that are predicted to be catalytically important. Computational modeling of the substrate-bound forms of both enzymes indicates the residues that are likely to be involved in substrate binding and catalysis. Both enzymes are selective for DHNA-CoA as a substrate, but this selectivity is achieved using divergent predicted binding strategies. The Slr0204 binding pocket is predominantly hydrophobic and closely conforms to DHNA, while that of AtDHNAT1 is more polar and solvent-exposed. Considered in light of the related 4-HBA-CoA thioesterases, these structures indicate that hotdog-fold thioesterases using either an active-site aspartate or glutamate diverged into distinct clades prior to the evolution of strong substrate specificity in these enzymes.

scholarly journals Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0255098
Author(s):  
Ekaterina Yu. Bezsudnova ◽  
Alena Yu. Nikolaeva ◽  
Alina K. Bakunova ◽  
Tatiana V. Rakitina ◽  
Dmitry A. Suplatov ◽  
...  
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Active Site ◽  
Active Sites ◽  
Site Directed Mutagenesis ◽  
Primary Amines ◽  
Active Site Residues ◽  
Type Iv ◽  
Fold Type

Creating biocatalysts for (R)-selective amination effectively is highly desirable in organic synthesis. Despite noticeable progress in the engineering of (R)-amine activity in pyridoxal-5’-phosphate-dependent transaminases of fold type IV, the specialization of the activity is still an intuitive task, as there is poor understanding of sequence-structure-function relationships. In this study, we analyzed this relationship in transaminase from Thermobaculum terrenum, distinguished by expanded substrate specificity and activity in reactions with L-amino acids and (R)-(+)-1-phenylethylamine using α-ketoglutarate and pyruvate as amino acceptors. We performed site-directed mutagenesis to create a panel of the enzyme variants, which differ in the active site residues from the parent enzyme to a putative transaminase specific to (R)-primary amines. The variants were examined in the overall transamination reactions and half-reaction with (R)-(+)-1-phenylethylamine. A structural analysis of the most prominent variants revealed a spatial reorganization in the active sites, which caused changes in activity. Although the specialization to (R)-amine transaminase was not implemented, we succeeded in understanding the role of the particular active site residues in expanding substrate specificity of the enzyme. We showed that the specificity for (R)-(+)-1-phenylethylamine in transaminase from T. terrenum arises without sacrificing the specificity for L-amino acids and α-ketoglutarate and in consensus with it.

scholarly journals How special is the biochemical function of native proteins?

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 207 ◽  
Author(s):  
Jeffrey Skolnick ◽  
Mu Gao ◽  
Hongyi Zhou
Keyword(s):  
Active Sites ◽  
Enzymatic Catalysis ◽  
Protein Structures ◽  
Dna Interactions ◽  
Native Proteins ◽  
Protein Library ◽  
Protein Dna Interactions ◽  
Biochemical Function ◽  
And Control ◽  
Intrinsic Feature

Native proteins perform an amazing variety of biochemical functions, including enzymatic catalysis, and can engage in protein-protein and protein-DNA interactions that are essential for life. A key question is how special are these functional properties of proteins. Are they extremely rare, or are they an intrinsic feature? Comparison to the properties of compact conformations of artificially generated compact protein structures selected for thermodynamic stability but not any type of function, the artificial (ART) protein library, demonstrates that a remarkable number of the properties of native-like proteins are recapitulated. These include the complete set of small molecule ligand-binding pockets and most protein-protein interfaces. ART structures are predicted to be capable of weakly binding metabolites and cover a significant fraction of metabolic pathways, with the most enriched pathways including ancient ones such as glycolysis. Native-like active sites are also found in ART proteins. A small fraction of ART proteins are predicted to have strong protein-protein and protein-DNA interactions. Overall, it appears that biochemical function is an intrinsic feature of proteins which nature has significantly optimized during evolution. These studies raise questions as to the relative roles of specificity and promiscuity in the biochemical function and control of cells that need investigation.

Abstract 237: Structural Analysis of Ang Peptide Specificity in Binding to ACE, ACE2, Neprilysin, and PRCP

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Jeremy W Prokop ◽  
Fabiano C Araujo ◽  
Ingrid K Watanabie ◽  
Dulce E Caserini ◽  
Amy Milsted
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Active Sites ◽  
Aromatic Amino Acids ◽  
Structural Data ◽  
Angiotensin Peptides ◽  
C Terminus ◽  
N Terminus ◽  
Natural Variants ◽  
Multiple Species

A combination of known biochemistry and physiology data on angiotensin converting enzyme (ACE), ACE2, neprilysin and prolylcarboxypeptidase (PRCP) with structural biochemistry and bioinformatics reveals several key amino acids contributing to the orientation of Ang peptides in the active site of the various enzymes. A total of four structures are known for the ACE N-terminus, twelve for the ACE C-terminus, eleven for ACE2, seven for neprilysin, and one for PRCP. Of all the known structures, none include angiotensin peptides bound into an active site. Autodock experiments reveal possible binding conformations of Ang I, Ang II, Ang-(1-9), Ang-(1-7), or Ang III into the active site of each enzyme. These autodock experiments were validated using combinations of disease associated natural variants, known mutagenesis data, conservation of amino acids in multiple species and inhibitor based structural data, all revealing amino acids of high functionality that interact with Ang peptides. Aromatic amino acids (Phe, Tyr, and Trp) tend to dictate the cleavage site of the Ang peptides through interactions with amino acid 4 (Tyr), 6 (His) or 8 (Phe) of angiotensin. The ACE C-terminus and N-terminus have three conserved aromatic amino acids 10 Å or less from the catalytic Zn, ACE2 has two 11 Å or less from the catalytic Zn, neprilysin has a Phe 8.4Å from the catalytic Zn, and PRCP has five aromatic amino acids 12Å or less from the active site. A high density of serine and threonine amino acids is found in the active sites of some enzymes when compared with total serines and threonines (ACE N-term=39%, ACE C-term=46%, ACE2=32%, neprilysin=15%, PRCP=10%), suggesting a strong likelihood of a hydrogen bonding network with angiotensin peptides in the active site, aiding in proper orientation for cleavage. Positions of serines and threonines (along with Lys, Arg, Val, Tyr, Phe, and Met) differ in the two active sites of ACE and are in contact with docked Ang I. This study shows enzyme differences and similarities in amino acids contacting the Ang peptides. Further, our results suggest that it may be possible to target multiple enzymes with the same (or a single) treatment in hypertensive patients, minimizing additive side effects of multidrug treatments.

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