Structural insights into the specificity and catalytic mechanism of mycobacterial nucleotide pool sanitizing enzyme MutT2.

Mis-incorporation of modified nucleotides, such as 5-methyl-dCTP or 8-oxo-dGTP, in DNA can be detrimental to genomic integrity. MutT proteins are sanitization enzymes which function by hydrolyzing such nucleotides and regulating the pool of free nucleotides in the cytoplasm. Mycobacterial genomes have a set of four MutT homologs, namely, MutT1, MutT2, MutT3 and MutT4. Mycobacterial MutT2 hydrolyzes 5 m-dCTP and 8-oxo-dGTP to their respective monophosphate products. Additionally, it can hydrolyze canonical nucleotides dCTP and CTP, with a suggested role in sustaining their optimal levels in the nucleotide pool. The structures of M. smegmatis MutT2 and its complexes with cytosine derivatives have been determined at resolutions ranging from 1.10 Što 1.73 Å. The apo enzyme and its complexes with products (dCMP, CMP and 5 m-dCMP) crystallize in space group P21212, while those involving substrates (dCTP, CTP and 5 m-dCTP) crystallize in space group P21. The molecule takes an α/ß/α sandwich fold arrangement, as observed in other MutT homologs. The nucleoside moiety of the ligands is similarly located in all the complexes, while the location of the remaining tail exhibits variability. This is the first report of a MutT2-type protein in complex with ligands. A critical interaction involving Asp116 confers the specificity of the enzyme towards cytosine moieties. A conserved set of enzyme-ligand interactions along with concerted movements of important water molecules provide insights into the mechanism of action.

RecGwed : A probable novel regulator in the resolution of branched DNA structures in mycobacteria.

Structure-specific helicases, such as RecG, play an important role in the resolution of recombination intermediates. A bioinformatic analysis of mycobacterial genomes led to the identification of a protein (RecGwed ) with a C-terminal "edge" domain, similar to the wedge domain of RecG. RecGwed is predominately found in the phylum Actinobacteria and in few human pathogens. Mycobacterium smegmatis RecGwed was able to bind branched DNA structures in vitro but failed to interact with single- or double-stranded DNA. The expression of recGwed in M. smegmatis cells was up-regulated during stationary phase/UV damage and down-regulated during MMS/H2 O2 treatment. These observations indicate the possible involvement of RecGwed in transactions during recombination events, that proceed though branched DNA intermediates. © 2018 IUBMB Life, 70(8):786-794, 2018.

Hydrolysis of diadenosine polyphosphates. Exploration of an additional role of Mycobacterium smegmatis MutT1.

Diadenosine polyphosphates (ApnA, n=2-6), particularly Ap4A, are involved in several important physiological processes. The substantial sequence identity of the Nudix hydrolase domain (domain 1) of Mycobacterium smegmatis MutT1 (MsMutT1) with a known Ap4A hydrolase suggested that MsMutT1 could also hydrolyse diadenosine polyphosphates. Biochemical experiments yielded results in conformity with this suggestion, with Ap4A as the best among the substrates. ATP is a product in all experiments; small amounts of ADP were also observed in the experiments involving Ap4A and Ap6A. Hydrolysis was inhibited by fluoride ions in all cases. The mechanism of action and its inhibition in relation to ApnA were explored through the X-ray analysis of the crystals of the MsMutT1 complexes with Ap5A; Ap5A and MnCl2; Ap4A; ATP; and ATP.NaF.MgCl2. The aggregation pattern of molecules in the first four crystals is similar to that found in a majority of MsMutT1-NTP crystals. Substrate molecules occupy the primary binding site and ATP occupies a site at an intermolecular interface, in the first two. ATP occupies both the sites in the third and fourth crystal. The protein-ligand interactions observed in these crystal structures lead to an explanation of the molecular mechanism of hydrolysis of ApnA by MsMutT1. The fifth crystal exhibits a new packing arrangement. The structure of the complex provides an explanation for the fluoride inhibition of the activity of the enzyme. It would thus appear that MutT1 has a major role involving the hydrolysis of diadenosine polyphosphates, which could be elucidated at the molecular level.

Cortisol plays a role in the high environmental ammonia associated suppression of the immune response in zebrafish.

Exposure to high environmental ammonia (HEA) levels increases the vulnerability of fishes to parasitic, viral and bacterial diseases. We tested the hypothesis that elevated plasma cortisol levels play a role in the HEA-mediated immunosuppression in fishes. To this end, we tested the effect of exogenous cortisol treatment on the lipopolysaccharide (LPS)-induced immune response in zebrafish (Danio rerio). Also, to test whether glucocorticoid receptor (GR) signaling is involved in HEA-mediated immunosuppression, zebrafish were treated with mifepristone, a GR antagonist, and the LPS-induced immune response assessed after HEA exposure. We evaluated a panel of important immunity-related genes including interleukin 1ß (il1b) and suppressor of cytokine signaling (socs-1a, 2, 3) and acute phase response genes [serum amyloid A (saa), transferrin (tfa), leukocyte cell-derived chemotaxin 2-like (lect2l), haptoglobin (hp), hepcidin (=hepatic anti-microbial peptide hamp), and complement component 3b (c3b)] by real-time quantitative PCR. Our results demonstrate that exogenous cortisol administration as well as elevated cortisol levels in response to HEA exposure modulate mRNA transcript levels of key mediators of the innate immune response in zebrafish. Mifepristone treatment reduced whole body cortisol levels and eliminated the HEA-mediated changes in transcript abundance of socs1a, il1b, as well as APR genes. Together, these results suggest that the HEA effect on the innate immune response is in part mediated by cortisol signaling, while the mode of action, including the receptors involved remains to be elucidated.

Structure of the second Single Stranded DNA Binding protein (SSBb) from Mycobacterium smegmatis.

All mycobacteria with sequenced genomes, except M. leprae, have a second Single Stranded DNA Binding protein (SSBb) in addition to the canonical one (SSBa). This paralogue from M. smegmatis (MsSSBb) has been cloned, expressed and purified. The protein, which is probably involved in stress response, has been crystallized and X-ray analyzed in the first structure elucidation of a mycobacterial SSBb. In spite of the low sequence identity between SSBas and SSBbs in mycobacteria, the tertiary and quaternary structure of the DNA binding domain of MsSSBb is similar to that observed in mycobacterial SSBas. In particular, the quaternary structure is 'clamped' using a C-terminal stretch of the N-domain, which endows the tetrameric molecule with additional stability and its characteristic shape. Comparison involving available, rather limited, structural data on SSBbs from other sources, appears to suggest that SSBbs could exhibit higher structural variability than SSBas do.

Archeal lectins: An identification through a genomic search.

Forty-six lectin domains which have homologues among well established eukaryotic and bacterial lectins of known three-dimensional structure, have been identified through a search of 165 archeal genomes using a multipronged approach involving domain recognition, sequence search and analysis of binding sites. Twenty-one of them have the 7-bladed ß-propeller lectin fold while 16 have the ß-trefoil fold and 7 the legume lectin fold. The remainder assumes the C-type lectin, the ß-prism I and the tachylectin folds. Acceptable models of almost all of them could be generated using the appropriate lectins of known three-dimensional structure as templates, with binding sites at one or more expected locations. The work represents the first comprehensive bioinformatic study of archeal lectins. The presence of lectins with the same fold in all domains of life indicates their ancient origin well before the divergence of the three branches. Further work is necessary to identify archeal lectins which have no homologues among eukaryotic and bacterial species.

Structural plasticity in Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng) and its functional implications.

17 independent crystal structures of family I uracil-DNA glycosylase from Mycobacterium tuberculosis (MtUng) and its complexes with uracil and its derivatives, distributed among five distinct crystal forms, have been determined. Thermodynamic parameters of binding in the complexes have been measured using isothermal titration calorimetry. The two-domain protein exhibits open and closed conformations, suggesting that the closure of the domain on DNA binding involves conformational selection. Segmental mobility in the enzyme molecule is confined to a 32-residue stretch which plays a major role in DNA binding. Uracil and its derivatives can bind to the protein in two possible orientations. Only one of them is possible when there is a bulky substituent at the 5' position. The crystal structures of the complexes provide a reasonable rationale for the observed thermodynamic parameters. In addition to providing fresh insights into the structure, plasticity and interactions of the protein molecule, the results of the present investigation provide a platform for structure-based inhibitor design.

Jacalin-carbohydrate interactions: distortion of the ligand molecule as a determinant of affinity.

Jacalin is among the most thoroughly studied lectins. Its carbohydrate-binding site has also been well characterized. It has been postulated that the lower affinity of ß-galactosides for jacalin compared with α-galactosides is caused by steric interactions of the substituents in the former with the protein. This issue has been explored energetically and structurally using different appropriate carbohydrate complexes of jacalin. It turns out that the earlier postulation is not correct. The interactions of the substituent with the binding site remain essentially the same irrespective of the anomeric nature of the substitution. This is achieved through a distortion of the sugar ring in ß-galactosides. The difference in energy, and therefore in affinity, is caused by a distortion of the sugar ring in ß-galactosides. The elucidation of this unprecedented distortion of the ligand as a strategy for modulating affinity is of general interest. The crystal structures also provide a rationale for the relative affinities of the different carbohydrate ligands for jacalin.

Identification of mycobacterial lectins from genomic data.

Sixty-four sequences containing lectin domains with homologs of known three-dimensional structure were identified through a search of mycobacterial genomes. They appear to belong to the ß-prism II, the C-type, the Microcystis virdis (MV), and the ß-trefoil lectin folds. The first three always occur in conjunction with the LysM, the PI-PLC, and the ß-grasp domains, respectively while mycobacterial ß-trefoil lectins are unaccompanied by any other domain. Thirty heparin binding hemagglutinins (HBHA), already annotated, have also been included in the study although they have no homologs of known three-dimensional structure. The biological role of HBHA has been well characterized. A comparison between the sequences of the lectin from pathogenic and nonpathogenic mycobacteria provides insights into the carbohydrate binding region of the molecule, but the structure of the molecule is yet to be determined. A reasonable picture of the structural features of other mycobacterial proteins containing one or the other of the four lectin domains can be gleaned through the examination of homologs proteins, although the structure of none of them is available. Their biological role is also yet to be elucidated. The work presented here is among the first steps towards exploring the almost unexplored area of the structural biology of mycobacterial lectins.

The sequence and structure of snake gourd (Trichosanthes anguina) seed lectin, a three-chain nontoxic homologue of type II RIPs.

The sequence and structure of snake gourd seed lectin (SGSL), a nontoxic homologue of type II ribosome-inactivating proteins (RIPs), have been determined by mass spectrometry and X-ray crystallography, respectively. As in type II RIPs, the molecule consists of a lectin chain made up of two ß-trefoil domains. The catalytic chain, which is connected through a disulfide bridge to the lectin chain in type II RIPs, is cleaved into two in SGSL. However, the integrity of the three-dimensional structure of the catalytic component of the molecule is preserved. This is the first time that a three-chain RIP or RIP homologue has been observed. A thorough examination of the sequence and structure of the protein and of its interactions with the bound methyl-α-galactose indicate that the nontoxicity of SGSL results from a combination of changes in the catalytic and the carbohydrate-binding sites. Detailed analyses of the sequences of type II RIPs of known structure and their homologues with unknown structure provide valuable insights into the evolution of this class of proteins. They also indicate some variability in carbohydrate-binding sites, which appears to contribute to the different levels of toxicity exhibited by lectins from various sources.

Cloning, expression, purification, crystallization and preliminary X-ray studies of argininosuccinate lyase (Rv1659) from Mycobacterium tuberculosis.

The last enzyme in the arginine-biosynthesis pathway, argininosuccinate lyase, from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized, and preliminary X-ray studies have been carried out on the crystals. The His-tagged tetrameric enzyme with a subunit molecular weight of 50.9 kDa crystallized with two tetramers in the asymmetric unit of the orthorhombic unit cell, space group P2(1)2(1)2(1). Molecular-replacement calculations and self-rotation calculations confirmed the space group and the tetrameric nature of the molecule.

Constructing Z-scheme g-C3N4/TiO2 heterostructure for promoting degradation of the hazardous dye pollutants.

The use of dyes and segments has increased widely in recent years, but it poses a serious health risk to ecosystems. In this work, TiO2 and two-dimensional g-C3N4 nanosheets (g-CN) were fabricated through co-precipitation and thermal polymerization technique, respectively. The g-CN-TiO2 photocatalyst (1: 3, 2: 2, 3: 1) in various weight percentages was prepared using a simple impregnation process. The photocatalytic behaviour of the g-CN, TiO2 NPs, and different weight percentages of g-CN-TiO2 photocatalyst was evaluated against methylene blue (MB) dye under UV-visible light illumination. Compared to pristine and other weight percentages of the g-CN-TiO2 nanocomposite, the optimized g-CN-TiO2 nanocomposite (3:1) showed promoted performance against MB dye. The enriched catalytic efficiency can be accredited to the low amount of TiO2 nanoparticles deposited on gCN nanosheets, possibly due to the boosted transport properties of the electron-hole pairs. The enriched photocatalytic behaviour can be attributed to the development of the Z-scheme system between TiO2 and g-CN. The current study is an outstanding demonstration of the development of maximum catalytic efficiency for destroying hazardous chemical dyes.

Structures of new crystal forms of Mycobacterium tuberculosis peptidyl-tRNA hydrolase and functionally important plasticity of the molecule.

The X-ray structures of new crystal forms of peptidyl-tRNA hydrolase from M. tuberculosis reported here and the results of previous X-ray studies of the enzyme from different sources provide a picture of the functionally relevant plasticity of the protein molecule. The new X-ray results confirm the connection deduced previously between the closure of the lid at the peptide-binding site and the opening of the gate that separates the peptide-binding and tRNA-binding sites. The plasticity of the molecule indicated by X-ray structures is in general agreement with that deduced from the available solution NMR results. The correlation between the lid and the gate movements is not, however, observed in the NMR structure.

Crystallization and preliminary X-ray studies of MutT1 (MSMEG_2390) from Mycobacterium smegmatis.

MutT1 (MSMEG_2390) from Mycobacterium smegmatis has been crystallized and the crystals have been characterized using X-ray diffraction. The crystals belonged to space group P2(1)2(1)2(1). The Matthews coefficient suggested the possibility of one protein molecule in the asymmetric unit of the orthorhombic unit cell. Solution of the structure using the known three-dimensional structure of a bacterial MutT1 is anticipated.

Characteristics of ovarian follicle steroidogenesis during vitellogenesis in an asynchronously ovulating stock of rainbow trout Oncorhynchus mykiss.

This study explored several physiological criteria that could be used to assess the steroidogenic condition of the ovarian follicles of individual fish of an asynchronously ovulating captive rainbow trout Oncorhynchus mykiss stock. In these fish, the date of sampling, morphological variables such as gonado-somatic index or ovarian follicle mass and visual assessment of the ovary provided accurate indications of the maturational condition of an individual. The physiological variables measured included the in vitro basal and cyclic adenosine monophosphate (cAMP)-stimulated synthesis by ovarian follicles of 17ß-oestradiol (E(2)) and testosterone (T); in addition, quantitative reverse-transcription (RT)-PCR was used to measure the relative expression of star and p450scc genes by ovarian follicles. The ratios of cAMP-stimulated E(2) and T synthesis to basal E(2) and T synthesis provided a reliable indication of differences in the steroidogenic status of the follicles of individual animals. On the basis of these criteria, together with the use of gene expression profiles, it was possible to classify individual fish as being at an early, mid or late-vitellogenic stage.

Silica nanoparticles derived from Arundo donax L. ash composite with Titanium dioxide nanoparticles as an efficient nanocomposite for photocatalytic degradation dye.

Water pollution is a serious problem that threatens both developed and developing countries. Several methods have been used to purify contaminated water, among which the photocatalytic decomposition approach is widely used to purify contaminated water from organic pollutants. In this work, biomass derived SiO2 nanoparticles composite with TiO2 semiconductors used as an efficient photocatalyst for degradation of RhB dye molecules under UV-visible light irradiation is proclaimed. The different weight percentages of Arundo donax L. ash-derived SiO2 nanoparticles combined with TiO2 nanoparticles were prepared through the wet impregnation method. The photocatalytic degradation ability of the as-prepared samples has been scrutinized against the degradation of Rh B dye in which the pronounced photocatalytic degradation efficiency 93.7% is successfully achieved on 50 wt % SiO2-50 wt % TiO2 nanocomposite photocatalyst. The catalytic performance of the nanocomposite decreases with an increase of 50%-75% in SiO2 nanoparticles. There could have been a decrease in degradation efficiency due to an excess amount of SiO2 covering TiO2 nanoparticles, which prevented photons from reaching the nanoparticles. The efficiency of cyclic decomposition of the 50 wt% SiO2-50 wt% TiO2 composite showed only a slight change in photocatalytic capacity compared to the first cycle, which ensures the durability of the sample. However, the hydroxyl radical species play the main role in the degradation process, which has been confirmed by the scavenger test. The probable reaction mechanism is also deliberated in detail. The high photocatalytic performance of novel eco-friendly SiO2-TiO2 photocatalyst make it ideal for water purification applications.

Location and conformation of pantothenate and its derivatives in Mycobacterium tuberculosis pantothenate kinase: insights into enzyme action.

Previous studies of complexes of Mycobacterium tuberculosis PanK (MtPanK) with nucleotide diphosphates and nonhydrolysable analogues of nucleoside triphosphates in the presence or the absence of pantothenate established that the enzyme has dual specificity for ATP and GTP, revealed the unusual movement of ligands during enzyme action and provided information on the effect of pantothenate on the location and conformation of the nucleotides at the beginning and the end of enzyme action. The X-ray analyses of the binary complexes of MtPanK with pantothenate, pantothenol and N-nonylpantothenamide reported here demonstrate that in the absence of nucleotide these ligands occupy, with a somewhat open conformation, a location similar to that occupied by phosphopantothenate in the `end' complexes, which differs distinctly from the location of pantothenate in the closed conformation in the ternary `initiation' complexes. The conformation and the location of the nucleotide were also different in the initiation and end complexes. An invariant arginine appears to play a critical role in the movement of ligands that takes place during enzyme action. The work presented here completes the description of the locations and conformations of nucleoside diphosphates and triphosphates and pantothenate in different binary and ternary complexes, and suggests a structural rationale for the movement of ligands during enzyme action. The present investigation also suggests that N-alkylpantothenamides could be phosphorylated by the enzyme in the same manner as pantothenate.

Crystallographic and modelling studies on Mycobacterium tuberculosis RuvA Additional role of RuvB-binding domain and inter species variability.

RuvA, along with RuvB, is involved in branch migration of heteroduplex DNA in homologous recombination. The structures of three new crystal forms of RuvA from Mycobacterium tuberculosis (MtRuvA) have been determined. The RuvB-binding domain is cleaved off in one of them. Detailed models of the complexes of octameric RuvA from different species with the Holliday junction have also been constructed. A thorough examination of the structures presented here and those reported earlier brings to light the hitherto unappreciated role of the RuvB-binding domain in determining inter-domain orientation and oligomerization. These structures also permit an exploration of the interspecies variability of structural features such as oligomerization and the conformation of the loop that carries the acidic pin, in terms of amino acid substitutions. These models emphasize the additional role of the RuvB-binding domain in Holliday junction binding. This role along with its role in oligomerization could have important biological implications.

X-ray and molecular-dynamics studies on Mycobacterium leprae single-stranded DNA-binding protein and comparison with other eubacterial SSB structures.

The crystal structures of two forms of Mycobacterium leprae single-stranded DNA-binding protein (SSB) have been determined at 2.05 and 2.8 Å resolution. Comparison of these structures with the structures of other eubacterial SSBs indicates considerable variation in their quaternary association, although the DNA-binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but could be related to variation in protein stability. Molecular-dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype Escherichia coli SSB and the individual subunits of both proteins. Together, the X-ray studies and molecular-dynamics simulations yield information on the relatively rigid and flexible regions of the molecule and on the effect of oligomerization on flexibility. The simulations provide insight into the changes in subunit structure on oligomerization. They also provide insight into the stability and time evolution of the hydrogen bonds/water bridges that connect the two pairs of monomers in the tetramer.

Crystallization and preliminary X-ray studies of a galactose-specific lectin from the seeds of bitter gourd (Momordica charantia).

A galactose-specific lectin from the seeds of bitter gourd (Momordica charantia) is a four-chain type II ribosome-inactivating protein (RIP) resulting from covalent association through a disulfide bridge between two identical copies of a two-chain unit. The available structural information on such four-chain RIPs is meagre. The bitter gourd lectin was therefore crystallized for structural investigation and the crystals have been characterized. It is anticipated that the structure of the orthorhombic crystals will be analysed using molecular replacement by taking advantage of its sequence, and presumably structural, homology to normal two-chain type II RIPs.