Analysis Of Substrate Binding Research Articles

Structure elucidation of a multi-modular recombinant endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus ATCC 27405 and its substrate binding analysis

Cellulases from GH9 family show endo-, exo- or processive endocellulase activity, but the reason behind the variation is unclear. A GH9 recombinant endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus was structurally characterized for conformation, binding and dynamics assessment. Modeled AtGH9C-CBM3A-CBM3B depicted (α/α)6-barrel structure with Asp98, Asp101 and Glu489 acting as catalytic triad. CD results revealed 25.2 % α-helix, 18.4 % β-sheet and rest 56.4 % of random coils, corroborating with predictions from PSIPRED and SOPMA. MD simulation of AtGH9C-CBM3A-CBM3B bound cellotetraose showed structural stability and global compactness with lowered RMSD values (1.5 nm) as compared with only AtGH9C-CBM3A-CBM3B (1.8 nm) for 200 ns. Higher fluctuation in RMSF values in far-positioned CBM3B pointed to its redundancy in substrate binding. Docking studies showed maximum binding with cellotetraose (ΔG = −5.05 kcal/mol), with reduced affinity towards ligands with degree of polymerization (DP) lower (DP < 4) or higher than 4 (DP > 4). Processivity index displayed the enzyme to be processive with loop 3 (342–379 aa) possibly blocking the non-reducing end of cellulose chain, resulting in cellotetraose release. SAXS analysis of AtGH9C-CBM3A-CBM3B at 5 mg/mL displayed monodispersed state with fist-and-elbow shape in solution. Negative zeta potential of −24 mV at 5 mg/mL indicated stability and free from aggregation.

Rational surface charge engineering of haloalkane dehalogenase for boosting the enzymatic performance in organic solvent solutions

Biocatalysis in organic solvents (OSs) has numerous important applications, but native enzymes in OSs often exhibit limited catalytic performance. Herein, we proposed a computation-aided surface charge engineering strategy to improve the catalytic performance of haloalkane dehalogenase DhaA in OSs based on the energetic analysis of substrate binding to the DhaA surface. Several variants with enhanced OS resistance were obtained by replacing negative charged residues on the surface with positive charged residue (Arg). Particularly, a four-substitution variant E16R/E93R/E121R/E257R exhibited the best catalytic performance (five-fold improvement in OS resistance and seven-fold half-life increase in 40% (vol) dimethylsulfoxide). As a result, the overall catalytic performance of the variant could be at least 26 times higher than the wild-type DhaA. Fluorescence spectroscopy and molecular dynamics simulation studies revealed that the residue substitution mainly enhanced OS resistance from four aspects: (a) improved the overall structural stability, (b) increased the hydrophobicity of the local microenvironment around the catalytic triad, (c) enriched the hydrophobic substrate around the enzyme molecule, and (d) lowered the contact frequency between OS molecules and the catalytic triad. Our findings validate that computation-aided surface charge engineering is an effective and ingenious rational strategy for tailoring enzyme performance in OSs.

Hydroxylation, Epoxidation, and Dehydrogenation of Capsaicin by a Microbial Promiscuous Cytochrome P450 105D7.

Cytochrome P450 enzymes (P450s) are versatile biocatalysts, which insert a molecular oxygen into inactivated C-H bonds under mild conditions. CYP105D7 from Streptomyces avermitilis has been reported as a bacterial substrate-promiscuous P450 which catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, compactin and steroids. In this study, CYP105D7 catalyzes hydroxylation, epoxidation and dehydrogenation of capsaicin, a pharmaceutical agent, revealing its functional diversity. The kinetic parameters of the CYP105D7 oxidation of capsaicin were determined as Km =311.60±87.30 μM and kcat =2.01±0.33 min-1 . In addition, we conducted molecular docking, mutagenesis and substrate binding analysis, indicating that Arg81 plays crucial role in the capsaicin binding and catalysis. To our best knowledge, this study presents the first report to illustrate that capsaicin can be catalyzed by prokaryotic P450s.

Homology Modelling and in Silico Substrate Binding Analysis of a Rhizobium sp. RC1 Haloalkanoic Acid Permease

Rhizobium sp. RC1 grows on haloalkanoic acid (haloacid) pollutants and expresses a haloacid permease (DehrP) which mediates the uptake of haloacids into the cells. For the first time, we report the homology model and docking analysis of DehrP and proposed its putative binding residues. The Protein Data Bank for protein of similar sequence. Ligand structures were retrieved from the ChemSpider database. The 3-dimensional (3-D) structure of DehrP was modelled based on the structure of Staphylococcus epidermidis glucose: H+ symporter (GlcPse) by Phyre2, refined by 3Drefine and evaluated by ProSA z-score, ERRAT and RAMPAGE. Docking of monobromoacetate, monochloroacetate, dibromoacetate, dichloroacetate, trichloroacetate, and 2,2-dichloropropionate ligands was done with AutoDock vina1.1.2. The 3-D structure of DehrP protein has twelve transmembrane helices. The overall quality factor of the model is ∼91%, with 93.6% of the residues in the favored region and the z-score is within the ≤ 10 limit. The putative H+ binding site residues are Gln133, Asp36, and Arg130. Docking analysis showed that Glu33, Trp34, Phe37, Phe38, Gln165, and Glu370 are potential haloacid interacting residues. DehrP-haloacid complexes had a binding affinity between -2.9 to -4.0 kcal/mol. DehrP has both putative H+ and haloacid binding sites that are most likely involved in the co-transport of H+ and haloacids. DehrP interacts with haloacids majorly through van der Waals and halogen bond interactions and has greater affinity for 2,2-dichloropropionate and could be a specialized chloropropionate uptake system. Site-directed mutagenasis of DehrP binding residues could improve its haloacid binding affinity.

Homology Modelling and in Silico Substrate Binding Analysis of a Rhizobium sp. RC1 Haloalkanoic Acid Permease

Rhizobium sp. RC1 grows on haloalkanoic acid (haloacid) pollutants and expresses a haloacid permease (DehrP) which mediates the uptake of haloacids into the cells. For the first time, we report the homology model and docking analysis of DehrP and proposed its putative binding residues. The Protein Data Bank for protein of similar sequence. Ligand structures were retrieved from the ChemSpider database. The 3-dimensional (3-D) structure of DehrP was modelled based on the structure of Staphylococcus epidermidis glucose: H+ symporter (GlcPse) by Phyre2, refined by 3Drefine and evaluated by ProSA z-score, ERRAT and RAMPAGE. Docking of monobromoacetate, monochloroacetate, dibromoacetate, dichloroacetate, trichloroacetate, and 2,2-dichloropropionate ligands was done with AutoDock vina1.1.2. The 3-D structure of DehrP protein has twelve transmembrane helices. The overall quality factor of the model is ∼91%, with 93.6% of the residues in the favored region and the z-score is within the ≤ 10 limit. The putative H+ binding site residues are Gln133, Asp36, and Arg130. Docking analysis showed that Glu33, Trp34, Phe37, Phe38, Gln165, and Glu370 are potential haloacid interacting residues. DehrP-haloacid complexes had a binding affinity between -2.9 to -4.0 kcal/mol. DehrP has both putative H+ and haloacid binding sites that are most likely involved in the co-transport of H+ and haloacids. DehrP interacts with haloacids majorly through van der Waals and halogen bond interactions and has greater affinity for 2,2-dichloropropionate and could be a specialized chloropropionate uptake system. Site-directed mutagenasis of DehrP binding residues could improve its haloacid binding affinity.

How substrate subsites in GH26 endo-mannanase contribute towards mannan binding

Comprehensive knowledge on the role of substrate subsites is a prerequisite to understand the interaction between glycoside hydrolase and its substrate. The present study delineates the role of individual substrate subsites present in ManB-1601 (GH26 endo-mannanase from Bacillus sp.) towards interaction with mannans. Isothermal titration calorimetry of catalytic mutant (E167A/E266A) of ManB-1601 with mannobiose to mannohexose revealed presence of six substrate subsites in ManB-1601. The amino acids present in substrate subsites of ManB-1601 were found to be highly conserved among GH26 endo-mannanases from Bacillus spp. Qualitative substrate binding analysis of subsite mutants by native affinity gel electrophoresis suggested that −3, −2, −1, +1 and + 2 subsites have a major role while, −4 subsite had minor role towards mannan binding. Affinity gels also pointed out the pivotal role of −1 subsite towards glucomannan binding. Quantitative substrate binding analysis using fluorescence titration revealed that −1 and −2 subsite mutants had 27- and 30-fold higher binding affinity (KD) for carob galactomannan when compared with catalytic mutant. The −1 subsite mutant also had highest KD values for glucomannan (13.6-fold) and ivory nut mannan (5-fold) among all mutants. The positive subsites contributed more towards binding with glucomannan (up to 10-fold higher KD) and ivory nut mannan (up to 4.3-fold higher KD) rather than carob galactomannan (up to 4-fold higher KD). Between distal subsites, −3 mutant displayed 10-fold higher KD for both carob galactomannan and glucomannan while, −4 mutant did not show any noticeable change in KD values when compared to catalytic mutant.

NMR analysis of substrate binding to a two-domain chitinase: Comparison between soluble and insoluble chitins

CJP-4 is a two-domain chitinase from Japanese cedar (Cryptomeria japonica) pollen, consisting of an N-terminal CBM18 domain and a GH19 catalytic domain. The substrate binding to an inactive mutant protein of full-length CJP-4, in which the catalytic acid Glu108 was mutated to glutamine, CJP-4(E108Q), was analyzed by NMR spectroscopy. Based on the chemical shift perturbations of 1H-15N HSQC signals of Gly26 (CBM18 domain) and Trp185 (GH19 domain), the association constants for individual domains of CJP-4(E108Q) toward soluble chitin hexamer (GlcNAc)6 were determined to be 2300 and 3500 M−1, respectively. Isothermal titration calorimetry provided a similar association constant for (GlcNAc)6 (1980 M−1) with the one-site binding model. One (GlcNAc)6 molecule appeared to bind to a single binding site of CJP-4(E108Q), spanning from CBM18 to GH19 domains. When chitin nanofibers, insoluble chitinase substrate, were added to the CJP-4(E108Q) solution, strong line-broadening was observed for the majority of the backbone resonances in CBM18 domain but not in GH19 domain, indicating a binding preference of CBM18 domain to the insoluble chitin. We here demonstrated importance of CBM18 domain in insoluble chitin recognition based on the NMR binding data obtained for full-length CJP-4. Chitin nanofibers were found to be useful for spectroscopic observation of insoluble chitin binding to proteins.

Papers of note in Nature 552 (7683)

This week’s articles highlight a potential therapeutic target for fibrosis; parasite receptors that contribute to malaria; a potential drawback of PD-1 inhibition for cancer immunotherapy; a noncoding RNA that regulates ribosome biogenesis; and a structural analysis of substrate binding by the kinase PINK1.

Insights into the structural characteristics and substrate binding analysis of chondroitin AC lyase (PsPL8A) from Pedobacter saltans

The structure of chondroitin AC lyase (PsPL8A) of family 8 polysaccharide lyase was characterized. Modeled PsPL8A structure showed, it contains N-terminal (α/α)6 incomplete toroidal fold and a layered β sandwich structure at C-terminal. Ramchandran plot displayed 98.5% residues in favoured and 1.2% in generously allowed region. Secondary structure of PsPL8A by CD revealed 27.31% α helices 22.7% β sheets and 49.9% random coils. Protein melting study showed, PsPL8A completely unfolds at 60°C. SAXS analysis showed, PsPL8A is fully folded in solution form. The ab initio derived dummy model of PsPL8A superposed well with its modeled structure excluding some α-helices and loop region. Structural superposition and docking analysis showed, N153, W105, H203, Y208, Y212, R266 and E349 were involved in catalysis. Mutants N153A, H203A, Y212F, R266A and E349A created by SDM revealed no residual activity. Isothermal titration calorimetry analysis of Y212F and H203A with C4S polysaccharide, showed moderate binding by Y212F (Ka=9.56±3.81×105) and no binding with H203A, showing active contribution of Y212 in substrate binding. Residues Y212 and H203 or R266 might act as general base and general acid respectively. Residues N153 and E349 are likely contributing in charge neutralization and stabilizing enolate anion intermediate during β-elimination.

Analysis of substrate binding in individual active sites of bifunctional human ATIC

Aminoimidazolecarboxamide ribonucleotide formyl transferase (AICARFT): Inosine monophosphate cyclohydrolase (IMPCH, collectively called ATIC) is a bifunctional enzyme that catalyses the penultimate and final steps in the purine de novo biosynthesis pathway. The bifunctional protein is dimeric and each monomer contains two different active sites both of which are capable of binding nucleotide substrates, this means to a potential total of four distinct binding events might be observed. Within this work we used a combination of site-directed and truncation mutants of ATIC to independently investigate the binding at these two sites using calorimetry. A single S10W mutation is sufficient to block the IMPCH active site allowing investigation of the effects of mutation on ligand binding in the AICARFT active site. The majority of nucleotide ligands bind selectively at one of the two active sites with the exception of xanthosine monophosphate, XMP, which, in addition to binding in both AICARFT and IMPCH active sites, shows evidence for cooperative binding with communication between symmetrically-related active sites in the two IMPCH domains. The AICARFT site is capable of independently binding both nucleotide and folate substrates with high affinity however no evidence for positive cooperativity in binding could be detected using the model ligands employed in this study.

The self-sufficient CYP102 family enzyme, Krac9955, from Ktedonobacter racemifer DSM44963 acts as an alkyl- and alkyloxy-benzoic acid hydroxylase

A self-sufficient CYP102 family encoding gene (Krac_9955) has been identified from the bacterium Ktedonobacter racemifer DSM44963 which belongs to the Chloroflexi phylum. The characterisation of the substrate range of this enzyme was hampered by low levels of production using E. coli. The yield and purity of the Krac9555 enzyme was improved using a codon optimised gene, the introduction of a tag and modification of the purification protocol. The heme domain was isolated and in vitro analysis of substrate binding and turnover was performed. Krac9955 was found to preferentially bind alkyl- and alkyloxy-benzoic acids (≥95% high spin, Kd < 3 μM) over saturated and unsaturated fatty acids. Unusually for a self-sufficient CYP102 family member Krac9955 showed low levels of NAD(P)H oxidation activity for all the substrates tested though product formation was observed for many. For nearly all substrates the preferred site of hydroxylation of Krac9955 was eight carbons away from the carboxylate group with certain reactions proceeding at ≥ 90% selectivity. Krac9955 differs from CYP102A1 (P450Bm3), and is the first self-sufficient member of the CYP102 family of P450 enzymes which is not optimised for fast fatty acid hydroxylation close to the ω-terminus.

Computational evaluation of glutamine synthetase as drug target against infectious diseases: molecular modeling, substrate-binding analysis, and molecular dynamics simulation studies

Glutamine synthetase is an enzyme that catalyzes the condensation of glutamate and ammonia to form glutamine in the presence of adenosine triphosphate. The wealth of structure-related information about glutamine synthetase across the species is endorsing it as emerging potential drug target. Owing to its well-characterized role in metabolism of Mycobacterium, various high throughput screening studies have been aimed at the identification of inhibitors against MtGS. The present work is focussed on comparative sequence and structural studies of glutamine synthetase and its evaluation as drug target against the infectious diseases. We have done molecular modeling studies of glutamine synthetase of Leishmania and Plasmodium. The structure models and molecular dynamics simulations studies shed light on to the binding modes of substrates viz. adenosine diphosphate, glutamate, ammonia, and metal ions. The comparative studies of MtGS, HsGS, LmGS, and PvGS helped in better understanding of prospects of structure-based inhibitor design. The results suggest that amino acid-binding site is highly conserved, whereas nucleotide-binding site possess subtle variations and thus offers opportunity for specific inhibitor design. Therefore, present study suggests that broad spectrum glutamine synthetase inhibition is feasible and it is potential drug target against infectious diseases.

Expression, Purification, and Characterization of a Sucrose Nonfermenting 1-Related Protein Kinases 2 of Arabidopsis thaliana in E. coli-Based Cell-Free System

The plant-specific sucrose nonfermenting 1-related protein kinase 2 (SnRK2) family is considered an important regulator of plant responses to abiotic stresses such as drought, cold, salinity, and nutrition deficiency. However, little information is available on how SnRK2s regulate sulfur deprivation responses in Arabidopsis. Large-scale production of SnRK2 kinases in vitro can help to elucidate the biochemical properties and physiological functions of this protein family. However, heterogenous expression of SnRK2s usually leads to inactive proteins. In this study, we expressed a recombinant Arabidopsis SnRK2.1 in a modified E. coli cell-free system, which combined two kinds of extracts allowing for a convenient and affordable protein preparation. The recombinant SnRK2.1 was produced in large-scale and the autophosphorylation activity of purified SnRK2.1 was characterized, allowing for further biochemical and substrate binding analysis in sulfur signaling. The application of this improved E. coli cell-free system provides us a promising and convenient platform to enhance expression of the target proteins economically.

Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residue Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the “170–179” loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential “hot-spots” for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. This work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications.

Identification of Redox Partners and Development of a Novel Chimeric Bacterial Nitric Oxide Synthase for Structure Activity Analyses

Production of nitric oxide (NO) by nitric oxide synthase (NOS) requires electrons to reduce the heme iron for substrate oxidation. Both FAD and FMN flavin groups mediate the transfer of NADPH derived electrons to NOS. Unlike mammalian NOS that contain both FAD and FMN binding domains within a single polypeptide chain, bacterial NOS is only composed of an oxygenase domain and must rely on separate redox partners for electron transfer and subsequent activity. Here, we report on the native redox partners for Bacillus subtilis NOS (bsNOS) and a novel chimera that promotes bsNOS activity. By identifying and characterizing native redox partners, we were also able to establish a robust enzyme assay for measuring bsNOS activity and inhibition. This assay was used to evaluate a series of established NOS inhibitors. Using the new assay for screening small molecules led to the identification of several potent inhibitors for which bsNOS-inhibitor crystal structures were determined. In addition to characterizing potent bsNOS inhibitors, substrate binding was also analyzed using isothermal titration calorimetry giving the first detailed thermodynamic analysis of substrate binding to NOS.

English

Background: Arabitol dehydrogenase (ArDH) is involved in the production of different sugar alcohols like arabitol, sorbitol, mannitol, erythritol and xylitol by using five carbon sugars as substrate. Arabinose, d-ribose, d-ribulose, xylose and d-xylulose are known substrate of this enzyme. ArDH is mainly produced by osmophilic fungi for the conversion of ribulose to arabitol under stress conditions. Recently this enzyme has been used by various industries for the production of pharmaceutically important sugar alcohols form cheap source than glucose. But the information at structure level as well as its binding energy analysis with different substrates was missing. Results: The present study was focused on sequence analysis, insilico characterization and substrate binding analysis of ArDH from a fungus specie candida albican. Sequence analysis and physicochemical properties showed that this protein is highly stable, negatively charged and having more hydrophilic regions, these properties made this enzyme to bind with number of five carbon sugars as substrate. The predicted 3D model will helpful for further structure based studies. Docking analysis provided free energies of binding of each substrate from a best pose as arabinose -9.8224calK/mol, dribose -11.3701Kcal/mol, d-ribulose -8.9230Kcal/mol, xylose -9.7007Kcal/mol and d-xylulose 9.7802Kcal/mol. Conclusion: Our study provided insight information of structure and interactions of ArDH with its substrate. These results obtained from this study clearly indicate that d-ribose is best substrate for ArDH for the production of sugar alcohols. This information will be helpful for better usage of this enzyme for hyper-production of sugar alcohols by different industries.

Biodegradation of Lignin by Laccase for Conversion of Biomass to Fuel: Analysis of Substrate Binding

Laccases play an important role in biodegradation of lignin, particularly by mediation with easily oxidizable phenolic compounds through a radical-catalyzed reaction. Such processes may potentially be considered for turning lignocellulosic biomass into fuel. In this work, in order to better understand effects of the phenolic compound bulkiness, binding propensity of six substrates that vary in steric hindrance was considered. Changes in binding affinity, also upon mutation, were analyzed for a range of laccases. The results reveal the potential for improved binding of various laccases and their mutations, to be considered in future work.

Biodegradation of Lignin by Laccase for Conversion of Biomass to Fuel: Analysis of Substrate Binding

not Available.

Two novel mutations in CYP11B1 and modeling the consequent alterations of the translated protein in classic congenital adrenal hyperplasia patients

Mutations in the 11β-hydroxylase (CYP11B1) gene are the second leading cause of congenital adrenal hyperplasia (CAH), an autosomal recessive disorder characterized by adrenal insufficiency, virilization of female external genitalia, and hypertension with or without hypokalemic alkalosis. Molecular analysis of CYP11B1 gene in CAH patients with 11β-hydroxylase deficiency was performed in this study. Cycle sequencing of 9 exons in CYP11B1 was performed in 5 unrelated families with 11β-hydroxylase deficient children. Three-dimensional models for the normal and mutant proteins and their affinity to their known substrates were examined. Analysis of the CYP11B1 gene revealed two novel mutations, a small insertion in exon 7 (InsAG393) and a small deletion in exon 2 (DelG766), and three previously known missense mutations (T318M, Q356X, and R427H). According to docking results, the affinity of the protein to its substrates is highly reduced by these novel mutations. DelG766 has more negative impact on the protein in comparison to InsAG393. The novel mutations, InsAG393 and DelG766, change the folding of the protein and disrupt the enzyme's active site as it was measured in the protein modeling and substrate binding analysis. Molecular modeling and sequence conservation were predictive of clinical severity of the disease and correlated with the clinical diagnosis of the patients.

Structure and Dynamics of the ATP-Bound Open Conformation of Hsp70 Chaperones

Central to the chaperone function of Hsp70s is the transition between open and closed conformations of their polypeptide substrate binding domain (SBD), which is regulated through an allosteric mechanism via ATP binding and hydrolysis in their nucleotide binding domain (NBD). Although the structure of the closed conformation of Hsp70s is well studied, the open conformation has remained elusive. Here, we report on the 2.4Å crystal structure of the ATP-bound open conformation of the Escherichia coli Hsp70 homolog DnaK. In the open DnaK structure, the β sheet and α-helical lid subdomains of the SBD are detached from one another and docked to different faces of the NBD. The contacts between the β sheet subdomain and the NBD reveal the mechanism of allosteric regulation. In addition, we demonstrate that docking of the β sheet and α-helical lid subdomains to the NBD is a sequential process influenced by peptide and protein substrates.