scholarly journals An Insight into the Proofreading Functions of Multisubunit DNA-Dependent RNA Polymerases and Their Catalytic Mechanism

2021 ◽  
pp. 15-59
Author(s):  
Peramachi Palanivelu
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Protein Sequence ◽  
Catalytic Mechanism ◽  
Rna Polymerases ◽  
Zinc Atom ◽  
Proton Acceptor ◽  
X Ray ◽  
Clustal Omega

Aim: To analyze the active sites of the proofreading (PR) functions in the multisubunit DNA-dependent RNA polymerases (MSU RNAPs) from prokaryotes, chloroplasts and eukaryotes, and propose a plausible unified catalytic mechanism for these enzymes. Study Design: Data collected on these enzymes from bioinformatics, biochemical, site-directed mutagenesis (SDM), X-ray crystallography and cryo-electron microscopy (cryo-EM) were used for the analyses. Methodology: The protein sequence data of MSU RNAPs from prokaryotes, prokaryotic-types (plant chloroplasts) and eukaryotes 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 from biochemical and SDM experiments, and X-ray crystallographic and cryo-EM data on these enzymes are also used to confirm the possible amino acids involved in the active site of the PR function in these MSU RNAPs Results: All the seven types of MSU RNAPs (I-VII) reported from prokaryotes to eukaryotes were analyzed by the multiple sequence alignment (MSA) software, Clustal Omega, to find out conservations among them. The MSA analysis showed many conserved amino acid motifs including small and large peptide regions from the MSU RNAPs of prokaryotes, eukaryotes and plant chloroplasts. Interestingly, the catalytic amino acid and template-binding pairs are highly conserved in all these polymerases, with a few exceptions. Most of them use a basic amino acid (R/K/H) for initiating catalysis and an -YG/FG- pair for template-binding. Some odd type of catalytic amino acids and template-binding pairs are observed in human pathogens, parasites and organisms which cannot ferment sugars. In all the MSU RNAPs, the proposed polymerase catalytic region also possessed three invariant Cs and an invariant H within it. The invariant Cs is shown to bind a zinc atom and proposed to involve in the PR function by excising any misincorporated nucleotide during the transcription process. In the plant-specific MSU RNAPs IV and V, which involve in transcriptional gene silencing in plants, the catalytic and template-binding pairs do not follow the regular distance conservations as observed with other five of the MSU RNAPs. Their polymerase/PR active site regions are similar to RNAP III rather than to RNAP II, as all three make only low molecular weight RNAs. Conclusions: All the known MSU RNAPs possess three invariant Cs and an invariant H embedded within the polymerase active site itself. The three invariant Cs are shown to bind a zinc atom and the invariant H could act as the proton acceptor from a metal-bound water molecule, for initiating excision of the mismatches by a Zn-mediated hydrolysis. Thus, the PR function in MSU RNAPs is integrated within the polymerase active site itself, which is in sharp contrast to the PR functions reported in DNA-dependent DNA polymerases and RNA-dependent RNA polymerases. Therefore, all the seven MSU RNAPs from prokaryotes and eukaryotes are proposed to follow a unified mechanism to excise the mismatches during transcription. The discovery of intrinsic self-correcting RNA transcription mechanism fulfils the missing link in molecular evolution.

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 Analyses of Homing Endonucleases and Mechanism of Action of CRISPR-Cas9 HNH Endonucleases

2020 ◽  
pp. 1-25
Author(s):  
Peramachi Palanivelu
Keyword(s):  
Amino Acids ◽  
Mechanism Of Action ◽  
Metal Binding ◽  
Binding Sites ◽  
Proton Acceptor ◽  
Homing Endonucleases ◽  
Multiple Sequence ◽  
Conserved Motifs ◽  
X Ray ◽  
Metal Binding Sites

Aim: To analyze different HNH endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 proteins for their conserved motifs, metal-binding sites and catalytic amino acids and propose a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 as the model enzyme. Study Design: Multiple sequence analysis (MSA) of homing endonucleases including the CRISPR-Cas9 using Clustal Omega was studied. Other biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data were also analyzed. Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India, between 2007 and 2013. Methodology: Bioinformatics, Biochemical, SDM and X-ray crystallographic data of the HNH endonucleases from different organisms including CRISPR-Cas9 enzymes were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of different HNH endonucleases from various sources. The conserved motifs identified by the bioinformatics analysis were analyzed further with the data already available from biochemical and SDM and X-ray crystallographic analyses of this group of enzymes and to confirm the possible amino acids involved in the active sites and catalysis. Results: Different types of homing endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 enzymes exhibit different catalytic regions and metal-binding sites. However, the catalytic amino acid, i.e., the proton acceptor histidine (His), is completely conserved in all homing endonucleases analyzed. From these data, a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 from Streptococcus pyogenes, as the model enzyme is proposed. Furthermore, multiple sequence alignment (MSA) of various homing endonucleases from different organisms showed many highly conserved motifs also among them. However, some of the HNH endonucleases showed consensus only around the active site regions. Possible catalytic amino acids identified among them belong to either -DH---N or -HH--N types. There are at least two types of metal-binding sites and bind Mg2+ or Zn2+ or both. The CRISPR-Cas9 enzyme from S. pyogenes belongs to the -DH- based HNH endonucleases and possesses –DxD- type metal-binding site where it possibly binds to a Mg2+ ion. The other HNH enzymes possess one or two invariant Zn binding CxxC/ CxxxC motifs. Conclusions: The CRISPR-Cas9 enzymes are found to be -DH- type where the first D is likely to involve in metal-binding and the second invariant H acts as the proton acceptor and the N in –HNH- Cas9 confers specificity by interacting with the nucleotide near the catalytic region. In this communication, a metal-bound water molecule is shown as the nucleophile initiating catalysis. Homing endonucleases may be used as novel DNA binding and cleaving reagents for a variety of genome editing applications and Zinc finger nucleases have already found applications in genome editing.

PREDICTION OF PATHOGENIC EFFECT FOR AMINO ACID SUBSTITUTIONS IN BRCA1 AND BRCA2 PROTEINS USING MNA DESCRIPTORS AND NAIVE BAYES CLASSIFIER

2020 ◽  
pp. 250-252
Author(s):  
D. Filimonov ◽  
A. Lagunin
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Sequence ◽  
Amino Acid Substitutions ◽  
Classification Models ◽  
Bayes Classifier ◽  
Brca1 And Brca2 ◽  
Chemical Structures ◽  
Pathogenic Effect ◽  
Pathogenic Effects

It is advisable to use data peptide's chemical structures with amino acids (AMA) substitution and the corresponding sections of the protein sequence without mutation to construct classification models predicting the pathogenic effects AMA substitutions based on MNA descriptors.

Sorption of α-amino-acids by copper montmorillonite

Clay Minerals ◽  
1967 ◽  
Vol 7 (2) ◽  
pp. 167-176 ◽  
Author(s):  
W. Bodenheimer ◽  
L. Heller
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Complex Formation ◽  
Glutamic Acid ◽  
Acid Complex ◽  
X Ray ◽  
Ray Methods

AbstractSorption of an acidic, amphoteric, sulphur containing and basic α-amino-acid (glutamic acid, glycine, methionine and lysine) by copper montmorillonite was studied by chemical and X-ray methods. With glutamic acid complex formation occurs only in solution but increasing basicity of the aminoacid favours complex formation in the clay interlayers.

PREDICTION OF THE THREE-DIMENSIONAL STRUCTURE OF THE ENZYMATIC PART OF T-PA

1987 ◽  
Author(s):  
A Heckel ◽  
K M Hasselbach
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Serine Proteases ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
Salt Bridges ◽  
Three Dimensional Structure ◽  
Insertions And Deletions

Up to now the three-dimensional structure of t-PA or parts of this enzyme is unknown. Using computer graphical methods the spatial structure of the enzymatic part of t-PA is predicted on the hypothesis, the three-dimensional backbone structure of t-PA being similar to that of other serine proteases. The t-PA model was built up in three steps:1) Alignment of the t-PA sequence with other serine proteases. Comparison of enzyme structures available from Brookhaven Protein Data Bank proved elastase as a basis for modeling.2) Exchange of amino acids of elastase differing from the t-PA sequence. The replacement of amino acids was performed such that backbone atoms overlapp completely and side chains superpose as far as possible.3) Modeling of insertions and deletions. To determine the spatial arrangement of insertions and deletions parts of related enzymes such as chymotrypsin or trypsin were used whenever possible. Otherwise additional amino acid sequences were folded to a B-turn at the surface of the proteine, where all insertions or deletions are located. Finally the side chain torsion angles of amino acids were optimised to prevent close contacts of neigh bouring atoms and to improve hydrogen bonds and salt bridges.The resulting model was used to explain binding of arginine 560 of plasminogen to the active site of t-PA. Arginine 560 interacts with Asp 189, Gly 19 3, Ser 19 5 and Ser 214 of t-PA (chymotrypsin numbering). Furthermore interaction of chromo-genic substrate S 2288 with the active site of t-PA was studied. The need for D-configuration of the hydrophobic amino acid at the N-terminus of this tripeptide derivative could be easily explained.

scholarly journals Structure based protein engineering to confer selectivity of guanine deaminase

2014 ◽  
Vol 70 (a1) ◽  
pp. C437-C437
Author(s):  
Aruna Bitra ◽  
Ruchi Anand
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
X Ray ◽  
Secondary Activity ◽  
Guanine Deaminase ◽  
Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

scholarly journals X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase

2003 ◽  
Vol 373 (3) ◽  
pp. 733-738 ◽  
Author(s):  
Peter T. ERSKINE ◽  
Leighton COATES ◽  
Danica BUTLER ◽  
James H. YOUELL ◽  
Amanda A. BRINDLEY ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Zinc Ion ◽  
Side Chain ◽  
Hydroxide Ion ◽  
X Ray ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Cyclic Intermediate ◽  
Aminolaevulinic Acid Dehydratase

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 Å) resolution. The cyclic intermediate is bound covalently to Lys263 with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion. The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen. The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C–C bond linking both substrates in the intermediate is formed before the C–N bond.

X-ray studies on crystalline complexes involving amino acids and peptides. XL. Conformational variability, recurring and new features of aggregation, and effect of chirality in the malonic acid complexes of DL- and L-arginine

2002 ◽  
Vol 58 (6) ◽  
pp. 1051-1056 ◽  
Author(s):  
N. T. Saraswathi ◽  
M. Vijayan
Keyword(s):  
Amino Acids ◽  
Hydrogen Bonding ◽  
Amino Acid ◽  
Crystal Structures ◽  
Malonic Acid ◽  
Conformational Flexibility ◽  
X Ray ◽  
Conformational Variability ◽  
Aggregation Pattern ◽  
Positively Charged

The crystal structures of the complexes of malonic acid with DL- and L-arginine, which contain positively charged argininium ions and negatively charged semimalonate ions, further demonstrate the conformational flexibility of amino acids. A larger proportion of folded conformations than would be expected on the basis of steric consideration appears to occur in arginine, presumably because of the requirements of hydrogen bonding. The aggregation pattern in the DL-arginine complex bears varying degrees of resemblance to patterns observed in other similar structures. An antiparallel hydrogen-bonded dimeric arrangement of arginine, and to a lesser extent lysine, is a recurring motif. Similarities also exist among the structures in the interactions with this motif and its assembly into larger features of aggregation. However, the aggregation pattern observed in the L-arginine complex differs from any observed so far, which demonstrates that all the general patterns of amino-acid aggregation have not yet been elucidated. The two complexes represent cases where the reversal of the chirality of half the amino-acid molecules leads to a fundamentally different aggregation pattern.

Nucleotide and Amino Acid Sequences of the Metallo-β-Lactamase, ImiS, from Aeromonas veronii bv. sobria

1998 ◽  
Vol 42 (2) ◽  
pp. 436-439 ◽  
Author(s):  
T. R. Walsh ◽  
W. A. Neville ◽  
M. H. Haran ◽  
D. Tolson ◽  
D. J. Payne ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Aeromonas Veronii ◽  
Putative Active Site ◽  
Lactamase Gene ◽  
Active Site Sequence

ABSTRACT The Aeromonas veronii bv. sobria metallo-β-lactamase gene, imiS, was cloned. The imiS open reading frame extends for 762 bp and encodes a protein of 254 amino acids with a secreted modified protein of 227 amino acids and a predicted pI of 8.1. To confirm the predicted sequence, purified ImiS was digested and the resulting peptides were identified, yielding an identical sequence for ImiS, with 98% identity to CphA. Both possessed the putative active-site sequence Asn-Tyr-His-Thr-Asp at positions 88 to 92, which is unique to the Aeromonas metallo-β-lactamases.

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