Genomic characterization and evolutionary analysis of dengue virus from Aedes mosquitoes in Telangana, India.

BACKGROUND & OBJECTIVES: Entomological surveillance for mosquito-borne viruses is vital for monitoring disease transmission and vector control programs. The vector control program is reliant not only on vector density but also on the timely detection of mosquito-borne infections. In the present study, we conducted an entomological surveillance in different locations of Hyderabad, Telangana, India during 2017-2018 and the collected mosquitoes were screened for dengue virus. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) was used for the identification and serotyping of the dengue virus. Bioinformatics analysis was performed using Mega 6.0 software. Followed by phylogenetic analysis, which was based on CprM structural genome sequence, was performed by using the Maximum-Likelihood method. RESULTS: The TaqMan RT-PCR assay was used to analyze the serotypes of 25 pools of Aedes mosquitoes and found that all four serotypes are circulating in Telangana. DENV1 (50%) was the most commonly detected serotype followed by DENV2 (16.6%), DENV3 (25%), and DENV4 (8.3%). Moreover, DENV1 has the highest MIR (16 per 1000 mosquitoes) compared with DENV2, 3, and 4. The CprM structural gene sequence was used for phylogenetic analysis, revealing that all four strains have a close relationship with strains isolated from India, Pakistan, China and Thailand. Similarly, two variations in amino acid sequence DENV1 at position 43 (K-R) and 86 (S-T) and a single mutation DENV2 at 111 amino acid position were observed. INTERPRETATION & CONCLUSION: The results of the study provide an in-depth transmission dynamic of the dengue virus and persistence of this emerging pathogen in Telangana, India that needs appropriate prevention programs.

Development of 2-chloroquinoline based heterocyclic frameworks as dual inhibitors of SARS-CoV-2 MPro and PLPro.

Evolution of new variants of SARS-CoV-2 warrant the need for the continued efforts in identifying target-oriented new drugs. Dual targeting agents against MPro and PLPro not only overcome the incomplete efficacy but also the drug resistance, which is common problem. Since both these are cysteine proteases, we designed 2-chloroquinoline based molecules with additional imine moiety in the middle as possible nucleophilic warheads. In the first round of design and synthesis, three molecules (C3, C4 and C5) inhibited (Ki < 2 µM) only MPro by binding covalently to C145 and one molecule (C10) inhibited both the proteases non-covalently (Ki < 2 µM) with negligible cytotoxicity. Further conversion of the imine in C10 to azetidinone (C11) improved the potency against both the enzymes in the nanomolar range (820 nM against MPro and 350 nM against PLPro) with no cytotoxicity. Conversion of imine to thiazolidinone (C12), reduced the inhibition by 3-5 folds against both the enzymes. Biochemical and computational studies suggest that C10-C12 bind in the substrate binding pocket of MPro and in the BL2 loop of the PLPro. Since these dual inhibitors have least cytotoxicity, they could be further explored as therapeutics against the SARS-CoV-2 and other analogous viruses.

Anti-inflammatory and antioxidant activities of Gymnema Sylvestre extract rescue acute respiratory distress syndrome in rats via modulating the NF-κB/MAPK pathway.

Acute respiratory distress syndrome (ARDS) is one of the major causes of mortality in COVID-19 patients, due to limited therapeutic options. This prompted us to explore natural sources to mitigate this condition. Gymnema Sylvestre (GS) is an ancient medicinal plant known to have various therapeutic effects. This investigation examined the therapeutic effect of hydroalcoholic extract of Gymnema Sylvestre (HAEGS) against lipopolysaccharide (LPS)-induced lung injury and ARDS in in vitro and in vivo models. UHPLC-HRMS/GC-MS was employed for characterizing the HAEGS and identified several active derivatives including gymnemic acid, gymnemasaponins, gymnemoside, gymnemasin, quercetin, and long fatty acids. Gene expression by RT-qPCR and DCFDA analysis by flow cytometry revealed that several inflammatory cytokine/chemokine, cell injury markers, and reactive oxygen species (ROS) levels were highly upregulated in LPS control and were significantly reduced upon HAEGS treatment. Consistent with the in vitro studies, we found that in LPS-induced ARDS model, pre-treatment with HAEGS significantly suppressed the LPS-induced elevation of inflammatory cell infiltrations, cytokine/chemokine marker expression, ROS levels, and lung injury in a dose-dependent manner. Further mechanistic studies demonstrated that HAEGS suppressed oxidative stress by modulating the NRF2 pathway and ameliorated the ARDS through the NF-κB/MAPK signalling pathway. Additional fractionation results revealed that fraction 6 which has the exclusive composition of gymnemic acid derivatives showed better anti-inflammatory effects (inhibition of IL-6 and IL-1ß) at lower concentrations compared to HAEGS. Overall, HAEGS significantly mitigated LPS-induced lung injury and ARDS by targeting the NF-κB/MAPK signalling pathway. Thus, our work unravels the protective role of HAEGS for the first time in managing ARDS.

A single amino acid difference between archaeal and human type 2 methionine aminopeptidases differentiates their affinity towards ovalicin.

In almost all living cells, methionine aminopeptidase (MetAP) co-translationally cleaves the initiator methionine in at least 70% of the newly synthesized polypeptides. MetAPs are typically classified into Type 1 and Type 2. While prokaryotes and archaea contain only either Type 1 or Type 2 MetAPs respectively, eukaryotes contain both types of enzymes. Almost all MetAPs published till date cleave only methionine from the amino terminus of the substrate peptides. Earlier experiments on crude Type 2a MetAP isolated from Pyrococcus furiosus (PfuMetAP2a) cosmid protein library was shown to cleave leucine in addition to methionine. Authors in that study have ruled out the PfuMetAP2a activity against leucine substrates and assumed it to be a background reaction contributed by other contaminating proteases. In the current paper, using the pure recombinant enzyme, we report that indeed activity against leucine is directly carried out by the PfuMetAP2a. In addition, the natural product ovalicin which is a specific covalent inhibitor of Type 2 MetAPs does not show efficient inhibition against the PfuMetAP2a. Bioinformatic analysis suggested that a glycine in eukaryotic MetAP2s (G222 in human MetAP2b) and asparagine (N53 in PfuMetAP2a) in archaeal MetAP2s positioned at the analogous position. N53 side chain forms a hydrogen bond with a conserved histidine (H62) at the entrance of the active site and alters its orientation to accommodate the ovalicin. This slight orientational difference of the H62, reduces affinity of the ovalicin by 300,000-fold when compared with the HsMetAP2b inhibition. This difference in the activity is partly reduced in the case of N53G mutation of the PfuMetAP2a.

Regioselectivity in inhibition of peptide deformylase from Haemophilus influenzae by 4- vs 5-azaindole hydroxamic acid derivatives: Biochemical, structural and antimicrobial studies.

Ribosome assisted protein synthesis in all prokaryotes begins with a formylated methionine. Deformylation and demethionylation of these newly synthesized proteins are critical co-translational events carried out by peptide deformylase (PDF) and methionine aminopeptidase (MetAP) in all living cells. Since the mechanism of N-terminal modification is common between the infectious microbes and the host human cells, it is a challenge to identify selective inhibitors. Given that both MetAP and PDF are metalloenzymes, and have strong affinity for hydroxamic acids, we reasoned that the azaindole-based hydroxamic acids could inhibit the PDF enzymes. In the present study we describe the screening of a 17-compound library with 4- and 5- substituted azaindole hydroxamic acid derivatives against PDF enzyme from H. influenzae (HiPDF), M. tuberculosis (MtPDF) and human PDF (HsPDF). Several of these molecules showed nanomolar inhibition against HiPDF enzyme, best at 21 nM (15). On the other hand, none of these compounds inhibited the human enzyme while only two molecules showed moderate inhibition against Mtb enzyme. Surprisingly only 5-substituted azaindole derivatives inhibited the PDF enzymes. Some of the 5-substituted azaindole compounds inhibited the growth of different microbes indicating their potential application in antimicrobial therapy. Crystallographic and modeling studies provided the mechanistic view of regioselective inhibition.

Design, Synthesis and Mechanistic Studies of Novel Isatin-Pyrazole Hydrazone Conjugates as Selective and Potent Bacterial MetAP Inhibitors.

Methionine aminopeptidases (MetAPs) are attractive drug targets due to their essential role in eukaryotes as well as prokaryotic cells. In this study, biochemical assays were performed on newly synthesized Isatin-pyrazole hydrazones (PS1-14) to identify potent and selective bacterial MetAPs inhibitors. Compound PS9 inhibited prokaryotic MetAPs, i.e., MtMetAP1c, EfMetAP1a and SpMetAP1a with Ki values of 0.31, 6.93 and 0.37 µM, respectively. Interestingly, PS9 inhibited the human analogue HsMetAP1b with Ki (631.7 µM) about ten thousand-fold higher than the bacterial MetAPs. The in vitro screening against Gram-positive (Enterococcus faecalis, Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa, Klebsiella pneumonia and Escherichia coli) bacterial strains also exhibited their antibacterial potential supported by minimum bactericidal concentration (MBC), disk diffusion assay, growth curve and time-kill curve experiments. Additionally, PS6 and PS9 had synergistic effects when combined with ampicillin (AMP) and ciprofloxacin (CIP) against selective bacterial strains. PS9 showed no significant cytotoxic effect on human RBCs, HEK293 cells and Galleria mellonella larvae in vivo. PS9 inhibited the growth of multidrug-resistant environmental isolates as it showed the MIC lower than the standard drugs used against selective bacterial strains. Overall, the study suggested PS9 could be a useful candidate for the development of antibacterial alternatives.

Selective inhibition of Helicobacter pylori methionine aminopeptidase by azaindole hydroxamic acid derivatives: Design, synthesis, in vitro biochemical and structural studies.

Methionine aminopeptidases (MetAPs) are an important class of enzymes that work co-translationally for the removal of initiator methionine. Chemical inhibition or gene knockdown is lethal to the microbes suggesting that they can be used as antibiotic targets. However, sequence and structural similarity between the microbial and host MetAPs has been a challenge in the identification of selective inhibitors. In this study, we have analyzed several thousands of MetAP sequences and established a pattern of variation in the S1 pocket of the enzyme. Based on this knowledge, we have designed a library of 17 azaindole based hydroxamic acid derivatives which selectively inhibited the MetAP from H. pylori compared to the human counterpart. Structural studies provided the molecular basis for the selectivity.

Exo-selective intermolecular Diels-Alder reaction by PyrI4 and AbnU on non-natural substrates.

The 100-year-old Diels-Alder reaction (DAr) is an atom economic and elegant organic chemistry transformation combining a 1,3-diene and a dienophile in a [4+2] cycloaddition leading to a set of products with several stereo centres and multiple stereoisomers. Stereoselective [4+2] cycloaddition is a challenge. Here, we describe two natural enzymes, PyrI4 and AbnU performing stereospecific intermolecular DAr on non-natural substrates. AbnU catalyses a single exo-stereoisomer by 32-fold higher than the background. PyrI4 catalyses the same stereoisomer (15-fold higher) as a major component (>50%). Structural, biochemical and fluorescence studies indicate that the dienophile enters first into the ß-barrel of the enzymes followed by the 1,3-diene, yielding a stereospecific product. However, if some critical interactions are disrupted to increase the catalytic efficiency, stereoselectivity is compromised. Since it is established that natural enzymes can carry out intermolecular DAr on non-natural substrates, several hundreds of Diels-Alderases available in nature could be explored.

Methionine aminopeptidases with short sequence inserts within the catalytic domain are differentially inhibited: Structural and biochemical studies of three proteins from Vibrio spp.

Methionine aminopeptidases (MetAPs) have been recognized as drug targets and have been extensively studied for discovery of selective inhibitors. MetAPs are essential enzymes in all living cells. While most prokaryotes contain a single gene, some prokaryotes and all eukaryotes including human have redundancy. Due to the similarity in the active sites of the MetAP enzyme between the pathogens and human limited the success of discovering selective inhibitors. We recently have discovered that MetAPs with small inserts within the catalytic domain to have different susceptibilities against some inhibitors compared to those that do not have. Using this clue we used bioinformatic tools to identify new variants of MetAPs with inserts in pathogenic species. Two new isoforms were identified in Vibrio species with two and three inserts in addition to an isoform without any insert. Multiple sequence alignment suggested that inserts are conserved in several of the Vibrio species. Two of the three inserts are common between two and three insert isoforms. One of the inserts is identified to have "NNKNN" motif that is similar to well-characterized quorum sensing peptide, "NNWNN". Another insert is predicted to have a posttranslational modification site. Three Vibrio proteins were cloned, expressed, purified, enzyme kinetics established and inhibitor screening has been performed. Several of the pyridinylpyrimidine derivatives selectively inhibited MetAPs with inserts compared to those that do not have, including the human enzyme. Crystal structure and molecular modeling studies provide the molecular basis for selective inhibition.

Puromycin, a selective inhibitor of PSA acts as a substrate for other M1 family aminopeptidases: Biochemical and structural basis.

Puromycin sensitive aminopeptidase (PSA or NPEPPS) is a M1 class aminopeptidase is selectively inhibited by the natural product puromycin, an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. The molecular basis for this selective inhibition has not been understood well. Here, we report the basis for selectivity of puromycin using biochemical, structural and molecular modeling tools on four different M1 family enzymes including human PSA. Except for PSA, the other three enzymes were not inhibited. Instead, the peptide bond in the puromycin is hydrolyzed to O-methyl-L-tyrosine (OMT) and puromycin aminonucleoside (PAN). Neither of the hydrolyzed products, individually or together inhibit any of the four enzymes. Crystal structure of ePepN using crystals that are incubated with puromycin contained the hydrolyzed products instead of intact puromycin. On the other hand, intact puromycin molecule was observed in the crystal structure of the inactive mutant ePepN (E298A)-puromycin complex. Surprisingly, puromycin does not enter the active site of the mutant enzyme but binds near the entrance. Comparison of puromycin binding region in ePepN mutant enzyme and molecular modeling studies suggest that PSA might be inhibited by similar mode of binding there by blocking the entrance of the active site.

Bengamides display potent activity against drug-resistant Mycobacterium tuberculosis.

Mycobacterium tuberculosis infects over 10 million people annually and kills more people each year than any other human pathogen. The current tuberculosis (TB) vaccine is only partially effective in preventing infection, while current TB treatment is problematic in terms of length, complexity and patient compliance. There is an urgent need for new drugs to combat the burden of TB disease and the natural environment has re-emerged as a rich source of bioactive molecules for development of lead compounds. In this study, one species of marine sponge from the Tedania genus was found to yield samples with exceptionally potent activity against M. tuberculosis. Bioassay-guided fractionation identified bengamide B as the active component, which displayed activity in the nanomolar range against both drug-sensitive and drug-resistant M. tuberculosis. The active compound inhibited in vitro activity of M. tuberculosis MetAP1c protein, suggesting the potent inhibitory action may be due to interference with methionine aminopeptidase activity. Tedania-derived bengamide B was non-toxic against human cell lines, synergised with rifampicin for in vitro inhibition of bacterial growth and reduced intracellular replication of M. tuberculosis. Thus, bengamides isolated from Tedania sp. show significant potential as a new class of compounds for the treatment of drug-resistant M. tuberculosis.

Unraveling structural insights of ribokinase from Leishmania donovani.

Ribokinase (RK) is an ATP dependent sugar kinase that enables the entry of ribose in the metabolism. Leishmania accumulates ribose into the cytosol through hydrolysis of nucleosides and by transport from the extracellular environment. Activation by RK is critical to mobilize the ribose into the metabolism of Leishmania. To understand the catalytic role, the crystal structure of RK (LdRK) from L. donovani was determined in the apo and complex forms with several nucleotides (ATP, AMPPCP and ADP) in the presence of Na+ ion. The dual insertion of five amino acid stretches makes LdRK structurally unique from other reported structures of RKs. The structure of LdRK-ATP provided the basis for positioning of γ-phosphate of ATP by conserved -GAGD- motif. Liganded and unliganded structures of LdRK exists in similar conformation, which suggests binding of nucleotides does not make any significant conformational changes in nucleotide-bound structures. Substitution of a conserved asparagine with phenylalanine in ribose binding pocket differentiates the LdRK from other RKs. Glycerol molecule bound in the substrate binding pocket mimics the enzyme-substrate interactions but in turn, hampers the binding of ribose to LdRK. Comparative structural analysis revealed the flexibility of γ-phosphate, which adopts multiple conformations in the absence of divalent metal ion and ribose. Similar to other RKs, LdRK is also dependent on monovalent as well as divalent cations for its catalytic activity.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis.

Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

Discovery of a new class of type 1 methionine aminopeptidases that have relaxed substrate specificity.

Methionine aminopeptidases (MetAPs) are a class of enzymes evolved to cleave initiator methionine in 60-70% of the total cellular proteins in all living cells. Based on their sequence differences, they are classified into Type 1 and Type 2. Type 1 is further divided into Type 1a, 1a', 1b, 1c and 1d. Irrespective of various classifications, all MetAPs reported till date displayed hydrolytic activity against peptides that contain only methionine on the N-terminus. A cysteine at the top of the active site in all the Type 1 structures is reported to be critical for the specificity. Mutation of this cysteine to serine or asparagine leads to loss of specificity. In the present study, we have identified a class of MetAPs in some of the proteobacteria that have an asparagine at this site. Most of the proteobacteria that contain MetAP1n are pathogenic in nature. Biochemical and structural studies on two proteins, one from each of V. coralliilyticus and K. pneumoniae confirm that these enzymes cleave leucine in addition to methionine. Crystallographic and homology modeling studies suggest that relaxed substrate specificity of this new class of enzymes could be due to the increased flexibility in the active site. Since this new class has an asparagine at the critical position that probably contributes for the relaxed substrate specificity and also differentiates them from other Type 1 MetAPs, we classified them as Type 1n.

Synthesis and mechanistic studies of diketo acids and their bioisosteres as potential antibacterial agents.

A series of diketo esters and their pertinent bioisosteres were designed and synthesized as potent antibacterial agents by targeting methionine amino peptidases (MetAPs). In the biochemical assay against purified MetAPs from Streptococcus pneumoniae (SpMetAP1a), Mycobacterium tuberculosis (MtMetAP1c), Enterococcus faecalis (EfMetAP1a) and human (HsMetAP1b), compounds 3a, 4a and 5a showed more than 85% inhibition of all the tested MetAPs at 100 µM concentration. Compounds 4a and 5a also exhibited antibacterial potential with MIC values 62.5 µg/mL (S. pneumoniae), 31.25 µg/mL (E. faecalis), 62.5 µg/mL (Escherichia coli) and 62.5 µg/mL (S. pneumoniae), 62.5 µg/mL (E. coli), respectively. Moreover, 5a also significantly inhibited the growth of multidrug resistant E. coli strains at 512 µg/mL conc., while showing no cytotoxic effect towards healthy CHO cells and thus being selected. Growth kinetics study showed significant inhibition of bacterial growth when treated with different conc. of 5a. TEM analysis also displayed vital damage to bacterial cells by 5a at MIC conc. Moreover, significant inhibition of biofilm formation was observed in bacterial cells treated with MIC conc. of 5a as visualized by SEM micrographs. Interestingly, 5a did not cause an alteration in the hemocyte density in Galleria mellonella larvae which is considered in vivo model for antimicrobial studies and was non-toxic up to a conc. of 2.5 mg/mL.

Aminobenzosuberone Scaffold as a Modular Chemical Tool for the Inhibition of Therapeutically Relevant M1 Aminopeptidases.

The synthesis of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochlorides was optimized to enhance reproducibility and increase the overall yield. In order to investigate their specificity, series of enzyme inhibition assays were carried out against a diversity of proteases, covering representative members of aspartic, cysteine, metallo and serine endopeptidases and including eight members of the monometallic M1 family of aminopeptidases as well as two members of the bimetallic M17 and M28 aminopeptidase families. This aminobenzosuberone scaffold indeed demonstrated selective inhibition of M1 aminopeptidases to the exclusion of other tested protease families; it was particularly potent against mammalian APN and its bacterial/parasitic orthologues EcPepN and PfAM1.

Discovery, Structural and Biochemical Studies of a rare Glu/Asp Specific M1 Class Aminopeptidase from Legionella pneumophila.

Aminopeptidases catalyze the hydrolysis of amino acids from the N-terminus of protein or peptide substrates. M1 family aminopeptidases are important for the pathogenicity of bacteria and play critical role in many physiological processes such as protein maturation, regulation of peptide hormone levels in humans. Most of the M1 family aminopeptidases reported till date display broad substrates specificity, mostly specific to basic and hydrophobic residues. In the current study we report the discovery of a novel M1 class aminopeptidase from Legionella pneumophila (LePepA), which cleaves only acidic residues. Biochemical and structural studies reveal that the S1 pocket is polar and positively charged. Bioinformatic analysis suggests that such active site is unique to only Legionella species and probably evolved for special needs of the microbe. Given its specific activity, LePepA could be useful in specific biotechnological applications.

Human Naa50 Protein Displays Broad Substrate Specificity for Amino-terminal Acetylation: DETAILED STRUCTURAL AND BIOCHEMICAL ANALYSIS USING TETRAPEPTIDE LIBRARY.

Amino-terminal acetylation is a critical co-translational modification of the newly synthesized proteins in a eukaryotic cell carried out by six amino-terminal acetyltransferases (NATs). All NATs contain at least one catalytic subunit, and some contain one or two additional auxiliary subunits. For example, NatE is a complex of Naa10, Naa50, and Naa15 (auxiliary). In the present study, the crystal structure of human Naa50 suggested the presence of CoA and acetylated tetrapeptide (AcMMXX) that have co-purified with the protein. Biochemical and thermal stability studies on the tetrapeptide library with variations in the first and second positions confirm our results from the crystal structure that a peptide with Met-Met in the first two positions is the best substrate for this enzyme. In addition, Naa50 acetylated all MXAA peptides except for MPAA. Transcriptome analysis of 10 genes that make up six NATs in humans from eight different cell lines suggests that components of NatE are transcribed in all cell lines, whereas others are variable. Because Naa10 is reported to acetylate all amino termini that are devoid of methionine and Naa50 acetylates all other peptides that are followed by methionine, we believe that NatE complex can be a major contributor for amino-terminal acetylation at the ribosome exit tunnel.

Expression, Functional Characterization and X-ray Analysis of HosA, A Member of MarR Family of Transcription Regulator from Uropathogenic Escherichia coli.

Regulators belonging to multiple antibiotic resistance regulator (MarR) family are widespread in prokaryotes and are involved in regulation of genes that are responsible for virulence and pathogenicity in most of the clinically important pathogens. Here we report the transcriptional, biophysical, and X-ray analyses of homologue of SlyA (HosA), a member of MarR family that is predominantly present in the pathogenic strains of Enterobacteriaceae family. The initiation of hosA transcription was observed to occur at two independent start sites and subsequent binding study has revealed that the purified HosA interacts with its upstream region suggesting a probable autoregulation. The secondary structure analysis through circular dichroism spectroscopy demonstrated that HosA is predominantly composed of the alpha helix with higher thermal stability. To further understand the three-dimensional structure, HosA was crystallized and the crystals were diffracted to maximum of 2.9 Çº on exposure to X-rays. Analysis of the X-ray crystallographic data suggested a primitive space group (P 6 ? 2 2), with unit cell parameters a = b = 64.19 Å and c = 244.25 Å. The solvent content and Matthews coefficient were 41 % and 2.11 Å(3) Da(-1), respectively, which indicated the existence of two molecules of HosA in the asymmetric unit of crystal.

The unique functional role of the C-HS hydrogen bond in the substrate specificity and enzyme catalysis of type 1 methionine aminopeptidase.

It is intriguing how nature attains recognition specificity between molecular interfaces where there is no apparent scope for classical hydrogen bonding or polar interactions. Methionine aminopeptidase (MetAP) is one such enzyme where this fascinating conundrum is at play. In this study, we demonstrate that a unique C-HS hydrogen bond exists between the enzyme methionine aminopeptidase (MetAP) and its N-terminal-methionine polypeptide substrate, which allows specific interaction between apparent apolar interfaces, imposing a strict substrate recognition specificity and efficient catalysis, a feature replicated in Type I MetAPs across all kingdoms of life. We evidence this evolutionarily conserved C-HS hydrogen bond through enzyme assays on wild-type and mutant MetAP proteins from Mycobacterium tuberculosis that show a drastic difference in catalytic efficiency. The X-ray crystallographic structure of the methionine bound protein revealed a conserved water bridge and short contacts involving the Met side-chain, a feature also observed in MetAPs from other organisms. Thermal shift assays showed a remarkable 3.3 °C increase in melting temperature for methionine bound protein compared to its norleucine homolog, where C-HS interaction is absent. The presence of C-HS hydrogen bonding was also corroborated by nuclear magnetic resonance spectroscopy through a change in chemical shift. Computational chemistry studies revealed the unique role of the electrostatic environment in facilitating the C-HS interaction. The significance of this atypical hydrogen bond is underscored by the fact that the function of MetAP is essential for any living cell.