Thirty years with three-dimensional structures of lipoxygenases.

The X-ray crystal structures of soybean lipoxygenase (LOX) and rabbit 15-LOX were reported in the 1990s. Subsequent 3D structures demonstrated a conserved U-like shape of the substrate cavities as reviewed here. The 8-LOX:arachidonic acid (AA) complex showed AA bound to the substrate cavity carboxylate-out with C10 at 3.4 Å from the iron metal center. A recent cryo-electron microscopy (EM) analysis of the 12-LOX:AA complex illustrated AA in the same position as in the 8-LOX:AA complex. The 15- and 12-LOX complexes with isoenzyme-specific inhibitors/substrate mimics confirmed the U-fold. 5-LOX oxidizes AA to leukotriene A4, the first step in biosynthesis of mediators of asthma. The X-ray structure showed that the entrance to the substrate cavity was closed to AA by Phe and Tyr residues of a partly unfolded α2-helix. Recent X-ray analysis revealed that soaking with inhibitors shifted the short α2-helix to a long and continuous, which opened the substrate cavity. The α2-helix also adopted two conformations in 15-LOX. 12-LOX dimers consisted of one closed and one open subunit with an elongated α2-helix. 13C-ENDOR-MD computations of the 9-MnLOX:linoleate complex showed carboxylate-out position with C11 placed 3.4 ± 0.1 Å from the catalytic water. 3D structures have provided a solid ground for future research.

Structural and Functional Characterization of OXA-48: Insight into Mechanism and Structural Basis of Substrate Recognition and Specificity.

Class D ß-lactamase OXA-48 is widely distributed among Gram-negative bacteria and is an important determinant of resistance to the last-resort carbapenems. Nevertheless, the detailed mechanism by which this ß-lactamase hydrolyzes its substrates remains poorly understood. In this study, the complex structures of OXA-48 and various ß-lactams were modeled and the potential active site residues that may interact with various ß-lactams were identified and characterized to elucidate their roles in OXA-48 substrate recognition. Four residues, namely S70, K73, S118, and K208 were found to be essential for OXA-48 to undergo catalytic hydrolysis of various penicillins and carbapenems both in vivo and in vitro. T209 was found to be important for hydrolysis of imipenem, whereas R250 played a major role in hydrolyzing ampicillin, imipenem, and meropenem most likely by forming a H-bond or salt-bridge between the side chain of these two residues and the carboxylate oxygen ions of the substrates. Analysis of the effect of substitution of alanine in two residues, W105 and L158, revealed their roles in mediating the activity of OXA-48. Our data show that these residues most likely undergo hydrophobic interaction with the R groups and the core structure of the ß-lactam ring in penicillins and the carbapenems, respectively. Unlike OXA-58, mass spectrometry suggested a loss of the C6-hydroxyethyl group during hydrolysis of meropenem by OXA-48, which has never been demonstrated in Class D carbapenemases. Findings in this study provide comprehensive knowledge of the mechanism of the substrate recognition and catalysis of OXA-type ß-lactamases.

Mechanistic studies on Pyrobaculum calidifontis porphobilinogen synthase (5-aminolevulinic acid dehydratase).

Porphobilinogen synthase (PBG synthase) gene from Pyrobaculum calidifontis was cloned and expressed in E. coli. The recombinant enzyme was purified as an octamer and was found by mass spectrometry to have a subunit Mr of 37676.59 (calculated, 37676.3). The enzyme showed high thermal stability and retained almost all of its activity after incubation at 70 °C for 16 h in the presence of ß-mercaptoethanol (ß-ME) and zinc chloride. However, in the absence of the latter the enzyme was inactivated after 16 h although it regained full activity in the presence of ß-ME and zinc chloride. The protein contained 4 mol of tightly bound zinc per octamer. Further, 4 mol of low affinity zinc could be incorporated following incubation with exogenous zinc salts. The enzyme was inactivated by incubation with levulinic acid followed by treatment with sodium borohydride. Tryptic digest of the modified enzyme and mass spectrometric analysis showed that Lys257 was the site of modification, which has previously been shown to be the site for the binding of 5-aminolevulinic acid giving rise to the propionate-half of porphobilinogen. P. calidifontis PBG synthase was inactivated by 5-chlorolevulinic acid and the residue modified was shown to be the central cysteine (Cys127) of the zinc-binding cysteine-triad, comprising Cys125, 127, 135. The present results in conjunction with earlier findings on zinc containing PBG synthases, are discussed which advocate that the catalytic role of zinc in the activation of the 5-aminolevulinic acid molecule forming the acetate-half of PBG is possible.

Evaluating the catalytic importance of a conserved Glu97 residue in triosephosphate isomerase.

Investigating enzyme activity is central to our understanding of biological function, and the design of biocatalysts continues to find applications in synthesis. While a role for active site residues can be proposed based on structure and mechanism, our understanding of the catalytic importance for residues surrounding the active site is less well understood. In triosephosphate isomerase (TIM), Glu97 is situated adjacent to the active site and is found in essentially all sequences. Prior studies reported mutation of Glu97 to Asp and Gln in TIM from Plasmodium falciparum (PfTIM) led to a 100- and 4000-fold decrease in activity, respectively, while the E97D mutation in TIM from Gallus gallus (cTIM) had no effect on activity. To investigate further the question of how mutations in essentially superimposable structures give different effects, we mutated E97 in TIM from Trypanosoma brucei brucei (TbbTIM), Saccharomyces cerevisiae (yTIM), and human (hTIM). The E97D, E97A, and E97Q mutations led to a ∼three-tenfold decrease in activity, a modest effect compared to the 102-103-fold effect in PfTIM. CD and fluorescence studies showed the overall structures for the mutants were essentially unchanged. Structural analysis shows that several residues surrounding E97 differ between PfTIM and TIM from the other organisms, and rearrangements or mispositioning of residues in PfTIM may lead to the different rate effects. The results illustrate the interplay of active site and surrounding residues in affecting catalysis and highlight that understanding of the role of residues surrounding the active site may aid in the incorporation of favorable or avoidance of unfavorable interactions when designing enzymes.

CSmetaPred: a consensus method for prediction of catalytic residues.

BACKGROUND: Knowledge of catalytic residues can play an essential role in elucidating mechanistic details of an enzyme. However, experimental identification of catalytic residues is a tedious and time-consuming task, which can be expedited by computational predictions. Despite significant development in active-site prediction methods, one of the remaining issues is ranked positions of putative catalytic residues among all ranked residues. In order to improve ranking of catalytic residues and their prediction accuracy, we have developed a meta-approach based method CSmetaPred. In this approach, residues are ranked based on the mean of normalized residue scores derived from four well-known catalytic residue predictors. The mean residue score of CSmetaPred is combined with predicted pocket information to improve prediction performance in meta-predictor, CSmetaPred_poc. RESULTS: Both meta-predictors are evaluated on two comprehensive benchmark datasets and three legacy datasets using Receiver Operating Characteristic (ROC) and Precision Recall (PR) curves. The visual and quantitative analysis of ROC and PR curves shows that meta-predictors outperform their constituent methods and CSmetaPred_poc is the best of evaluated methods. For instance, on CSAMAC dataset CSmetaPred_poc (CSmetaPred) achieves highest Mean Average Specificity (MAS), a scalar measure for ROC curve, of 0.97 (0.96). Importantly, median predicted rank of catalytic residues is the lowest (best) for CSmetaPred_poc. Considering residues ranked ≤20 classified as true positive in binary classification, CSmetaPred_poc achieves prediction accuracy of 0.94 on CSAMAC dataset. Moreover, on the same dataset CSmetaPred_poc predicts all catalytic residues within top 20 ranks for ~73% of enzymes. Furthermore, benchmarking of prediction on comparative modelled structures showed that models result in better prediction than only sequence based predictions. These analyses suggest that CSmetaPred_poc is able to rank putative catalytic residues at lower (better) ranked positions, which can facilitate and expedite their experimental characterization. CONCLUSIONS: The benchmarking studies showed that employing meta-approach in combining residue-level scores derived from well-known catalytic residue predictors can improve prediction accuracy as well as provide improved ranked positions of known catalytic residues. Hence, such predictions can assist experimentalist to prioritize residues for mutational studies in their efforts to characterize catalytic residues. Both meta-predictors are available as webserver at: http://14.139.227.206/csmetapred/ .

In Silico Discovery of Potential Uridine-Cytidine Kinase 2 Inhibitors from the Rhizome of Alpinia mutica.

Uridine-cytidine kinase 2 is implicated in uncontrolled proliferation of abnormal cells and it is a hallmark of cancer, therefore, there is need for effective inhibitors of this key enzyme. In this study, we employed the used of in silico studies to find effective UCK2 inhibitors of natural origin using bioinformatics tools. An in vitro kinase assay was established by measuring the amount of ADP production in the presence of ATP and 5-fluorouridine as a substrate. Molecular docking studies revealed an interesting ligand interaction with the UCK2 protein for both flavokawain B and alpinetin. Both compounds were found to reduce ADP production, possibly by inhibiting UCK2 activity in vitro. In conclusion, we have identified flavokawain B and alpinetin as potential natural UCK2 inhibitors as determined by their interactions with UCK2 protein using in silico molecular docking studies. This can provide information to identify lead candidates for further drug design and development.

Roles of conserved active site residues in the IscS cysteine desulfurase reaction.

Escherichia coli cysteine desulfurase (CD), IscS, modifies basal metabolism by transferring sulphur (S) from L-cysteine to numerous cellular pathways, whereas NFS1, a human CD, is active only in the formation of the [Acp]2:[ISD11]2:[NFS1]2 complex. Despite the accumulation of red-coloured IscS in E. coli cells as a result of the deficiency of accessible iron, as revealed in our previous studies, the mechanism of the potential enzymatic reaction remains unclear. In this study, the N-terminus of IscS was fused with the C-terminus of NFS1, which was reported to be almost fully active as IscS and exhibits a pyridoxal 5'-phosphate (PLP) absorption peak at 395 nm. Moreover, SUMO-EH-IscS exhibited significant growth recovery and NADH-dehydrogenase I activity in the iscS mutant cells. Furthermore, through in vitro and in vivo experiments combined with high-performance liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry, it was shown that the new absorption peaks of the IscS H104Q, IscS Q183E, IscS K206A, and IscS K206A&C328S variants at 340 and 350 nm may correspond to the enzyme reaction intermediates, Cys-ketimine and Cys-aldimine, respectively. However, after mutation of the conserved active-site residues, additional absorption peaks at 420 and 430 nm were associated with PLP migration in the active-site pocket. Additionally, the corresponding absorption peaks of Cys-quinonoid, Ala-ketimine, and Ala-aldimine intermediates in IscS were 510, 325, and 345 nm, respectively, as determined by site-directed mutagenesis and substrate/product-binding analyses during the CD reaction process. Notably, red IscS formed in vitro by incubating IscS variants (Q183E and K206A) with excess L-alanine and sulphide under aerobic conditions produced an absorption peak similar to the wild-type IscS, at 510 nm. Interestingly, site-directed mutation of IscS with hydrogen bonds to PLP at Asp180 and Gln183 resulted in a loss of enzymatic activity followed by an absorption peak consistent with NFS1 (420 nm). Furthermore, mutations at Asp180 or Lys206 inhibited the reaction of IscS in vitro with L-cysteine (substrate) and L-alanine (product). These results suggest that the conserved active site residues (His104, Asp180, and Gln183) and their hydrogen bond with PLP in the N-terminus of IscS play a key role in determining whether the L-cysteine substrate can enter the active-site pocket and regulate the enzymatic reaction process. Therefore, our findings provide a framework for evaluating the roles of conserved active-site residues, motifs, and domains in CDs.

Erratum: Cytoplasmic Lipases-A Novel Class of Fungal Defense Proteins Against Nematodes.

[This corrects the article DOI: 10.3389/ffunb.2021.696972.].

Cytoplasmic Lipases-A Novel Class of Fungal Defense Proteins Against Nematodes.

Fungi are an attractive food source for predators such as fungivorous nematodes. Several fungal defense proteins and their protective mechanisms against nematodes have been described. Many of these proteins are lectins which are stored in the cytoplasm of the fungal cells and bind to specific glycan epitopes in the digestive tract of the nematode upon ingestion. Here, we studied two novel nematotoxic proteins with lipase domains from the model mushroom Coprinopsis cinerea. These cytoplasmically localized proteins were found to be induced in the vegetative mycelium of C. cinerea upon challenge with fungivorous nematode Aphelenchus avenae. The proteins showed nematotoxicity when heterologously expressed in E. coli and fed to several bacterivorous nematodes. Site-specific mutagenesis of predicted catalytic residues eliminated the in-vitro lipase activity of the proteins and significantly reduced their nematotoxicity, indicating the importance of the lipase activity for the nematotoxicity of these proteins. Our results suggest that cytoplasmic lipases constitute a novel class of fungal defense proteins against predatory nematodes. These findings improve our understanding of fungal defense mechanisms against predators and may find applications in the control of parasitic nematodes in agriculture and medicine.

Glutathione S-transferase: a versatile protein family.

Glutathione-S transferase (GST) is a most ancient protein superfamily of multipurpose roles and evolved principally from gene duplication of an ancestral GSH binding protein. They have implemented in diverse plant functions such as detoxification of xenobiotic, secondary metabolism, growth and development, and majorly against biotic and abiotic stresses. The vital structural features of GSTs like highly divergent functional topographies, conserved integrated architecture with separate binding pockets for substrates and ligand, the stringent structural fidelity with high Tm values (50º-60º), and stress-responsive cis-regulatory elements in the promoter region offer this protein as most flexible plant protein for plant breeding approaches, biotechnological applications, etc. This review article summarizes the recent information of GST evolution, and their distribution and structural features with emphasis on the assorted roles of Ser and Cys GSTs with the signature motifs in their active sites, alongside their recent biotechnological application in the area of agriculture, environment, and nanotechnology have been highlighted.

Identification, Biological Characteristics, and Active Site Residues of 3-Ketosteroid Δ1-Dehydrogenase Homologues from Arthrobacter simplex.

3-Ketosteroid Δ1-dehydrogenase (KsdD) is the key enzyme responsible for Δ1-dehydrogenation, which is one of the most valuable reactions for steroid catabolism. Arthrobacter simplex has been widely used in the industry due to its superior bioconversion efficiency, but KsdD information is not yet fully clear. Here, five KsdD homologues were identified in A. simplex CGMCC 14539. Bioinformatic analysis indicated their distinct properties and structures. Each KsdD was functionally confirmed by transcriptional response, overexpression, and heterologous expression. The substantial difference in substrate profiles might be related to the enzyme loop structure. Two promising enzymes (KsdD3 and KsdD5) were purified and characterized, exhibiting strong organic solvent tolerance and clear preference for 4-ene-3-oxosteroids. KsdD5 seemed to be more versatile due to good activity on substrates with or without a substituent at C11 and high optimal temperature and also possessed unique residues. It is the first time that KsdDs have been comprehensively disclosed in the A. simplex industrial strain.

Sequence- and structure-based analysis of proteins involved in miRNA biogenesis.

miRNA biogenesis is a multistage process for the generation of a mature miRNA and involves several different proteins. In this work, we have carried out both sequence- and structure-based analysis for crucial proteins involved in miRNA biogenesis, namely Dicer, Drosha, Argonaute (Ago), and Exportin-5 to understand evolution of these proteins in animal kingdom and also to identify key sequence and structural features that are determinants of their function. Our analysis reveals that in animals the miRNA biogenesis pathway first originated in molluscs. The phylogeny of Dicer and Ago indicated evolution through gene duplication followed by sequence divergence that resulted in functional divergence. Our detailed structural analysis also revealed that RIIIDb domains of Drosha and Dicer, share significant similarity in sequence, structure, and substrate-binding pocket. On the other hand, PAZ domains of Dicer and Ago show only conservation of the substrate-binding pockets in the catalytic sites despite significant divergence in sequence and overall structure. Based on a comparative structural analysis of all four human Ago proteins (hAgo1-4) and their known biochemical activity, we have also attempted to identify key residues in Ago2 which are responsible for the unique slicer activity of hAgo2 among all isoforms. We have identified six key residues in N domain of hAgo2, which are located far away from the catalytic pocket, but might be playing a major role in slicer activity of hAgo2 protein because of their involvement in mRNA binding.

Physiochemical and Thermodynamic Characterization of Highly Active Mutated Aspergillus niger ß-glucosidase for Lignocellulose Hydrolysis.

BACKGROUND: Cellulose represents a major source of fermentable sugars in lignocellulosic biomass and a combined action of hydrolytic enzymes (exoglucanases , endoglucanases and ß-glucosidases) is required to effectively convert cellulose to glucose that can be fermented to bio-ethanol. However, in-order to make the production of bio-ethanol an economically feasible process, the costs of the enzymes to be used for hydrolysis of the raw material need to be reduced and an increase in specific activity or production efficiency of cellulases is required. Among the cellulases, ß-glucosidase not only hydrolyzes cellobiose to glucose but it also reduces the cellobiose inhibition, resulting in efficient functioning of endo- and exo-glucanases. Therefore, in the current study kinetic and thermodynamic characteristics of highly active ß-glucosidase from randomly mutated Aspergillus niger NIBGE-06 have been evaluated for its industrial applications. OBJECTIVE: The main objective of this study was the identification of mutations and determination of their effect on the physiochemical, kinetic and thermodynamic characteristics of ß-glucosidase activity and stability. METHODS: Pure cultures of Aspergillus niger NIBGE and its 2-Deoxy-D-glucose resistant γ-rays mutant Aspergillus niger NIBGE-06 were grown on Vogel's medium containing wheat bran (3% w/v), at 30±1 °C for 96-108 h. Crude enzymes from both strains were subjected to ammonium sulfate precipitation and column chromatography on Fast Protein Liquid Chromatography (FPLC) system. The purified ß-glucosidases from both fungal sources were characterized for their native and subunit molecular mass through FPLC and SDS-PAGE, respectively. The purified enzymes were then comparatively characterized for their optimum temperature, activation energy (Ea), temperature quotient (Q10), Optimum pH, Heat of ionization (ΔHI) of active site residues , Michaelis-Menten constants (Vmax, Km, kcat and kcat/Km) and thermodynamics of irreversible inactivation through various enzyme assays. The genomic DNA from both fungal strains was also extracted by SDS-method and full length ß- glucosidase genes (bgl) were amplified through PCR. The PCR products were cloned in TA cloning vector followed by the sequencing of potentially full length clones using the commercial services of Macrogen, Korea. The in silico analyses of the sequences thus obtained were also performed using various online tools such as blastn, blastp, GeneWise, SignalP, Inter- ProScan. RESULTS: The extracellular ß-glucosidases (BGL) from both fungal sources were purified to homogeneity level by ammonium sulfate precipitation and FPLC system. The BGLs from both strains were dimeric in nature, with subunit and native molecular masses of 130 kDa and 252 kDa, respectively. The comparative analysis of nucleotides of bgl genes revealed 8 point mutations. Significant improvement was observed in the kinetic properties of the mutant BGL relative to the wild type enzyme. Arrhenius plot for energy of activation (Ea) showed a biphasic trend and ES-complex formation required Ea of 50 and 42 kJ mol-1 by BGL from parent and mutant, respectively. The pKa1 and pKa2 of the active site residues were 3.4 & 5.5 and 3.2 & 5.6, respectively. The heat of ionization for the acidic limb (ΔHI-AL) and the basic limb (ΔHI-BL) of BGL from both strains were equal to 56 & 41 and 71 & 45 kJ mol-1, respectively. Kinetic constants of cellobiose hydrolysis for BGL from both strains were determined as follows: kcat = 2,589 and 4,135 s-1, Km = 0.24 and 0.26 mM cellobiose, kcat/Km = 10,872 and 15,712 s-1 mM-1 cellobiose, respectively. Thermodynamic parameters for cellobiose hydrolysis also suggested that mutant BGL is more efficient compared to the parent enzyme. Comparative analysis of Ea(d), ΔH* and ΔG* for irreversible thermostability indicated that the thermostabilization of mutant enzyme was due to higher functional energy (free energy), which enabled the enzyme to resist against unfolding of its transition state. CONCLUSION: Physiochemical and thermodynamic characterization of extracellular ß-glucosidases (BGL) from 2-Deoxy-Dglucose resistant mutant derivative of A. niger showed that mutagenesis did not greatly affect the physiochemical properties of the BGL enzyme, like temperature optima, pH optima and molecular mass, while the catalytic efficiency for cellobiose hydrolysis was significantly improved (High kcat and kcat/Km). Furthermore, the mutant BGL was more thermostable than the parent enzyme. This shows that random mutagenesis has changed the BGL structural gene, resulting in improvement within its stability- function characteristics. Hence, directed evolution or random mutagenesis with careful selection can result in the engineering of highly efficient enzymes for intended industrial applications.

Homology modeling and assigned functional annotation of an uncharacterized antitoxin protein from Streptomyces xinghaiensis.

Streptomyces xinghaiensis is a Gram-positive, aerobic and non-motile bacterium. The bacterial genome is known. Therefore, it is of interest to study the uncharacterized proteins in the genome. An uncharacterized protein (gi|518540893|86 residues) in the genome was selected for a comprehensive computational sequence-structure-function analysis using available data and tools. Subcellular localization of the targeted protein with conserved residues and assigned secondary structures is documented. Sequence homology search against the protein data bank (PDB) and non-redundant GenBank proteins using BLASTp showed different homologous proteins with known antitoxin function. A homology model of the target protein was developed using a known template (PDB ID: 3CTO:A) with 62% sequence similarity in HHpred after assessment using programs PROCHECK and QMEAN6. The predicted active site using CASTp is analyzed for assigned anti-toxin function. This information finds specific utility in annotating the said uncharacterized protein in the bacterial genome.