scholarly journals Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

2021 ◽  
Author(s):  
Jaya Joshi ◽  
Qiang Li ◽  
Jorge D. García-García ◽  
Bryan J. Leong ◽  
You Hu ◽  
...  
Keyword(s):  
Active Site ◽  
Data Use ◽  
Comparative Genomic ◽  
Aerobic Conditions ◽  
Single Turnover ◽  
Catalytic Function ◽  
Metal Cofactor ◽  
Catalytic Metal ◽  
And Function ◽  
Sulfur Donor

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multiple-turnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been biochemically characterized – but only from archaeal methanogens that are anaer­obic, O2-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2-sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

scholarly journals Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

2021 ◽  
Author(s):  
Jaya Joshi ◽  
Qiang Li ◽  
Jorge D. Garcia-Garcia ◽  
Bryan J. Leong ◽  
You Hu ◽  
...  
Keyword(s):  
Active Site ◽  
Molecular Basis ◽  
Structure And Function ◽  
Comparative Genomic ◽  
Aerobic Conditions ◽  
Single Turnover ◽  
Metal Cofactor ◽  
Catalytic Metal ◽  
And Function ◽  
Sulfur Donor

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single–turnover suicide reaction that uses an active–site Cys residue as sulfur donor. Multiple turnover (i.e. catalytic) THI4s lacking an active–site Cys (non–Cys THI4s) that use sulfide as sulfur donor have been characterized—but only from archaeal methanogens that are anaerobic, O2–sensitivehyperthermophiles from sulfide–rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non–Cys THI4s in aerobic mesophiles from sulfide–poor habitats, suggesting that multiple–turnover THI4 operation is possible in aerobic, mild, low–sulfide conditions. This was confirmed by testing 23 representative non–Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were active, and more so when intracellular sulfidelevel was raised by supplying Cys, demonstrating that they function in the presence of O2 at mild temperatures and indicating they use sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non–Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2–sensitive methanogens but is consistent with an alternative catalytic metal. Together with complementation data, the use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

scholarly journals Modulating Enzyme Function via Dynamic Allostery within Biliverdin Reductase B

2021 ◽  
Vol 8 ◽  
Author(s):  
Jasmina S. Redzic ◽  
Michael R. Duff ◽  
Ashley Blue ◽  
Todd M. Pitts ◽  
Pratul Agarwal ◽  
...  
Keyword(s):  
Active Site ◽  
Redox Regulation ◽  
Nmr Relaxation ◽  
Functional Coupling ◽  
Global Dynamics ◽  
Biliverdin Reductase ◽  
Coenzyme Binding ◽  
Catalytic Function ◽  
Order Of Magnitude ◽  
And Function

The biliverdin reductase B (BLVRB) class of enzymes catalyze the NADPH-dependent reduction of multiple flavin substrates and are emerging as critical players in cellular redox regulation. However, the role of dynamics and allostery have not been addressed, prompting studies here that have revealed a position 15 Å away from the active site within human BLVRB (T164) that is inherently dynamic and can be mutated to control global micro-millisecond motions and function. By comparing the inherent dynamics through nuclear magnetic resonance (NMR) relaxation approaches of evolutionarily distinct BLVRB homologues and by applying our previously developed Relaxation And Single Site Multiple Mutations (RASSMM) approach that monitors both the functional and dynamic effects of multiple mutations to the single T164 site, we have discovered that the most dramatic mutagenic effects coincide with evolutionary changes and these modulate coenzyme binding. Thus, evolutionarily changing sites distal to the active site serve as dynamic “dials” to globally modulate motions and function. Despite the distal dynamic and functional coupling modulated by this site, micro-millisecond motions span an order of magnitude in their apparent kinetic rates of motions. Thus, global dynamics within BLVRB are a collection of partially coupled motions tied to catalytic function.

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.

scholarly journals Fe(2)OG: an integrated HMM profile-based web server to predict and analyze putative non-haem iron(II)- and 2-oxoglutarate-dependent dioxygenase function in protein sequences

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Siddhartha Kundu
Keyword(s):  
Amino Acid ◽  
Water Molecule ◽  
Active Site ◽  
Ferrous Iron ◽  
Web Server ◽  
Protein Sequences ◽  
Diverse Group ◽  
And Function ◽  
Functionally Diverse ◽  
Haem Iron

Abstract Objective Non-haem iron(II)- and 2-oxoglutarate-dependent dioxygenases (i2OGdd), are a taxonomically and functionally diverse group of enzymes. The active site comprises ferrous iron in a hexa-coordinated distorted octahedron with the apoenzyme, 2-oxoglutarate and a displaceable water molecule. Current information on novel i2OGdd members is sparse and relies on computationally-derived annotation schema. The dissimilar amino acid composition and variable active site geometry thereof, results in differing reaction chemistries amongst i2OGdd members. An additional need of researchers is a curated list of sequences with putative i2OGdd function which can be probed further for empirical data. Results This work reports the implementation of $$Fe\left(2\right)OG$$ F e 2 O G , a web server with dual functionality and an extension of previous work on i2OGdd enzymes $$\left(Fe\left(2\right)OG\equiv \{H2OGpred,DB2OG\}\right)$$ F e 2 O G ≡ { H 2 O G p r e d , D B 2 O G } . $$Fe\left(2\right)OG$$ F e 2 O G , in this form is completely revised, updated (URL, scripts, repository) and will strengthen the knowledge base of investigators on i2OGdd biochemistry and function. $$Fe\left(2\right)OG$$ F e 2 O G , utilizes the superior predictive propensity of HMM-profiles of laboratory validated i2OGdd members to predict probable active site geometries in user-defined protein sequences. $$Fe\left(2\right)OG$$ F e 2 O G , also provides researchers with a pre-compiled list of analyzed and searchable i2OGdd-like sequences, many of which may be clinically relevant. $$Fe(2)OG$$ F e ( 2 ) O G , is freely available (http://204.152.217.16/Fe2OG.html) and supersedes all previous versions, i.e., H2OGpred, DB2OG.

In Vivo Consequences of Putative Active Site Mutations in Yeast DNA Polymerases α, ε, δ, and ζ

Genetics ◽  
2001 ◽  
Vol 159 (1) ◽  
pp. 47-64 ◽  
Author(s):  
Youri I Pavlov ◽  
Polina V Shcherbakova ◽  
Thomas A Kunkel
Keyword(s):  
Active Site ◽  
Dna Polymerases ◽  
Base Substitution ◽  
Loss Of Function ◽  
Chromosomal Dna ◽  
Strong Base ◽  
Dna Mismatch ◽  
Catalytic Function ◽  
Base Substitutions

Abstract Several amino acids in the active site of family A DNA polymerases contribute to accurate DNA synthesis. For two of these residues, family B DNA polymerases have conserved tyrosine residues in regions II and III that are suggested to have similar functions. Here we replaced each tyrosine with alanine in the catalytic subunits of yeast DNA polymerases α, δ, ε, and ζ and examined the consequences in vivo. Strains with the tyrosine substitution in the conserved SL/MYPS/N motif in region II in Polδ or Polε are inviable. Strains with same substitution in Rev3, the catalytic subunit of Polζ, are nearly UV immutable, suggesting severe loss of function. A strain with this substitution in Polα (pol1-Y869A) is viable, but it exhibits slow growth, sensitivity to hydroxyurea, and a spontaneous mutator phenotype for frameshifts and base substitutions. The pol1-Y869A/pol1-Y869A diploid exhibits aberrant growth. Thus, this tyrosine is critical for the function of all four eukaryotic family B DNA polymerases. Strains with a tyrosine substitution in the conserved NS/VxYG motif in region III in Polα, -δ, or -ε are viable and a strain with the homologous substitution in Rev3 is UV mutable. The Polα mutant has no obvious phenotype. The Polε (pol2-Y831A) mutant is slightly sensitive to hydroxyurea and is a semidominant mutator for spontaneous base substitutions and frameshifts. The Polδ mutant (pol3-Y708A) grows slowly, is sensitive to hydroxyurea and methyl methanesulfonate, and is a strong base substitution and frameshift mutator. The pol3-Y708A/pol3-Y708A diploid grows slowly and aberrantly. Mutation rates in the Polα, -δ, and -ε mutant strains are increased in a locus-specific manner by inactivation of PMS1-dependent DNA mismatch repair, suggesting that the mutator effects are due to reduced fidelity of chromosomal DNA replication. This could result directly from relaxed base selectivity of the mutant polymerases due to the amino acid changes in the polymerase active site. In addition, the alanine substitutions may impair catalytic function to allow a different polymerase to compete at the replication fork. This is supported by the observation that the pol3-Y708A mutation is recessive and its mutator effect is partially suppressed by disruption of the REV3 gene.

scholarly journals The Electronic Structure of FeV-cofactor in Vanadium-Dependent Nitrogenase

2021 ◽  
Author(s):  
Zhi-yong Yang ◽  
Emilio Jimenez-Vicente ◽  
Hayden Kallas ◽  
Dmitriy A Lukoyanov ◽  
Hao Yang ◽  
...  
Keyword(s):  
Electronic Structure ◽  
Active Site ◽  
Resting State ◽  
Metal Cofactor ◽  
Open Question

The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2...

A unique aromatic cluster near the active site of H. pylori CPA is essential for catalytic function

2021 ◽  
Author(s):  
Ditsa Sarkar ◽  
Ramachandran Vijayan ◽  
Samudrala Gourinath ◽  
Apurba Kumar Sau
Keyword(s):  
Active Site ◽  
Catalytic Function ◽  
H Pylori ◽  
Aromatic Cluster

scholarly journals Three Conserved Regions in Baculovirus Sulfhydryl Oxidase P33 Are Critical for Enzymatic Activity and Function

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Wenhua Kuang ◽  
Huanyu Zhang ◽  
Manli Wang ◽  
Ning-Yi Zhou ◽  
Fei Deng ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Active Site ◽  
Salt Bridge ◽  
Sulfhydryl Oxidase ◽  
Dimer Interface ◽  
Conserved Regions ◽  
Occlusion Bodies ◽  
Budded Virus ◽  
And Function ◽  
Substrate Proteins

ABSTRACT Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo. Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity. IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

scholarly journals Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme

RNA ◽  
2018 ◽  
Vol 24 (11) ◽  
pp. 1542-1554 ◽  
Author(s):  
Neil A. White ◽  
Minako Sumita ◽  
Victor E. Marquez ◽  
Charles G. Hoogstraten
Keyword(s):  
Active Site ◽  
Conformational Dynamics ◽  
Catalytic Function
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