scholarly journals Eukaryotic Multi-subunit DNA dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

2019 ◽  
pp. 1-60
Author(s):  
Peramachi Palanivelu
Keyword(s):  
Amino Acids ◽  
Metal Binding ◽  
Active Sites ◽  
Dna Polymerases ◽  
Rna Polymerases ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Conserved Motifs ◽  
X Ray ◽  
Eukaryotic Organisms

Aim: To analyze the most complex multi-subunit (MSU) DNA dependent RNA polymerases (RNAPs) of eukaryotic organisms and find out conserved motifs, metal binding sites and catalytic regions and propose a plausible mechanism of action for these complex eukaryotic MSU RNAPs, using yeast (Saccharomyces cerevisiae) RNAP II, as a model enzyme. Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis and X-ray crystallographic data were analyzed. Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India, between 2007- 2013. Methodology: Bioinformatics, Biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data of the enzyme were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNAPs from various eukaryotic sources. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical and SDM experiments and X-ray crystallographic analysis of these enzymes were used to confirm the possible amino acids involved in the active sites and catalysis. Results: Multiple sequence alignment (MSA) of RNAPs from different eukaryotic organisms showed a large number of highly conserved motifs among them.  Possible catalytic regions in the catalytic subunits of the yeast Rpb2 (= β in eubacteria) and Rpb1 (= β’ in eubacteria) consist of an absolutely conserved amino acid R, in contrast to a K that was reported for DNA polymerases and single subunit (SSU) RNAPs. However, the invariant ‘gatekeeper/DNA template binding’ YG pair that was reported in all SSU RNAPs, prokaryotic MSU RNAPs and DNA polymerases is also highly conserved in eukaryotic Rpb2 initiation subunits, but unusually a KG pair is found in higher eukaryotes including the human RNAPs. Like the eubacterial initiation subunits of MSU RNAPs, the eukaryotic initiation subunits, viz. Rpb2, exhibit very similar active site and catalytic regions but slightly different distance conservations between the template binding YG/KG pair and the catalytic R. In the eukaryotic initiation subunits, the proposed catalytic R is placed at the -9th position from the YG/KG pair and an invariant R is placed at -5 which are implicated to play a role in nucleoside triphosphate (NTP) selection as reported for SSU RNAPs (viral family) and DNA polymerases. Similarly, the eukaryotic elongation subunits (Rpb1) are also found to be very much homologous to the elongation subunits (β’) of prokaryotes. Interestingly, the catalytic regions are highly conserved, and the metal binding sites are absolutely conserved as in prokaryotic MSU RNAPs. In eukaryotes, the template binding YG pair is replaced with an FG pair. Another interesting observation is, similar to the prokaryotic β’ subunits, in the eukaryotic Rpb1 elongation subunits also, the proposed catalytic R is placed double the distance, i.e., -18 amino acids downstream from the FG pair unlike in the SSU RNAPs and DNA polymerases where the distance is only -8 amino acids downstream from the YG pair. Thus, the completely conserved FG pair, catalytic R with an invariant R, at -6th position are proposed to play a crucial role in template binding, NTP selection and polymerization reactions in the elongation subunits of eukaryotic MSU RNAPs. Moreover, the Zn binding motif with the three completely conserved Cs is also highly conserved in the eukaryotic elongation subunits. Another important difference is that the catalytic region is placed very close to the N-terminal region in eukaryotes. Conclusions: Unlike reported for the DNA polymerases and SSU RNA polymerases, the of eukaryotic MSU RNAPs use an R as the catalytic amino acid and exhibit a different distance conservation in the initiation and elongation subunits. An invariant Zn2+ binding motif found in the Rpb1 elongation subunits is proposed to participate in proof-reading function. Differences in the active sites of bacterial and human RNA polymerases may pave the way for the design of new and effective drugs for many bacterial infections, including the multidrug resistant strains which are a global crisis at present.

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 RNA Dependent RNA Polymerases of Severe Acute Respiratory Syndrome-Related Coronaviruses- An Insight into their Active Sites and Mechanism of Action

2020 ◽  
pp. 29-52
Author(s):  
Peramachi Palanivelu
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Active Site ◽  
Metal Binding ◽  
Rna Polymerases ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Viral Pathogens ◽  
Binding Motifs ◽  
X Ray

Aim: To analyze the RNA-dependent RNA polymerases (RdRps) of Severe Acute Respiratory Syndrome (SARS)-related coronaviruses (CoVs) to find out the conserved motifs, metal binding sites and catalytic amino acids and propose a plausible mechanism of action for these enzymes, using SARS-CoV-2 RdRp as a model enzyme. Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis (SDM), X-ray crystallographic and cryo-Electron microscopic (cryo-EM) data were analyzed. Methodology: Bioinformatics, Biochemical, Site-directed mutagenesis, X-ray crystallographic and cryo-EM data of these enzymes from RNA viral pathogens were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the RdRps. Results: Multiple sequence alignment (MSA) of RdRps from different SARS-related CoVs show a large number of highly conserved motifs among them. Though the RdRp from the Middle Eastern Respiratory syndrome (MERS)-CoV differed in many conserved regions yet the active site regions are completely conserved. Possible catalytic regions consist of an absolutely conserved amino acid K, as in single subunit (SSU) RNA polymerases and most of the DNA dependent DNA polymerases (DdDps). The invariant ‘gatekeeper/DNA template binding’ YG pair that was reported in all SSU DNA dependent RNA polymerases (DdRps), prokaryotic multi-subunit (MSU) DdRps and DdDps is also highly conserved in the RdRps of SARS-CoVs. The universal metal binding motif –GDD- and an additional motif–SDD- are also found in all SARS-CoV RdRps. In stark contrast, the (–) strand RNA viral pathogens like Ebola, rabies, etc. use –GDN- rather than –GDD- for catalytic metal binding. An invariant YA pair (instead of an YG pair) is found in the primases of the SARS-CoVs. The SARS-CoVs RdRps and primases exhibit very similar active site and catalytic regions with almost same distance conservations between the template binding YG/YA pair and the catalytic K. In SARS-CoV RdRps an invariant R is placed at -5 which is shown to play a role in nucleoside triphosphate (NTP) selection and is in close agreement with SSU DdRps (viral family) and DdDps. In primases no such invariant R/K/H is found very close to the catalytic K in the downstream region, as found in RdRp and Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) domains. An invariant YA pair is placed in the NiRAN domain instead of an YG pair, and an invariant H is placed at -5 position. Moreover, the Zn binding motif with the completely conserved Cs and a few DxD/DxxD type metal binding motifs are found in the RdRps and NiRAN domain. However, the primases contained only the DXD type metal binding motifs. Conclusions: The SARS and SARS-related CoV RdRps are very similar as large peptide regions are highly conserved among them. The closer identity between the RdRps of palm civet-CoV and SARS-CoV (CoV-1) suggest their possible link as found between their spike proteins also. The invariant YG and KL pairs may play a role in template binding and catalysis in SARS-CoV RdRps as reported in DdDps. An additional invariant –YAN- motif found in SARS-CoV RdRps may play a crucial role in nucleotide discrimination.

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.

scholarly journals Comparative Analyses of ACE2 Receptor Binding Corona Viruses that Cause Mild versus Severe Acute Respiratory Syndrome in Humans

2021 ◽  
pp. 64-85
Author(s):  
Peramachi Palanivelu
Keyword(s):  
Amino Acids ◽  
Severe Acute Respiratory Syndrome ◽  
Receptor Binding ◽  
Metal Binding ◽  
Protein Sequence ◽  
Active Sites ◽  
Protein Sequence Analysis ◽  
X Ray ◽  
Glycosylation Sites ◽  
Spike Proteins

Aim: To analyze the spike proteins and Replication-Transcription Complexes (RTCs) of the Mild and Severe Acute Respiratory Syndrome (SARS) and SARS-related coronaviruses (CoVs) to find out the similarities and differences between them, as both of groups bind to angiotensin-converting enzyme 2 (ACE2) receptor for human cell entry. Study Design: Bioinformatics, Biochemical, Site-Directed Mutagenesis (SDM), X-ray crystallographic, cryo-Electron microscopic (cryo-EM) and Mass Spectrometric (MS) data were analyzed. Methodology: The protein sequence data for spike proteins and the proteins of the RTCs, viz. the RNA- dependent RNA polymerases (RdRps), primases and the nonstructural protein 7 (NSP7) 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 and SDM experiments and X-ray crystallographic and cryo-EM  studies on these  proteins were used to confirm the possible amino acids involved in ACE2 receptor binding and active sites of the RTCs. For identification of probable N-linked and O-linked glycosylation sites, NetNGlyc 1.0 and NetOGlyc 4.0 tools of Technical University of Denmark were used. ExPASy tool was used for pI analysis. Results: The spike protein of human CoV (HCoV)-NL63 is ~90 amino acids longer than the spike proteins of SARS and SARS-related CoVs. The additions are mostly found in the N-terminal regions and few insertions are also found in the crucial receptor binding domain (RBD). The SARS and SARS-related CoVs and HCoV-NL63 showed several conserved residues, motifs and large peptide regions. The most important aspect between the recent pandemic causing SARS-CoV-2 and HCoV-NL63 is a unique but different tetrapeptide insertions very close to the S1/S2 cleavage region, i.e., -PRRA-  and  -IPVR-, respectively. The next cleavage point S2’ and the transmembrane domains are conserved between the two groups. The RdRps are highly conserved between the two groups. The catalytic regions, catalytic amino acids and the NTP selection tripeptide regions are completely conserved between SARS-CoVs and HCoV-NL63.  However, one of the metal binding sites, viz. the universal –GDD- reported in all RdRps is aligning with– KDG- in the RdRp of HCoV-NL63. The other metal binding site, viz. –SDD- is completely conserved in both the groups. The NiRAN domains of the RdRps differed from the possible catalytic amino acid and NTP selection tripeptide regions. The primases (NSP8) and the NSP7 subunits of the RTC are highly conserved in both the groups. The NSP8 and NSP7 subunits exhibit closer similarities between the MERS-CoV and HCoV-NL63. Unlike other SARS and SARS-related CoVs, the HCoV-NL63 possesses only a single accessory protein. Interestingly, a large number of amino acids are replaced with Ns in the spike proteins (which is also reflected in the number of N-linked glycosylation sites in it) as well as in the RTC. Conclusions: Detailed analysis revealed several unique features in the HCoV-NL63 pathogen. As all the pandemic strains like SARS-CoV-1, SARS-CoV-2 and the milder HCoV-NL63 strain, use the same ACE2 receptor for entry into human cells, the frequent infection of humans by HCoV-NL63, especially in children, suggests that there is an ample opportunity for highly pathogenic variants to evolve in the future.

scholarly journals Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif

1993 ◽  
Vol 13 (1) ◽  
pp. 123-132
Author(s):  
A D Sharrocks ◽  
H Gille ◽  
P E Shaw
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Amino Acid Peptide ◽  
Serum Response

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.

scholarly journals Characterization of iron-binding motifs in Candida albicans high-affinity iron permease CaFtr1p by site-directed mutagenesis

2002 ◽  
Vol 368 (2) ◽  
pp. 641-647 ◽  
Author(s):  
Hao-Ming FANG ◽  
Yue WANG
Keyword(s):  
Amino Acids ◽  
Candida Albicans ◽  
Iron Uptake ◽  
Iron Transport ◽  
Site Directed Mutagenesis ◽  
Detectable Effect ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Iron Binding ◽  
Direct Binding

A peptide motif Glu-Xaa-Xaa-Glu has been implicated in direct binding of ferric iron in several proteins involved in iron transport, sensing or storage. However, it is not known whether the motif alone is sufficient for iron binding and whether functional replacement of the conserved residues by other amino acids with similar properties is possible. We previously identified a Candida albicans iron permease, CaFtr1p, which contains five Glu-Xaa-Xaa-Glu motifs [Ramanan and Wang (2000) Science 288, 1062—1065]. In this study, we investigated the role of each of these motifs in iron uptake by site-directed mutagenesis. Substitution of Ala for any one of the two Glu residues in Glu-Gly-Leu-Glu158—161 abolished iron-uptake activity, while the same substitution in any of the other four motifs had little effect, indicating that only the motif at position 158—161 is required for iron transport. We then evaluated the importance of each of the residues within and immediately adjacent to this motif in iron uptake. The permease remained active when any one of the Glu residues was replaced by Asp, while it became inactive when both were replaced. We also found that the amino acid immediately in front of Glu-Gly-Leu-Glu158—161 must be either Arg or Lys. In addition, substitution of any of the two residues in the middle with several structurally distinct amino acids had no detectable effect on iron uptake. Here we propose to extend the iron-binding motif to Arg/Lys-Glu/Asp-Xaa-Xaa-Glu or Arg/Lys-Glu-Xaa-Xaa-Glu/Asp, which may serve as a guide for the identification of potential iron-binding sites in proteins.

scholarly journals Functional Analysis of Keto-Acid Reductoisomerase ILVC in the Entomopathogenic Fungus Metarhizium robertsii

2021 ◽  
Vol 7 (9) ◽  
pp. 737
Author(s):  
Yulong Wang ◽  
Shihong Liu ◽  
Xuebing Yin ◽  
Deshui Yu ◽  
Xiangyun Xie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Yeast Extract ◽  
Active Sites ◽  
Reductase Activity ◽  
Fungal Growth ◽  
Conidial Germination ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Metarhizium Robertsii ◽  
Branched Chain

Ketol-acid reductoisomerase (ILVC) is the second enzyme in the branched-chain amino acid (BCAA) biosynthesis, which regulates many physiological activities in a variety of organisms from bacteria to fungi and plants. In this work, function mechanisms of ILVC in Metarhizium robertsii Metchnikoff (Hypocreales: Clavicipitaceae) were explored with site-directed mutagenesis, reductase activity assays and transcriptomics analysis. The reductase activity assays showed that ILVC from phytopathogenic fungi exhibited significantly higher activities than those from entomopathogenic fungi but lower than those from yeast. Site-directed mutagenesis and enzymatic activities of MrILVC with different active-site mutants (Arg-113, Ser-118, Asp-152, Asp-260, and Glu-264) confirmed that active sites of MrILVC are conserved with plant and bacterial ILVCs. Deleting MrilvC causes the complete failures of vegetative growth and conidial germination, feeding with branched-chain amino acids (BCAAs) recovers the fungal growth but not conidial germination, while both characteristics are restored when supplemented with yeast extract. Compared to ΔMrilvC cultured in czapek agar (CZA), plenty of genes involved in the biosynthesis of antibiotics and amino acids were up- or down-regulated in the wild type or ΔMrilvC feeding with either BCAAs or yeast extract. Further analysis showed some genes, such as catalase A, participate in mycelial growth and conidial germination was down-regulated in ΔMrilvC from CZA, revealing that MrILVC might control the fungal development by gene regulation and BCAAs or yeast extract could play partial roles of MrILVC. This study will advance our understanding of ILVC function mechanisms in fungi.

scholarly journals Epimerase (Msed_0639) and Mutase (Msed_0638 and Msed_2055) Convert (S)-Methylmalonyl-Coenzyme A (CoA) to Succinyl-CoA in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Cycle

2012 ◽  
Vol 78 (17) ◽  
pp. 6194-6202 ◽  
Author(s):  
Yejun Han ◽  
Aaron S. Hawkins ◽  
Michael W. W. Adams ◽  
Robert M. Kelly
Keyword(s):  
Metal Binding ◽  
Active Sites ◽  
Coenzyme A ◽  
Divalent Cations ◽  
Growth Conditions ◽  
Binding Motif ◽  
Sequence Alignments ◽  
Content Type ◽  
Metallosphaera Sedula ◽  
Coenzyme B

ABSTRACTCrenarchaeotal genomes encode the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle for carbon dioxide fixation. Of the 13 enzymes putatively comprising the cycle, several of them, including methylmalonyl-coenzyme A (CoA) epimerase (MCE) and methylmalonyl-CoA mutase (MCM), which convert (S)-methylmalonyl-CoA to succinyl-CoA, have not been confirmed and characterized biochemically. In the genome ofMetallosphaera sedula(optimal temperature [Topt], 73°C), the gene encoding MCE (Msed_0639) is adjacent to that encoding the catalytic subunit of MCM-α (Msed_0638), while the gene for the coenzyme B12-binding subunit of MCM (MCM-β) is located remotely (Msed_2055). The expression of all three genes was significantly upregulated under autotrophic compared to heterotrophic growth conditions, implying a role in CO2fixation. Recombinant forms of MCE and MCM were produced inEscherichia coli; soluble, active MCM was produced only if MCM-α and MCM-β were coexpressed. MCE is a homodimer and MCM is a heterotetramer (α2β2) with specific activities of 218 and 2.2 μmol/min/mg, respectively, at 75°C. The heterotetrameric MCM differs from the homo- or heterodimeric orthologs in other organisms. MCE was activated by divalent cations (Ni2+, Co2+, and Mg2+), and the predicted metal binding/active sites were identified through sequence alignments with less-thermophilic MCEs. The conserved coenzyme B12-binding motif (DXHXXG-SXL-GG) was identified inM. sedulaMCM-β. The two enzymes together catalyzed the two-step conversion of (S)-methylmalonyl-CoA to succinyl-CoA, consistent with their proposed role in the 3-HP/4-HB cycle. Based on the highly conserved occurrence of single copies of MCE and MCM inSulfolobaceaegenomes, theM. sedulaenzymes are likely to be representatives of these enzymes in the 3-HP/4-HB cycle in crenarchaeal thermoacidophiles.

scholarly journals Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif.

1993 ◽  
Vol 13 (1) ◽  
pp. 123-132 ◽  
Author(s):  
A D Sharrocks ◽  
H Gille ◽  
P E Shaw
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Amino Acid Peptide ◽  
Serum Response

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.

scholarly journals Copper(I)-mediated protein–protein interactions result from suboptimal interaction surfaces

2009 ◽  
Vol 422 (1) ◽  
pp. 37-42 ◽  
Author(s):  
Lucia Banci ◽  
Ivano Bertini ◽  
Vito Calderone ◽  
Nunzia Della-Malva ◽  
Isabella C. Felli ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Metal Binding ◽  
Site Directed Mutagenesis ◽  
Protein Protein Interactions ◽  
X Ray ◽  
X Ray Crystallography ◽  
Metal Binding Domain ◽  
Modelling Techniques ◽  
Cellular Pathways ◽  
P Type

The homoeostasis of metal ions in cells is the result of the contribution of several cellular pathways that involve transient, often weak, protein–protein interactions. Metal transfer typically implies the formation of adducts where the metal itself acts as a bridge between proteins, by co-ordinating residues of both interacting partners. In the present study we address the interaction between the human copper(I)-chaperone HAH1 (human ATX1 homologue) and a metal-binding domain in one of its partners, namely the P-type copper-transporting ATPase, ATP7A (ATPase, Cu+ transporting, α polypeptide). The adduct was structurally characterized in solution, in the presence of copper(I), and through X-ray crystallography, upon replacing copper(I) with cadmium(II). Further insight was obtained through molecular modelling techniques and site-directed mutagenesis. It was found that the interaction involves a relatively small interface (less than 1000 Å2, 1 Å=0.1 nm) with a low fraction of non-polar atoms. These observations provide a possible explanation for the low affinity of the two apoproteins. It appears that electrostatics is important in selecting which domain of the ATPase is able to form detectable amounts of the metal-mediated adduct with HAH1.

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