Comparison of metal‐bound and unbound structures of aminopeptidase B proteins from Escherichia coli and Yersinia pestis

Protein degradation by aminopeptidases is involved in bacterial responses to stress. Escherichia coli produces two metal‐dependent M17 family leucine aminopeptidases (LAPs), aminopeptidase A (PepA) and aminopeptidase B (PepB). Several structures have been solved for PepA as well as other bacterial M17 peptidases. Herein, we report the first structures of a PepB M17 peptidase. The E. coli PepB protein structure was determined at a resolution of 2.05 and 2.6 Å. One structure has both Zn²⁺ and Mn²⁺, while the second structure has two Zn²⁺ ions bound to the active site. A 2.75 Å apo structure is also reported for PepB from Yersinia pestis. Both proteins form homohexamers, similar to the overall arrangement of PepA and other M17 peptidases. However, the divergent N‐terminal domain in PepB is much larger resulting in a tertiary structure that is more expanded. Modeling of a dipeptide substrate into the C‐terminal LAP domain reveals contacts that account for PepB to uniquely cleave after aspartate.     

Idiosyncrasy and identity in the prokaryotic Phe-system: crystal structure of E. coli phenylalanyl-tRNA synthetase complexed with phenylalanine and AMP

The crystal structure of Phenylalanyl‐tRNA synthetase from E. coli (EcPheRS), a class II aminoacyl‐tRNA synthetase, complexed with phenylalanine and AMP was determined at 3.05 Å resolution. EcPheRS is a (αβ)₂ heterotetramer: the αβ heterodimer of EcPheRS consists of 11 structural domains. Three of them: the N‐terminus, A1 and A2 belong to the α‐subunit and B1‐B8 domains to the β subunit. The structure of EcPheRS revealed that architecture of four helix‐bundle interface, characteristic of class IIc heterotetrameric aaRSs, is changed: each of the two long helices belonging to CLM transformed into the coil‐short helix structural fragments. The N‐terminal domain of the α‐subunit in EcPheRS forms compact triple helix domain. This observation is contradictory to the structure of the apo form of TtPheRS, where N‐terminal domain was not detected in the electron density map. Comparison of EcPheRS structure with TtPheRS has uncovered significant rearrangements of the structural domains involved in tRNA^Phe binding/translocation. As it follows from modeling experiments, to achieve a tighter fit with anticodon loop of tRNA, a shift of ～5 Å is required for C‐terminal domain B8, and of ～6 to 7 Å for the whole N terminus. EcPheRSs have emerged as an important target for the incorporation of novel amino acids into genetic code. Further progress in design of novel compounds is anticipated based on the structural data of EcPheRS.     

Crystal structure of β‐glucosidase 1A from Thermotoga neapolitana and comparison of active site mutants for hydrolysis of flavonoid glucosides

The β‐glucosidase TnBgl1A catalyses hydrolysis of O‐linked terminal β‐glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4′ and 7 glucosylation in flavonoids, and although conversion of 3‐glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure‐function relationships, the crystal structure of the nucleophile mutant TnBgl1A‐E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin‐3‐glucoside (Q3), as well as for the model substrate para‐nitrophenyl‐β‐d ‐glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3‐glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid‐3‐glucosides, improving the molecular understanding of activity in GH1 enzymes. Proteins 2017; 85:872–884. © 2016 Wiley Periodicals, Inc.     

Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short‐chain acyl esters (C2–C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4‐nitrophenyl‐β‐D ‐xylopyranoside monoacetates as substrates in a β‐xylosidase‐coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine‐substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic α/β‐hydrolase fold. TM0077 assembles into a doughnut‐shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Structural and functional characterization of a promiscuous feruloyl esterase (Est1E) from the rumen bacterium Butyrivibrio proteoclasticus

The release of polysaccharide from the plant cell wall is a key process to release the stored energy from plant biomass. Within the ruminant digestive system, a host of commensal microorganisms speed the breakdown of plant cell matter releasing fermentable sugars. The presence of phenolic compounds, most notably ferulic acid (FA), esterified within the cell wall is thought to pose a significant impediment to the degradation of the plant cell wall. The structure of a FA esterase from the ruminant bacterium Butyrivibrio proteoclasticus has been determined in two different space groups, in both the apo‐form, and the ligand bound form with FA located in the active site. The structure reveals a new lid domain that has no structural homologues in the PDB. The flexibility of the lid domain is evident by the presence of three different conformations adopted by different molecules in the crystals. In the FA‐bound structures, these conformations show sequential binding and closing of the lid domain over the substrate. Enzymatic activity assays demonstrate a broad activity against plant‐derived hemicellulose, releasing at least four aromatic compounds including FA, coumaric acid, coumarin‐3‐carboxylic acid, and cinnamic acid. The rumen is a complex ecosystem that efficiently degrades plant biomass and the genome of B. proteoclasticus contains greater than 130 enzymes, which are potentially involved in this process of which Est1E is the first to be well characterized. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Structure of WbdD: a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coli O9a

The Escherichia coli serotype O9a O‐antigen polysaccharide (O‐PS) is a model for glycan biosynthesis and export by the ATP‐binding cassette transporter‐dependent pathway. The polymannose O9a O‐PS is synthesized as a polyprenol‐linked glycan by mannosyltransferase enzymes located at the cytoplasmic membrane. The chain length of the O9a O‐PS is tightly regulated by the WbdD enzyme. WbdD first phosphorylates the terminal non‐reducing mannose of the O‐PS and then methylates the phosphate, stopping polymerization. The 2.2 Å resolution structure of WbdD reveals a bacterial methyltransferase domain joined to a eukaryotic kinase domain. The kinase domain is again fused to an extended C‐terminal coiled‐coil domain reminiscent of eukaryotic DMPK (Myotonic Dystrophy Protein Kinase) family kinases such as Rho‐associated protein kinase (ROCK). WbdD phosphorylates 2‐α‐d ‐mannosyl‐d ‐mannose (2α‐MB), a short mimic of the O9a polymer. Mutagenesis identifies those residues important in catalysis and substrate recognition and the in vivo phenotypes of these mutants are used to dissect the termination reaction. We have determined the structures of co‐complexes of WbdD with two known eukaryotic protein kinase inhibitors. Although these are potent inhibitors in vitro, they do not show any in vivo activity. The structures reveal new insight into O‐PS chain‐length regulation in this important model system.     

Apo raver1 structure reveals distinct RRM domain orientations

Raver1 is a multifunctional protein that modulates both alternative splicing and focal adhesion assembly by binding to the nucleoplasmic splicing repressor polypyrimidine tract protein (PTB) or to the cytoskeletal proteins vinculin and α‐actinin. The amino‐terminal region of raver1 has three RNA recognition motif (RRM1, RRM2, and RRM3) domains, and RRM1 interacts with the vinculin tail (Vt) domain and vinculin mRNA. We previously determined the crystal structure of the raver1 RRM1–3 domains in complex with Vt at 2.75 Å resolution. Here, we report crystal structure of the unbound raver1 RRM1–3 domains at 2 Å resolution. The apo structure reveals that a bound sulfate ion disrupts an electrostatic interaction between the RRM1 and RRM2 domains, triggering a large relative domain movement of over 30°. Superposition with other RNA‐bound RRM structures places the sulfate ion near the superposed RNA phosphate group suggesting that this is the raver1 RNA binding site. While several single and some tandem RRM domain structures have been described, to the best of our knowledge, this is the second report of a three‐tandem RRM domain structure.     

The macro domain as fusion tag for carrier‐driven crystallization

Obtaining well‐ordered crystals remains a significant challenge in protein X‐ray crystallography. Carrier‐driven crystallization can facilitate crystal formation and structure solution of difficult target proteins. We obtained crystals of the small and highly flexible SPX domain from the yeast vacuolar transporter chaperone 4 (Vtc4) when fused to a C‐terminal, non‐cleavable macro tag derived from human histone macroH2A1.1. Initial crystals diffracted to 3.3 Å resolution. Reductive protein methylation of the fusion protein yielded a new crystal form diffracting to 2.1 Å. The structures were solved by molecular replacement, using isolated macro domain structures as search models. Our findings suggest that macro domain tags can be employed in recombinant protein expression in E. coli, and in carrier‐driven crystallization.     

The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005 provides insights into the reaction mechanism of enzymes in its original family

Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H‐dependent reduction of maleylacetate, at a carbon–carbon double bond, to 3‐oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005, GraC, has been elucidated by the X‐ray diffraction method at 1.5 Å resolution. GraC is a homodimer, and each subunit consists of two domains: an N‐terminal NADH‐binding domain adopting an α/β structure and a C‐terminal functional domain adopting an α‐helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase‐like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate‐binding state and in the ligand‐free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase‐like superfamily. Proteins 2016; 84:1029–1042. © 2016 Wiley Periodicals, Inc.     

Structural conservation of the B subunit in the ammonia monooxygenase/particulate methane monooxygenase superfamily

The ammonia monooxygenase (AMO)/particulate methane monooxygenase (pMMO) superfamily is a diverse group of membrane‐bound enzymes of which only pMMO has been characterized on the molecular level. The pMMO active site is believed to reside in the soluble N‐terminal region of the pmoB subunit. To understand the degree of structural conservation within this superfamily, the crystal structure of the corresponding domain of an archaeal amoB subunit from Nitrosocaldus yellowstonii has been determined to 1.8 Å resolution. The structure reveals a remarkable conservation of overall fold and copper binding site location as well as several notable differences that may have implications for function and stability. Proteins 2014; 82:2263–2267. © 2014 Wiley Periodicals, Inc.     

Crystal structure of a novel two domain GH78 family α‐rhamnosidase from Klebsiella oxytoca with rhamnose bound

The crystal structure of the GH78 family α‐rhamnosidase from Klebsiella oxytoca (KoRha) has been determined at 2.7 Å resolution with rhamnose bound in the active site of the catalytic domain. Curiously, the putative catalytic acid, Asp 222, is preceded by an unusual non‐proline cis‐peptide bond which helps to project the carboxyl group into the active centre. This KoRha homodimeric structure is significantly smaller than those of the other previously determined GH78 structures. Nevertheless, the enzyme displays α‐rhamnosidase activity when assayed in vitro, suggesting that the additional structural domains found in the related enzymes are dispensible for function. Proteins 2015; 83:1742–1749. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism

Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that hydrolyze extracellular nucleotides to the respective monophosphate nucleotides. In the past 20 years, NTPDases belonging to mammalian, parasitic and prokaryotic domains of life have been discovered, cloned and characterized. We reveal the first structures of NTPDases from the legume plant species Trifolium repens (7WC) and Vigna unguiculata subsp. cylindrica (DbLNP). Four crystal structures of 7WC and DbLNP were determined at resolutions between 1.9 and 2.6 Å. For 7WC, structures were determined for an ‐apo form (1.89 Å) and with the product AMP (2.15 Å) and adenine and phosphate (1.76 Å) bound. For DbLNP, a structure was solved with phosphate and manganese bound (2.60 Å). Thorough kinetic data and analysis is presented. The structure of 7WC and DbLNP reveals that these NTPDases can adopt two conformations depending on the molecule and co‐factor bound in the active site. A central hinge region creates a “butterfly‐like” motion of the domains that reduces the width of the inter‐domain active site cleft upon molecule binding. This phenomenon has been previously described in Rattus norvegicus and Legionella pneumophila NTPDaseI and Toxoplasma gondii NTPDaseIII suggesting a common catalytic mechanism across the domains of life.     

The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu

Sufu (Suppressor of Fused), a two‐domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu‐like proteins have previously been identified based on sequence similarity to the N‐terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu‐like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu‐like protein. The structure revealed a striking similarity to the N‐terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ～15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu‐like proteins that are present in ～200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam.     

Crystal structure of BinB: A receptor binding component of the binary toxin from Lysinibacillus sphaericus

The binary toxin (Bin), produced by Lysinibacillus sphaericus, is composed of BinA (42 kDa) and BinB (51 kDa) proteins, which are both required for full toxicity against Culex and Anopheles mosquito larvae. Specificity of Bin toxin is determined by the binding of BinB component to a receptor present on the midgut epithelial membranes, while BinA is proposed to be a toxic component. Here, we determined the first crystal structure of the active form of BinB at a resolution of 1.75 Å. BinB possesses two distinct structural domains in its N‐ and C‐termini. The globular N‐terminal domain has a β‐trefoil scaffold which is a highly conserved architecture of some sugar binding proteins or lectins, suggesting a role of this domain in receptor‐binding. The BinB β‐rich C‐terminal domain shares similar three‐dimensional folding with aerolysin type β‐pore forming toxins, despite a low sequence identity. The BinB structure, therefore, is a new member of the aerolysin‐like toxin family, with probably similarities in the cytolytic mechanism that takes place via pore formation. Proteins 2014; 82:2703–2712. © 2014 Wiley Periodicals, Inc.     

Atomic resolution structures of Escherichia coli and Bacillus anthracis malate synthase A: Comparison with isoform G and implications for structure‐based drug discovery

Enzymes of the glyoxylate shunt are important for the virulence of pathogenic organisms such as Mycobacterium tuberculosis and Candida albicans. Two isoforms have been identified for malate synthase, the second enzyme in the pathway. Isoform A, found in fungi and plants, comprises ～530 residues, whereas isoform G, found only in bacteria, is larger by ～200 residues. Crystal structures of malate synthase isoform G from Escherichia coli and Mycobacterium tuberculosis were previously determined at moderate resolution. Here we describe crystal structures of E. coli malate synthase A (MSA) in the apo form (1.04 Å resolution) and in complex with acetyl‐coenzyme A and a competitive inhibitor, possibly pyruvate or oxalate (1.40 Å resolution). In addition, a crystal structure for Bacillus anthracis MSA at 1.70 Å resolution is reported. The increase in size between isoforms A and G can be attributed primarily to an inserted α/β domain that may have regulatory function. Upon binding of inhibitor or substrate, several active site loops in MSA undergo large conformational changes. However, in the substrate bound form, the active sites of isoforms A and G from E. coli are nearly identical. Considering that inhibitors bind with very similar affinities to both isoforms, MSA is as an excellent platform for high‐resolution structural studies and drug discovery efforts.     

Structural features of the Nostoc punctiforme debranching enzyme reveal the basis of its mechanism and substrate specificity

The debranching enzyme Nostoc punctiforme debranching enzyme (NPDE) from the cyanobacterium Nostoc punctiforme (PCC73102) hydrolyzes the α‐1,6 glycosidic linkages of malto‐oligosaccharides. Despite its high homology to cyclodextrin/pullulan (CD/PUL)‐hydrolyzing enzymes from glycosyl hydrolase 13 family (GH‐13), NPDE exhibits a unique catalytic preference for longer malto‐oligosaccharides (>G8), performing hydrolysis without the transgylcosylation or CD‐hydrolyzing activities of other GH‐13 enzymes. To investigate the molecular basis for the property of NPDE, we determined the structure of NPDE at 2.37‐Å resolution. NPDE lacks the typical N‐terminal domain of other CD/PUL‐hydrolyzing enzymes and forms an elongated dimer in a head‐to‐head configuration. The unique orientation of residues 25–55 in NPDE yields an extended substrate binding groove from the catalytic center to the dimeric interface. The substrate binding groove with a lengthy cavity beyond the ?1 subsite exhibits a suitable architecture for binding longer malto‐oligosaccharides (>G8). These structural results may provide a molecular basis for the substrate specificity and catalytic function of this cyanobacterial enzyme, distinguishing it from the classical neopullulanases and CD/PUL‐hydrolyzing enzymes. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

A mutation in an exoglucanase of Xanthomonas oryzae pv. oryzae,which confers an endo mode of activity,affects bacterial virulence,but not the induction of immune responses,in rice

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Xoo secretes a repertoire of cell wall‐degrading enzymes, including cellulases, xylanases and pectinases, to degrade various polysaccharide components of the rice cell wall. A secreted Xoo cellulase, CbsA, is not only a key virulence factor of Xoo, but is also a potent inducer of innate immune responses of rice. In this study, we solved the crystal structure of the catalytic domain of the CbsA protein to a resolution of 1.86 Å. The core structure of CbsA shows a central distorted TIM barrel made up of eight β strands with N‐ and C‐terminal loops enclosing the active site, which is a characteristic structural feature of an exoglucanase. The aspartic acid at the 131st position of CbsA was predicted to be important for catalysis and was therefore mutated to alanine to study its role in the catalysis and biological functions of CbsA. Intriguingly, the D131A CbsA mutant protein displayed the enzymatic activity of a typical endoglucanase. D131A CbsA was as proficient as wild‐type (Wt) CbsA in inducing rice immune responses, but was deficient in virulence‐promoting activity. This indicates that the specific exoglucanase activity of the Wt CbsA protein is required for this protein to promote the growth of Xoo in rice.     

Crystal structure of the functional region of Uro-adherence factor A from Staphylococcus saprophyticus reveals participation of the B domain in ligand binding

Staphylococci use cell wall‐anchored proteins as adhesins to attach to host tissues. Staphylococcus saprophyticus, a uropathogenic species, has a unique cell wall‐anchored protein, uro‐adherence factor A (UafA), which shows erythrocyte binding activity. To investigate the mechanism of adhesion by UafA, we determined the crystal structure of the functional region of UafA at 1.5 Å resolution. The structure was composed of three domains, designated as the N2, N3, and B domains, arranged in a triangular relative configuration. Hemagglutination inhibition assay with domain‐truncated mutants indicated that both N and B domains were necessary for erythrocyte binding. Based on these results, a novel manner of ligand binding in which the B domain acts as a functional domain was proposed as the adhesion mechanism of S. saprophyticus.