Structures of the Neisseria meningitides methionine‐binding protein MetQ in substrate‐free form and bound to l‐ and d‐methionine isomers

The bacterial periplasmic methionine‐binding protein MetQ is involved in the import of methionine by the cognate MetNI methionine ATP binding cassette (ABC) transporter. The MetNIQ system is one of the few members of the ABC importer family that has been structurally characterized in multiple conformational states. Critical missing elements in the structural analysis of MetNIQ are the structure of the substrate‐free form of MetQ, and detailing how MetQ binds multiple methionine derivatives, including both l ‐ and d ‐methionine isomers. In this study, we report the structures of the Neisseria meningitides MetQ in substrate‐free form and in complexes with l ‐methionine and with d ‐methionine, along with the associated binding constants determined by isothermal titration calorimetry. Structures of the substrate‐free (N238A) and substrate‐bound N. meningitides MetQ are related by a “Venus‐fly trap” hinge‐type movement of the two domains accompanying methionine binding and dissociation. l ‐ and d ‐methionine bind to the same site on MetQ, and this study emphasizes the important role of asparagine 238 in ligand binding and affinity. A thermodynamic analysis demonstrates that ligand‐free MetQ associates with the ATP‐bound form of MetNI ～40 times more tightly than does liganded MetQ, consistent with the necessity of dissociating methionine from MetQ for transport to occur.     

Probing the active site of the sugar isomerase domain from E. coli arabinose-5-phosphate isomerase via X-ray crystallography

Lipopolysaccharide (LPS) biosynthesis represents an underexploited target pathway for novel antimicrobial development to combat the emergence of multidrug‐resistant bacteria. A key player in LPS synthesis is the enzyme D ‐arabinose‐5‐phosphate isomerase (API), which catalyzes the reversible isomerization of D ‐ribulose‐5‐phosphate to D ‐arabinose‐5‐phosphate, a precursor of 3‐deoxy‐D ‐manno‐octulosonate that is an essential residue of the LPS inner core. API is composed of two main domains: an N‐terminal sugar isomerase domain (SIS) and a pair of cystathionine‐β‐synthase domains of unknown function. As the three‐dimensional structure of an enzyme is a prerequisite for the rational development of novel inhibitors, we present here the crystal structure of the SIS domain of a catalytic mutant (K59A) of E. coli D ‐arabinose‐5‐phosphate isomerase at 2.6‐Å resolution. Our structural analyses and comparisons made with other SIS domains highlight several potentially important active site residues. In particular, the crystal structure allowed us to identify a previously unpredicted His residue (H88) located at the mouth of the active site cavity as a possible catalytic residue. On the basis of such structural data, subsequently supported by biochemical and mutational experiments, we confirm the catalytic role of H88, which appears to be a generally conserved residue among two‐domain isomerases.     

Probing the catalytic mechanism of a C-3'-methyltransferase involved in the biosynthesis of D-tetronitrose

D ‐Tetronitrose is a nitro‐containing tetradeoxysugar found attached to the antitumor and antibacterial agent tetrocarcin A. The biosynthesis of this highly unusual sugar in Micromonospora chalcea requires 10 enzymes. The fifth step in the pathway involves the transfer of a methyl group from S‐adenosyl‐L ‐methionine (SAM) to the C‐3′ carbon of dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐D ‐glucose. The enzyme responsible for this transformation is referred to as TcaB9. It is a monomeric enzyme with a molecular architecture based around three domains. The N‐terminal motif contains a binding site for a structural zinc ion. The middle‐ and C‐terminal domains serve to anchor the SAM and dTDP–sugar ligands, respectively, to the protein, and the active site of TcaB9 is wedged between these two regions. For this investigation, the roles of Tyr 76, His 181, Tyr 222, Glu 224, and His 225, which form the active site of TcaB9, were probed by site‐directed mutagenesis, kinetic analyses, and X‐ray structural studies. In addition, two ternary complexes of the enzyme with bound S‐adenosyl‐L ‐homocysteine and either dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐D ‐glucose or dTDP‐3‐amino‐2,3,6‐trideoxy‐D ‐galactose were determined to 1.5 or 1.6 Å resolution, respectively. Taken together, these investigations highlight the important role of His 225 in methyl transfer. In addition, the structural data suggest that the methylation reaction occurs via retention of configuration about the C‐3′ carbon of the sugar.     

The Allosteric Regulatory Mechanism of the Escherichia coli MetNI Methionine ATP Binding Cassette (ABC) Transporter

The MetNI methionine importer of Escherichia coli, an ATP binding cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of d- and l-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of the MetNI mechanism has thus far been limited to a series of crystal structures in an inward-facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by l-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well.     

Crystal structure of decaprenylphosphoryl‐β‐ D‐ribose 2'‐epimerase from Mycobacterium smegmatis

Decaprenylphosphoryl‐β‐D ‐ribose 2'‐epimerase (DprE1) is an essential enzyme in the biosynthesis of cell wall components and a target for development of anti‐tuberculosis drugs. We determined the crystal structure of a truncated form of DprE1 from Mycobacterium smegmatis in two crystal forms to up to 2.35 Å resolution. The structure extends from residue 75 to the C‐terminus and shares homology with FAD‐dependent oxidoreductases of the vanillyl‐alcohol oxidase family including the DprE1 homologue from M. tuberculosis. The M. smegmatis DprE1 structure reported here provides further insights into the active site geometry of this tuberculosis drug target. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Crystal structure and fluorescence studies reveal the role of helical dimeric interface of staphylococcal FabG1 in positive cooperativity for NADPH

Crystal structure of Staphylococcal β‐ketoacyl‐ACP reductase 1 (SaFabG1) complexed with NADPH is determined at 2.5 Å resolution. The enzyme is essential in FAS‐II pathway and utilizes NADPH to reduce β‐ketoacyl‐ACP to (S)‐β‐hydroxyacyl‐ACP. Unlike the tetrameric FabGs, dimeric SaFabG1 shows positive homotropic cooperativity towards NADPH. Analysis of FabG:NADPH binary crystal structure endorses that NADPH interacts directly with the helices α4 and α5 those are present on a dimerization interface. A steady shift in tryptophan (of α4 helix) emission peak upon steady increment of NADPH concentration reveals that the dimeric interface is formed by α4‐α4′ and α5‐α5′ helices. This dimeric interface imparts positive homotropic cooperativity towards NADPH. PEG, a substrate mimicking molecule is also found near the active site of the enzyme. Proteins 2012; © 2011 Wiley Periodicals, Inc.     

The crystal structure of the Y140F mutant of ADP-l-glycero-d-manno-heptose 6-epimerase bound to ADP-��-d-mannose suggests a one base mechanism

Bacteria synthesize a wide array of unusual carbohydrate molecules, which they use in a variety of ways. The carbohydrate L ‐glycero‐D ‐manno‐heptose is an important component of lipopolysaccharide and is synthesized in a complex series of enzymatic steps. One step involves the epimerization at the C6″ position converting ADP‐D ‐glycero‐D ‐manno‐heptose into ADP‐L ‐glycero‐D ‐manno‐heptose. The enzyme responsible is a member of the short chain dehydrogenase superfamily, known as ADP‐L ‐glycero‐D ‐manno‐heptose 6‐epimerase (AGME). The structure of the enzyme was known but the arrangement of the catalytic site with respect to the substrate is unclear. We now report the structure of AGME bound to a substrate mimic, ADP‐β‐D ‐mannose, which has the same stereochemical configuration as the substrate. The complex identifies the key residues and allows mechanistic insight into this novel enzyme.     

Efficient synthesis of a long carbohydrate chain alkyl glycoside catalyzed by cyclodextrin glycosyltransferase (CGTase)

Alkyl glycosides with long carbohydrate groups are surfactants with attractive properties but they are very difficult to synthesize. Here, a method for extension of the carbohydrate group of commercially available dodecyl‐β‐d ‐maltoside (DDM) is presented. DDM was converted to dodecyl‐β‐d ‐maltooctaoside (DDMO) in a single step by using a CGTase as catalyst and α‐cyclodextrin (α‐CD) as glycosyl donor. The coupling reaction is under kinetic control and the maximum yield depends on the selectivity of the enzyme. The Bacillus macerans CGTase favored the coupling reaction while the Thermoanaerobacter enzyme also catalyzed disproportionation reactions leading to a broader product range. A high ratio α‐CD/DDM favored a high yield of DDMO and yields up to 80% were obtained using the B. macerans enzyme as catalyst. Biotechnol. Bioeng. 2009; 104: 854–861. © 2009 Wiley Periodicals, Inc.     

Structural insight into the catalytic mechanism of gluconate 5‐dehydrogenase from Streptococcus suis: Crystal structures of the substrate‐free and quaternary complex enzymes

Gluconate 5‐dehydrogenase (Ga5DH) is an NADP(H)‐dependent enzyme that catalyzes a reversible oxidoreduction reaction between D ‐gluconate and 5‐keto‐D ‐gluconate, thereby regulating the flux of this important carbon and energy source in bacteria. Despite the considerable amount of physiological and biochemical knowledge of Ga5DH, there is little physical or structural information available for this enzyme. To this end, we herein report the crystal structures of Ga5DH from pathogenic Streptococcus suis serotype 2 in both substrate‐free and liganded (NADP⁺/D ‐gluconate/metal ion) quaternary complex forms at 2.0 Å resolution. Structural analysis reveals that Ga5DH adopts a protein fold similar to that found in members of the short chain dehydrogenase/reductase (SDR) family, while the enzyme itself represents a previously uncharacterized member of this family. In solution, Ga5DH exists as a tetramer that comprised four identical ～29 kDa subunits. The catalytic site of Ga5DH shows considerable architectural similarity to that found in other enzymes of the SDR family, but the S. suis protein contains an additional residue (Arg104) that plays an important role in the binding and orientation of substrate. The quaternary complex structure provides the first clear crystallographic evidence for the role of a catalytically important serine residue and also reveals an amino acid tetrad RSYK that differs from the SYK triad found in the majority of SDR enzymes. Detailed analysis of the crystal structures reveals important contributions of Ca²⁺ ions to active site formation and of specific residues at the C‐termini of subunits to tetramer assembly. Because Ga5DH is a potential target for therapy, our findings provide insight not only of catalytic mechanism, but also suggest a target of structure‐based drug design.     

Structure‐based investigation into the functional roles of the extended loop and substrate‐recognition sites in an endo‐β‐1,4‐d‐mannanase from the Antarctic springtail,Cryptopygus antarcticus

Endo‐β‐1,4‐d ‐mannanase from the Antarctic springtail, Cryptopygus antarcticus (CaMan), is a cold‐adapted β‐mannanase that has the lowest optimum temperature (30°C) of all known β‐mannanases. Here, we report the apo‐ and mannopentaose (M5) complex structures of CaMan. Structural comparison of CaMan with other β‐mannanases from the multicellular animals reveals that CaMan has an extended loop that alters topography of the active site. Structural and mutational analyses suggest that this extended loop is linked to the cold‐adapted enzymatic activity. From the CaMan‐M5 complex structure, we defined the mannose‐recognition subsites and observed unreported M5 binding site on the surface of CaMan. Proteins 2014; 82:3217–3223. © 2014 Wiley Periodicals, Inc.     

Over‐expression of specific HvCslF cellulose synthase‐like genes in transgenic barley increases the levels of cell wall (1,3;1,4)‐β‐d‐glucans and alters their fine structure

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)‐β‐d ‐glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)‐β‐d ‐glucans in the Poaceae is mediated, in part at least, by the cellulose synthase‐like CslF family of genes. Over‐expression of the barley CslF6 gene under the control of an endosperm‐specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)‐β‐d ‐glucan content in grain of transgenic barley. Analyses of (1,3;1,4)‐β‐d ‐glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)‐β‐d ‐glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)‐β‐d ‐glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)‐β‐d ‐glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)‐β‐d ‐glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)‐β‐d ‐glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)‐β‐d ‐glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.     

Metamorphic changes in localization of sugars in skin of the leopard frog,Rana pipiens

A lectin histochemical study was carried out to determine the distribution of specific sugars in glycoconjugates within an important osmoregulatory organ, amphibian skin. Paraffin sections were made of Rana pipiens skin from dorsal and ventral regions of aquatic larvae in representative developmental stages as well as from several body regions of semiaquatic adult frogs. Sections were incubated with horseradish peroxidase (HRP)‐conjugated lectins, which bind to specific terminal sugar residues of glycoconjugates. Such sites were visualized by DAB‐H₂O₂. The following HRP‐lectins were used: UEA‐1 for α‐L ‐fucose, SBA for N‐acetyl‐D ‐galactosamine, WGA for N‐acetyl‐β‐D ‐glucosamine, PNA for β‐galactose, and Con A for α‐mannose. We found that lectin binding patterns in larvae change during metamorphic climax as the skin undergoes extensive histological remodeling; this results in adult skin with staining patterns that are specific for each lectin and are similar in all body regions. Such findings in R. pipiens provide additional insight into the localization of molecules involved in osmoregulation in amphibian skin. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Glycoside‐specific glycosyltransferases catalyze regio‐selective sequential glucosylations for a sesame lignan,sesaminol triglucoside

Sesame (Sesamum indicum) seeds contain a large number of lignans, phenylpropanoid‐related plant specialized metabolites. (+)‐Sesamin and (+)‐sesamolin are major hydrophobic lignans, whereas (+)‐sesaminol primarily accumulates as a water‐soluble sesaminol triglucoside (STG) with a sugar chain branched via β1→2 and β1→6‐O‐glucosidic linkages [i.e. (+)‐sesaminol 2‐O‐β‐d ‐glucosyl‐(1→2)‐O‐β‐d ‐glucoside‐(1→6)‐O‐β‐d ‐glucoside]. We previously reported that the 2‐O‐glucosylation of (+)‐sesaminol aglycon and β1→6‐O‐glucosylation of (+)‐sesaminol 2‐O‐β‐d ‐glucoside (SMG) are mediated by UDP‐sugar‐dependent glucosyltransferases (UGT), UGT71A9 and UGT94D1, respectively. Here we identified a distinct UGT, UGT94AG1, that specifically catalyzes the β1→2‐O‐glucosylation of SMG and (+)‐sesaminol 2‐O‐β‐d ‐glucosyl‐(1→6)‐O‐β‐d ‐glucoside [termed SDG(β1→6)]. UGT94AG1 was phylogenetically related to glycoside‐specific glycosyltransferases (GGTs) and co‐ordinately expressed with UGT71A9 and UGT94D1 in the seeds. The role of UGT94AG1 in STG biosynthesis was further confirmed by identification of a STG‐deficient sesame mutant that predominantly accumulates SDG(β1→6) due to a destructive insertion in the coding sequence of UGT94AG1. We also identified UGT94AA2 as an alternative UGT potentially involved in sugar–sugar β1→6‐O‐glucosylation, in addition to UGT94D1, during STG biosynthesis. Yeast two‐hybrid assays showed that UGT71A9, UGT94AG1, and UGT94AA2 were found to interact with a membrane‐associated P450 enzyme, CYP81Q1 (piperitol/sesamin synthase), suggesting that these UGTs are components of a membrane‐bound metabolon for STG biosynthesis. A comparison of kinetic parameters of these UGTs further suggested that the main β‐O‐glucosylation sequence of STG biosynthesis is β1→2‐O‐glucosylation of SMG by UGT94AG1 followed by UGT94AA2‐mediated β1→6‐O‐glucosylation. These findings together establish the complete biosynthetic pathway of STG and shed light on the evolvability of regio‐selectivity of sequential glucosylations catalyzed by GGTs.     

Crystal structure of D‐glycero‐Β‐D‐manno‐heptose‐1‐phosphate adenylyltransferase from Burkholderia pseudomallei

The crystal structure of HldC from B. pseudomallei (BpHldC), the fourth enzyme of the heptose biosynthesis pathway, has been determined. BpHldC converts ATP and d ‐glycero‐β‐d ‐manno‐heptose‐1‐phosphate into ADP‐d ‐glycero‐β‐d ‐manno‐heptose and pyrophosphate. The crystal structure of BpHldC belongs to the nucleotidyltransferase α/β phosphodiesterase superfamily sharing a common Rossmann‐like α/β fold with a conserved T/HXGH sequence motif. The invariant catalytic key residues of BpHldC indicate that the core catalytic mechanism of BpHldC may be similar to that of other closest homologues. Intriguingly, a reorientation of the C‐terminal helix seems to guide open and close states of the active site for the catalytic reaction.     

Catabolism of l–methionine in the formation of sulfur and other volatiles in melon (Cucumis melo L.) fruit

Sulfur‐containing aroma volatiles are important contributors to the distinctive aroma of melon and other fruits. Melon cultivars and accessions differ in the content of sulfur‐containing and other volatiles. l –methionine has been postulated to serve as a precursor of these volatiles. Incubation of melon fruit cubes with ¹³C‐ and ²H‐labeled l –methionine revealed two distinct catabolic routes into volatiles. One route apparently involves the action of an l ‐methionine aminotransferase and preserves the main carbon skeleton of l ‐methionine. The second route apparently involves the action of an l ‐methionine‐γ–lyase activity, releasing methanethiol, a backbone for formation of thiol‐derived aroma volatiles. Exogenous l ‐methionine also generated non‐sulfur volatiles by further metabolism of α–ketobutyrate, a product of l ‐methionine‐γ–lyase activity. α–Ketobutyrate was further metabolized into l –isoleucine and other important melon volatiles, including non‐sulfur branched and straight‐chain esters. Cell‐free extracts derived from ripe melon fruit exhibited l ‐methionine‐γ–lyase enzymatic activity. A melon gene (CmMGL) ectopically expressed in Escherichia coli, was shown to encode a protein possessing l ‐methionine‐γ–lyase enzymatic activity. Expression of CmMGL was relatively low in early stages of melon fruit development, but increased in the flesh of ripe fruits, depending on the cultivar tested. Moreover, the levels of expression of CmMGL in recombinant inbred lines co‐segregated with the levels of sulfur‐containing aroma volatiles enriched with +1 m/z unit and postulated to be produced via this route. Our results indicate that l ‐methionine is a precursor of both sulfur and non‐sulfur aroma volatiles in melon fruit.     

Nucleotide binding to the homodimeric MJ0796 protein: a computational study of a prokaryotic ABC transporter NBD dimer

Transport by ABC proteins requires a cycle of ATP-driven conformational changes of the nucleotide binding domains (NBDs). We compare three molecular dynamics simulations of dimeric MJ0796: with ATP was present at both NBDs; with ATP at one NBD but ADP at the other; and without any bound ATP. In the simulation with ATP present at both NBDs, the dimeric protein interacts with the nucleotides in a symmetrical manner. However, if ADP is present at one binding site then both NBD-NBD and protein-ATP interactions are enhanced at the opposite site.     

Mechanistic Studies of dUTPases

The essential enzyme dUTPase is responsible for preventive DNA repair via exclusion of uracil. Lack or inhibition of the enzyme induces thymine‐less cell death in cells performing active DNA synthesis, serving therefore as an important chemotherapeutic target. In the present work, employing differential circular dichroism spectroscopy, we show that D. mel. dUTPase, a recently described eukaryotic model, has a similar affinity of binding towards α,β‐imino‐dUTP as compared to the prokaryotic E. coli enzyme. However, in contrast to the prokaryotic dUTPase, the nucleotide exerts significant protection against tryptic digestion at a specific tryptic site 20 Å far from the active site in the fly enzyme. This result indicates that binding of the nucleotide in the active site induces an allosteric conformational change within the central threefold channel of the homotrimer exclusively in the eukaryotic enzyme. Nucleotide binding induced allosterism in the D. mel. dUTPase, but not in the E. coli enzyme, might be associated with the altered hydropathy of subunit interfaces in these two proteins.     

Crystal stuctures of MglB‐2 (TP0684), a topologically variant d‐glucose‐binding protein from Treponema pallidum,reveal a ligand‐induced conformational change

Previously, we determined the crystal structure of apo‐TpMglB‐2, a d ‐glucose‐binding component of a putative ABC transporter from the syphilis spirochete Treponema pallidum. The protein had an unusual topology for this class of proteins, raising the question of whether the d ‐glucose‐binding mode would be different in TpMglB‐2. Here, we present the crystal structures of a variant of TpMglB‐2 with and without d ‐glucose bound. The structures demonstrate that, despite its aberrant topology, the protein undergoes conformational changes and binds d ‐glucose similarly to other Mgl‐type proteins, likely facilitating d ‐glucose uptake in T. pallidum.     

A Salmonella Typhi homologue of bacteriophage muramidases controls typhoid toxin secretion

Unlike other Salmonella , which can infect a broad range of hosts causing self‐limiting infection , Salmonella Typhi is an exclusively human pathogen that causes typhoid fever, a life‐threatening systemic disease. Typhoid toxin is a unique virulence factor of Salmonella Typhi, which is expressed when the bacteria are within mammalian cells. Here, we report that an N ‐acetyl‐β‐D ‐muramidase similar to phage endolysins encoded within the same pathogenicity islet as the toxin is required for typhoid toxin secretion. Genetic and functional analysis of TtsA revealed unique amino acids at its predicted peptidoglycan‐binding domain that are essential for protein secretion and that distinguishes this protein from other homologues. We propose that TtsA defines a new protein secretion mechanism recently evolved from the machine that mediates phage release.