Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) bifunctional enzyme from Candidatus Liberibacer asiaticus.

The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 µM and Vmax, 0.95 µmol/min/mg) and AICAR (Km, 34.81 µM and Vmax, 0.56 µmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 µM and Vmax, 2.87 µmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 µM and 34.2 µM, respectively) compared to AICAR (Kd, 83.4 µM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.

Characterization of Type1 Lipid Transfer Protein from Citrus sinensis: Unraveling its potential as an antimicrobial and insecticidal agent.

Lipid Transfer Protein1 (LTP1) is a cationic, multifaceted protein belonging to the pathogenesis-related protein (PR14) family. Despite being involved in diverse physiological processes and defense mechanisms, the precise in-vivo role of LTP1 remains undiscovered. This work presents the characterization of recombinant Citrus sinensis LTP1 (CsLTP1) along with lipid binding studies through in-silico and in-vitro approaches. CsLTP1 demonstrated great thermal and pH stability with a huge biotechnological potential. It showed in-vitro binding capacity with jasmonic acid and lipids involved in regulating plant immune responses. Gene expression profiling indicated a significant upregulation of CsLTP1 in Candidatus-infected Citrus plants. CsLTP1 disrupted the cell membrane integrity of various pathogens, making it a potent antimicrobial agent. Further, in-vivo antimicrobial and insecticidal properties of CsLTP1 have been explored. The impact of exogenous CsLTP1 treatment on rice crop metabolism for managing blight disease has been studied using GC-MS. CsLTP1 triggered crucial metabolic pathways in rice plants while controlling the blight disease. CsLTP1 effectively inhibited Helicoverpa armigera larvae by impeding mid-gut α-amylase activity and obstructing its developmental stages. This study highlights the pivotal role of CsLTP1 in plant defense by offering insights for developing multi-target therapeutic agent or disease-resistant varieties to comprehensively tackle the challenges towards crop protection.

Characterization of haloacid dehalogenase superfamily acid phosphatase from Staphylococcus lugdunensis.

The haloacid dehalogenase superfamily implicated in bacterial pathogenesis comprises different enzymes having roles in many metabolic pathways. Staphylococcus lugdunensis, a Gram-positive bacterium, is an opportunistic human pathogen causing infections in the central nervous system, urinary tract, bones, peritoneum, systemic conditions and cutaneous infection. The haloacid dehalogenase superfamily proteins play a significant role in the pathogenicity of certain bacteria, facilitating invasion, survival, and proliferation within host cells. The genome of S. lugdunensis encodes more than ten proteins belonging to this superfamily. However, none of them have been characterized. The present work reports the characterization of one of the haloacid dehalogenase superfamily proteins (SLHAD1) from Staphylococcus lugdunensis. The functional analysis revealed that SLHAD1 is a metal-dependent acid phosphatase, which catalyzes the dephosphorylation of phosphorylated metabolites of cellular pathways, including glycolysis, gluconeogenesis, nucleotides, and thiamine metabolism. Based on the substrate specificity and genomic analysis, the physiological function of SLHAD1 in thiamine metabolism has been tentatively assigned. The crystal structure of SLHAD1, lacking 49 residues at the C-terminal, was determined at 1.7 Å resolution with a homodimer in the asymmetric unit. It was observed that SLHAD1 exhibited time-dependent cleavage at a specific point, occurring through a self-initiated process. A combination of bioinformatics, biochemical, biophysical, and structural studies explored unique features of SLHAD1. Overall, the study revealed a detailed characterization of a critical enzyme of the human pathogen Staphylococcus lugdunensis, associated with several life-threatening infections.

Unraveling antiviral efficacy of multifunctional immunomodulatory triterpenoids against SARS-COV-2 targeting main protease and papain-like protease.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be over, but its variants continue to emerge, and patients with mild symptoms having long COVID is still under investigation. SARS-CoV-2 infection leading to elevated cytokine levels and suppressed immune responses set off cytokine storm, fatal systemic inflammation, tissue damage, and multi-organ failure. Thus, drug molecules targeting the SARS-CoV-2 virus-specific proteins or capable of suppressing the host inflammatory responses to viral infection would provide an effective antiviral therapy against emerging variants of concern. Evolutionarily conserved papain-like protease (PLpro) and main protease (Mpro) play an indispensable role in the virus life cycle and immune evasion. Direct-acting antivirals targeting both these viral proteases represent an attractive antiviral strategy that is also expected to reduce viral inflammation. The present study has evaluated the antiviral and anti-inflammatory potential of natural triterpenoids: azadirachtin, withanolide_A, and isoginkgetin. These molecules inhibit the Mpro and PLpro proteolytic activities with half-maximal inhibitory concentrations (IC50 ) values ranging from 1.42 to 32.7 µM. Isothermal titration calorimetry (ITC) analysis validated the binding of these compounds to Mpro and PLpro. As expected, the two compounds, withanolide_A and azadirachtin, exhibit potent anti-SARS-CoV-2 activity in cell-based assays, with half-maximum effective concentration (EC50 ) values of 21.73 and 31.19 µM, respectively. The anti-inflammatory roles of azadirachtin and withanolide_A when assessed using HEK293T cells, were found to significantly reduce the levels of CXCL10, TNFα, IL6, and IL8 cytokines, which are elevated in severe cases of COVID-19. Interestingly, azadirachtin and withanolide_A were also found to rescue the decreased type-I interferon response (IFN-α1). The results of this study clearly highlight the role of triterpenoids as effective antiviral molecules that target SARS-CoV-2-specific enzymes and also host immune pathways involved in virus-mediated inflammation.

Mechanistic and thermodynamic characterization of antiviral inhibitors targeting nucleocapsid N-terminal domain of SARS-CoV-2.

The nucleocapsid (N) protein of SARS-CoV-2 plays a pivotal role in encapsulating the viral genome. Developing antiviral treatments for SARS-CoV-2 is imperative due to the diminishing immunity of the available vaccines. This study targets the RNA-binding site located in the N-terminal domain (NTD) of the N-protein to identify the potential antiviral molecules against SARS-CoV-2. A structure-based repurposing approach identified the twelve high-affinity molecules from FDA-approved drugs, natural products, and the LOPAC1280 compound libraries that precisely bind to the RNA binding site within the NTD. The interaction of these potential antiviral agents with the purified NTD protein was thermodynamically characterized using isothermal titration calorimetry (ITC). A fluorescence-based plate assay to assess the RNA binding inhibitory activity of small molecules against the NTD has been employed, and the selected compounds exhibited significant RNA binding inhibition with calculated IC50 values ranging from 8.8 µM to 15.7 µM. Furthermore, the antiviral efficacy of these compounds was evaluated using in vitro cell-based assays targeting the replication of SARS-CoV-2. Remarkably, two compounds, Telmisartan and BMS-189453, displayed potential antiviral activity against SARS-CoV-2, with EC50 values of approximately 1.02 µM and 0.98 µM, and a notable selective index of >98 and > 102, respectively. This study gives valuable insight into developing therapeutic interventions against SARS-CoV-2 by targeting the N-protein, a significant effort given the global public health concern posed due to the virus re-emergence and long COVID-19 disease.

Sulisobenzone is a potent inhibitor of the global transcription factor Cra.

Transcription is carried out by the RNA polymerase and is regulated through a series of interactions with transcription factors. Catabolite activator repressor (Cra), a LacI family transcription factor regulates the virulence gene expression in Enterohaemorrhagic Escherichia coli (EHEC) and thus is a promising drug target for the discovery of antivirulence molecules. Here, we report the crystal structure of the effector molecule binding domain of Cra from E. coli (EcCra) in complex with HEPES molecule. Based on the EcCra-HEPES complex structure, ligand screening was performed that identified sulisobenzone as an potential inhibitor of EcCra. The electrophoretic mobility shift assay (EMSA) and in vitro transcription assay validated the sulisobenzone binding to EcCra. Moreover, the isothermal titration calorimetry (ITC) experiments demonstrated a 40-fold higher binding affinity of sulisobenzone (KD 360 nM) compared to the HEPES molecule. Finally, the sulisobenzone bound EcCra complex crystal structure was determined to elucidate the binding mechanism of sulisobenzone to the effector binding pocket of EcCra. Together, this study suggests that sulisobenzone may be a promising candidate that can be studied and developed as an effective antivirulence agent against EHEC.

Identification and evaluation of potential inhibitor molecules against TcyA from Candidatus Liberibacter asiaticus.

Of the two putative amino acid binding periplasmic receptors of ABC transporter family in Candidatus Liberibacter asiaticus (CLas), cystine binding receptor (CLasTcyA) has been shown to mainly express in phloem of citrus plant and is a target for inhibitor development. The crystal structure of CLasTcyA in complex with substrates has been reported earlier. The present work reports the identification and evaluation of potential candidates for their inhibitory potential against CLasTcyA. Among many compounds, selected through virtual screening, and MD simulation, pimozide, clidinium, sulfasalazine and folic acid showed significantly higher affinities and stability in complex with CLasTcyA. The SPR studies with CLasTcyA revealed significantly higher binding affinities for pimozide and clidinium (Kd, 2.73 nM and 70 nM, respectively) as compared to cystine (Kd, 1.26 µM). The higher binding affinities could be attributed to significantly increased number of interactions in the binding pocket as evident from the crystal structures of CLasTcyA in complex with pimozide and clidinium as compared to cystine. The CLasTcyA possess relatively large binding pocket where bulkier inhibitors fit quite well. In planta studies, carried out to assess the effect of inhibitors on HLB infected Mosambi plants, showed significant reduction in CLas titre in plants treated with inhibitors as compared to control plants. The results showed that pimozide exhibited higher efficiency as compared to clidinium in reducing CLas titre in treated plants. Our results showed that the inhibitor development against critical proteins like CLasTcyA can be an important strategy in management of HLB.

Biochemical characterization and structure-based in silico screening of potent inhibitor molecules against the 1 cys peroxiredoxin of bacterioferritin comigratory protein family from Candidatus Liberibacter asiaticus.

Bacterioferritin comigratory protein family 1 Cys peroxiredoxin from Candidatus Liberibacter asiaticus (CLaBCP) is an important antioxidant defense protein that participates in the reduction of ROS, free radicals, and peroxides. In the present study, we report the biochemical studies and in silico screening of potent antibacterial molecules against CLaBCP. The CLaBCP showed enzymatic activity with the Km value 54.43, 94.34, 120.6 µM, and Vmax of 59.37, 69.37, 70.0 µM min-1 for H2O2, TBHP, CHP respectively. The residual peroxidase activity of CLaBCP was analyzed at different ranges of pH and temperatures. The CLaBCP showed structural changes and unfolding in the presence of its substrates and guanidinium chloride by CD and fluorescence. The structure-based drug design method was employed to screen and identify the more efficient molecule against CLaBCP. The validated CLaBCP model was used for the virtual screening of potent antibacterial molecules. The docking was performed at CLaBCP active site to evaluate the binding energy of the top five molecules (LAS 34150849, BDE 33184869, LAS 51497689, BDE 33672484, and LAS 34150966). All identified molecule has a higher binding affinity than adenanthin analyzed by molecular docking. Molecular dynamics studies such as RMSD, Rg, SASA, and PCA results showed that the CLaBCP inhibitor(s) complex is more stable than the CLaBCP-adenanthin complex. MMPBSA results suggested that the identified molecule could form a lower energy CLaBCP-inhibiter(s) complex than the CLaBCP-adenanthin complex. The screened molecules may pave the route for the development of potent antibacterial molecules against CLa.Communicated by Ramaswamy H. Sarma.

In silico identification of potential phytochemical inhibitors targeting farnesyl diphosphate synthase of cotton bollworm (Helicoverpa armigera).

Helicoverpa armigera (Ha), a polyphagous pest, causes significant damage to several crop plants, including cotton. The control of this cosmopolitan pest is largely challenging due to the development of resistance to existing management practices. The Juvenile Hormone (JH) plays a pivotal role in the life cycle of insects by regulating their morphogenetic and gonadotropic development. Hence, enzymes involved in JH biosynthesis are an attractive target for the development of selective insecticides. Farnesyl diphosphate synthase (FPPS), a member protein of (E)-prenyl-transferases, is one of the most crucial enzymes in the biosynthetic pathway of JHs. It catalyzes the condensation of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP), forming farnesyl diphosphate (FPP), a precursor of JH. The study was designed to identify an effective small inhibitory molecule that could inhibit the activity of Helicoverpa armigera - FPPS (HaFPPS) for an effective pest control intervention. Therefore, a 3D model of FPPS protein was generated using homology modeling. The FooDB database library of small molecules was selected for virtual screening, following which binding affinities were evaluated using docking studies. Three top-scored molecules were analyzed for various pharmacophore properties. Further, molecular dynamics (MD) simulation analysis showed that the identified molecules (mitraphylline-ZINC1607834, chlorogenic acid-ZINC2138728 and llagate-ZINC3872446) had a reasonably acceptable binding affinity for HaFPPS and resulted in the formation of a stable HaFPPS-inhibitor(s) complex. The identified phytochemical molecules may be used as potent inhibitors of HaFPPS thus, paving the way for further developing environment-friendly insect growth regulator(s). Communicated by Ramaswamy H. Sarma.

Biophysical and modeling-based approach for the identification of inhibitors against DOHH from Leishmania donovani.

The amino acid hypusine (Nε-4-amino-2-hydroxybutyl(lysine)) occurs only in isoforms of eukaryotic translation factor 5A (eIF5A) and has a role in initiating protein translation. Hypusinated eIF5A promotes translation and modulates mitochondrial function and oxygen consumption rates. The hypusination of eIF5A involves two enzymes, deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH is the second enzyme that completes the synthesis of hypusine and the maturation of eIF5A. Our current study aims to identify inhibitors against DOHH from Leishmania donovani (LdDOHH), an intracellular protozoan parasite causing Leishmaniasis in humans. The LdDOHH protein was produced heterologously in Escherichia coli BL21(DE3) cells and characterized biochemically. The three-dimensional structure was predicted, and the compounds folic acid, scutellarin and homoarbutin were selected as top hits in virtual screening. These compounds were observed to bind in the active site of LdDOHH stabilizing the structure by making hydrogen bonds in the active site, as observed by the docking and molecular dynamics simulation studies. These results pave the path for further investigation of these molecules for their anti-leishmanial activities.

Structural insights at acidic pH of dye-decolorizing peroxidase from Bacillus subtilis.

Dye-decolorizing peroxidases (DyPs), a type of heme-containing oxidoreductase enzymes, catalyze the peroxide-dependent oxidation of various industrial dyes as well as lignin and lignin model compounds. In our previous work, we have recently reported the crystal structures of class A-type DyP from Bacillus subtilis at pH 7.0 (BsDyP7), exposing the location of three binding sites for small substrates and high redox-potential substrates. The biochemical studies revealed the optimum acidic pH for enzyme activity. In the present study, the crystal structure of BsDyP at acidic pH (BsDyP4) reveals two-monomer units stabilized by intermolecular salt bridges and a hydrogen bond network in a homo-dimeric unit. Based on the monomeric structural comparison of BsDyP4 and BsDyP7, minor differences were observed in the loop regions, that is, LI (Ala64-Gln71), LII (Glu96-Lys108), LIII (Pro117-Leu124), and LIV (Leu295-Asp303). Despite these differences, BsDyP4 adopts similar heme architecture as well as three substrate-binding sites to BsDyP7. In BsDyP4, a shift in Asp187, heme pocket residue discloses the plausible reason for optimal acidic pH for BsDyP activity. This study provides insight into the structural changes in BsDyP at acidic pH, where BsDyP is biologically active.

Drug-Repurposing Approach To Combat Staphylococcus aureus: Biomolecular and Binding Interaction Study.

Staphylococcus aureus is considered as one of the most widespread bacterial pathogens and continues to be a prevalent cause of mortality and morbidity across the globe. FmtA is a key factor linked with methicillin resistance in S. aureus. Consequently, new antibacterial compounds are crucial to combat S. aureus resistance. Here, we present the virtual screening of a set of compounds against the available crystal structure of FmtA. The findings indicate that gemifloxacin, paromomycin, streptomycin, and tobramycin were the top-ranked potential drug molecules based on the binding affinity. Furthermore, these drug molecules were analyzed with molecular dynamics simulations, which showed that the identified molecules formed highly stable FmtA-inhibitor(s) complexes. Molecular mechanics Poisson-Boltzmann surface area and quantum mechanics/molecular mechanics calculations suggested that the active site residues (Ser127, Lys130, Tyr211, and Asp213) of FmtA are crucial for the interaction with the inhibitor(s) to form stable protein-inhibitor(s) complexes. Moreover, fluorescence- and isothermal calorimetry-based binding studies showed that all the molecules possess dissociation constant values in the micromolar scale, revealing a strong binding affinity with FmtAΔ80, leading to stable protein-drug(s) complexes. The findings of this study present potential beginning points for the rational development of advanced, safe, and efficacious antibacterial agents targeting FmtA.

Multi-target direct-acting SARS-CoV-2 antivirals against the nucleotide-binding pockets of virus-specific proteins.

The nucleotide-binding pockets (NBPs) in virus-specific proteins have proven to be the most successful antiviral targets for several viral diseases. Functionally important NBPs are found in various structural and non-structural proteins of SARS-CoV-2. In this study, the first successful multi-targeting attempt to identify effective antivirals has been made against NBPs in nsp12, nsp13, nsp14, nsp15, nsp16, and nucleocapsid (N) proteins of SARS-CoV-2. A structure-based drug repurposing in silico screening approach with ADME analysis identified small molecules targeting NBPs in SARS-CoV-2 proteins. Further, isothermal titration calorimetry (ITC) experiments validated the binding of top hit molecules to the purified N-protein. Importantly, cell-based antiviral assays revealed antiviral potency for INCB28060, darglitazone, and columbianadin with EC50 values 15.71 µM, 5.36 µM, and 22.52 µM, respectively. These effective antivirals targeting multiple proteins are envisioned to direct the development of antiviral therapy against SARS-CoV-2 and its emerging variants.

RNA splicing: a dual-edged sword for hepatocellular carcinoma.

RNA splicing is the fundamental process that brings diversity at the transcriptome and proteome levels. The spliceosome complex regulates minor and major processes of RNA splicing. Aberrant regulation is often associated with different diseases, including diabetes, stroke, hypertension, and cancer. In the majority of cancers, dysregulated alternative RNA splicing (ARS) events directly affect tumor progression, invasiveness, and often lead to poor survival of the patients. Alike the rest of the gastrointestinal malignancies, in hepatocellular carcinoma (HCC), which alone contributes to ~ 75% of the liver cancers, a large number of ARS events have been observed, including intron retention, exon skipping, presence of alternative 3'-splice site (3'SS), and alternative 5'-splice site (5'SS). These events are reported in spliceosome and non-spliceosome complexes genes. Molecules such as MCL1, Bcl-X, and BCL2 in different isoforms can behave as anti-apoptotic or pro-apoptotic, making the spliceosome complex a dual-edged sword. The anti-apoptotic isoforms of such molecules bring in resistance to chemotherapy or cornerstone drugs. However, in contrast, multiple malignant tumors, including HCC that target the pro-apoptotic favoring isoforms/variants favor apoptotic induction and make chemotherapy effective. Herein, we discuss different splicing events, aberrations, and antisense oligonucleotides (ASOs) in modulating RNA splicing in HCC tumorigenesis with a possible therapeutic outcome.

In-silico functional and structural annotation of hypothetical protein from Klebsiella pneumonia: A potential drug target.

Klebsiella pneumonia is known to cause several nosocomial infections in immunocompromised patients. It has developed resistance against a broad range of presently available antibiotics, resulting in high mortality rates in patients and declared an urgent threat. Therefore, exploration of possible novel drug targets against this opportunistic bacteria needs to be undertaken. In the present study, we performed an extensive in-silico analysis for functional and structural annotation and characterized HP CP995_08280 from K. pneumonia as a drug target and aimed to identify potent drug candidates. The functional and structural studies using several bioinformatics tools and databases predicted that HP CP995_08280 is a cytosolic protein that belongs to the ß-lactamase family and shares structural similarity with FmtA protein from Staphylococcus aureus (PDB ID: 5ZH8). The structure of HP CP995_08280 was successfully modeled followed by structure-based virtual screening, docking, molecular dynamics, and Molecular mechanic/Poisson-Boltzmann surface area (MMPBSA) were performed to identify the potential compounds. We have found five potent antibacterial molecules, namely BDD 24083171, BDD 24085737, BDE 25098678, BDE 33638819, and BDE 33672484, which exhibited high binding affinity (>-7.5 kcal/mol) and were stabilized by hydrogen bonding and hydrophobic interactions with active site residues (Ser42, Lys45, Tyr126, and Asp128) of protein. Molecular dynamics and MMPBSA revealed that HP CP995_08280 - ligand(s) complexes were less dynamic and more stable than native HP CP995_08280. Hence, the present study may serve as a potential lead for developing inhibitors against drug-resistant Klebsiella pneumonia.

Crystal structure and molecular characterization of NADP+-farnesol dehydrogenase from cotton bollworm, Helicoverpaarmigera.

Farnesol dehydrogenase (FDL) orchestrates the oxidation reaction catalyzing farnesol to farnesal, a key step in the juvenile hormone (JH) biosynthesis pathway of insects and hence, represents a lucrative target for developing insect growth regulators (IGRs). However, information on the structural and functional characterization of JH-specific farnesol dehydrogenase in insects remains elusive. Herein, we identified a transcript that encodes farnesol dehydrogenase (HaFDL) from Helicoverpa armigera, a major pest of cotton. The investigations of molecular assembly, biochemical analysis and spatio-temporal expression profiling showed that HaFDL exists as a soluble homo-tetrameric form, exhibits a broad substrate affinity and is involved in the JH-specific farnesol oxidation in H. armigera. Additionally, the study presents the first crystal structure of the HaFDL-NADP enzyme complex determined at 1.6 Å resolution. Structural analysis revealed that HaFDL belongs to the NADP-specific cP2 subfamily of the classical short-chain dehydrogenase/reductase (SDR) family and exhibits typical structural features of those enzymes including the conserved nucleotide-binding Rossman-fold. The isothermal titration calorimetry (ITC) showed a high binding affinity (dissociation constant, Kd, 3.43 µM) of NADP to the enzyme. Comparative structural analysis showed a distinct substrate-binding pocket (SBP) loop with a spacious and hydrophobic substrate-binding pocket in HaFDL, consistent with the biochemically observed promiscuous substrate specificity. Finally, based on the crystal structure, substrate modeling and structural comparison with homologs, a two-step reaction mechanism is proposed. Overall, the findings significantly impact and contribute to our understanding of farnesol dehydrogenase functional properties in JH biosynthesis in H. armigera.

Conformational flexibility enables catalysis of phthalate cis-4,5-dihydrodiol dehydrogenase.

Phthalate cis-4,5-dihydrodiol dehydrogenase (PhtC), the second enzyme of the phthalate catabolic pathway, catalyzes the dehydrogenation of cis-4,5-dihydrodiol phthalate (DDP). Here, we report the structural and biochemical characterization of PhtC from Comamonas testosteroni KF1 (PhtCKF1). With biochemical experiments, we have determined the enzyme's catalytic efficiency (kcat/Km) with DDP as 2.6 ± 0.5 M-1s-1, over 10-fold higher than with cis-3,4-dihydrodiol phthalate (CDP). To understand the structural basis of these reactions, the crystal structures of PhtCKF1 in apo-form, the binary complex with NAD+, and the ternary complex with NAD+ and 3-hydroxybenzoate (3HB) were determined. These crystal structures reveal that the binding of 3HB induces a conformational change in the substrate-binding loop. This conformational change causes the opening of the NAD + binding site while trapping the 3HB. The PhtCKF1 crystal structures show that the catalytic domain of PhtCKF1 is larger than that of other structurally characterized homologs and does not align with other cis-diol dehydrogenases. Structural and mutational analysis of the substrate-binding loop residues, Arg164 and Glu167 establish that conformational flexibility of this loop is necessary for positioning the substrate in a catalytically competent pose, as substitution of either of these residues to Ala did not yield the dehydrogenation activity. Further, based on the crystal structures of PhtCKF1 and related structural homologs, a reaction mechanism is proposed. Finally, with the biochemical analysis of a variant M251LPhtCKF1, the broader substrate specificity of this enzyme is explained.

Biochemical and molecular insights on the bioactivity and binding interactions of Bacillus australimaris NJB19 L-asparaginase.

L-asparaginase, an antileukemic enzyme, is indispensable to the treatment of Acute Lymphoblastic Leukemia (ALL). However, the intrinsic glutaminase activity entails various side effects to the patients; thus, an improved version of the enzyme lacking glutaminase activity would be a requisite for effective treatment management of ALL. The present study highlights the biochemical and molecular characteristics of the recombinant glutaminase-free L-asparaginase from Bacillus australimaris NJB19 (BaAsp). Investigation of the active site architecture of the protein unraveled the binding interactions of BaAsp with its substrate. Comparative analysis of the L-asparaginase sequences revealed few substitutions of key amino acids in the BaAsp that could construe its substrate selectivity and specificity. The purified heterologously expressed protein (42 kDa) displayed maximum L-asparaginase activity at 35-40 °C and pH 8.5-9, with no observed L-glutaminase activity. The kinetic parameters, Km and Vmax, were determined as 45.6 µM and 0.16 µmoles min-1, respectively. Furthermore, in silico analysis revealed a conserved zinc-binding site in the protein, which is generally implicated in inhibiting the L-asparaginase activity. However, BaAsp was not inhibited by zinc at 1 mM concentration. Therefore, the findings provide insights on the biochemical and molecular details of BaAsp, which could be valuable in formulating it for alternate antileukemic drug therapy.

Quantum Mechanics/Molecular Mechanics Studies on the Catalytic Mechanism of a Novel Esterase (FmtA) of Staphylococcus aureus.

FmtA is a novel esterase that shares the penicillin-binding protein (PBP) core structural folding but found to hydrolyze the removal of d-Ala from teichoic acids. Molecular docking, dynamics, and MM-GBSA of FmtA and its variants S127A, K130A, Y211A, D213A, and K130AY211A, in the presence or absence of wall teichoic acid (WTA), suggest that active site residues S127, K130, Y211, D213, N343, and G344 play a role in substrate binding. Quantum mechanics (QM)/molecular mechanics (MM) calculations reveal that during WTA catalysis, K130 deprotonates S127, and the nucleophilic S127 attacks the carbonyl carbon of d-Ala bound to WTA. The tetrahedral intermediate (TI) complex is stabilized by hydrogen bonding to the oxyanion holes. The TI complex displays a high energy gap and collapses to an energetically favorable acyl-enzyme complex.

Biochemical and structural basis for Moraxella catarrhalis enoyl-acyl carrier protein reductase (FabI) inhibition by triclosan and estradiol.

The enoyl-acyl carrier protein reductase (ENR) is an established drug target and catalyzes the last reduction step of the fatty acid elongation cycle. Here, we report the crystal structures of FabI from Moraxella catarrhalis (McFabI) in the apo form, binary complex with NAD+ and ternary complex with NAD + -triclosan (TCL) determined at 2.36, 2.12 and 2.22 Å resolutions, respectively. The comparative study of these three structures revealed three different conformational states for the substrate-binding loop (SBL), including an unstructured intermediate, a structured intermediate and a closed conformation in the apo, binary and ternary complex forms, respectively; indicating the flexibility of SBL during the ligand binding. Virtual screening has suggested that estradiol cypionate may be a potential inhibitor of McFabI. Subsequently, estradiol (EST), the natural form of estradiol cypionate, was assessed for its FabI-binding and -inhibition properties. In vitro studies demonstrated that TCL and EST bind to McFabI with high affinity (KD = 0.038 ± 0.004 and 5 ± 0.06 µM respectively) and inhibit its activity (Ki = 62.93 ± 3.95 nM and 25.97 ± 1.93 µM respectively) and suppress the growth of M. catarrhalis. These findings reveal that TCL and EST inhibit the McFabI activity and thereby affect cell growth. This study suggests that estradiol may be exploited as a novel scaffold for the designing and development of more potential FabI inhibitors.