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When administrated orally, the vasodilating drug diltiazem can be metabolized into diacetyl diltiazem in the presence of Bacteroides thetaiotaomicron , a human gut microbe. The removal of acetyl group from the parent drug is carried out by the GDSL/SGNH-family hydrolase BT4096. Here the crystal structure of the enzyme was solved by mercury soaking and single-wavelength anomalous diffraction. The protein folds into two parts. The N-terminal part comprises the catalytic domain which is similar to other GDSL/SGNH hydrolases. The flanking C-terminal part is made up of a β-barrel subdomain and an α-helical subdomain. Structural comparison shows that the catalytic domain is most akin to acetyl-xylooligosaccharide esterase and allows a plausible binding mode of diltiazem to be proposed. The β-barrel subdomain is similar in topology to the immunoglobulin-like domains, including some carbohydrate-binding modules, of various bacterial glycoside hydrolases. Consequently, BT4096 might originally function as an oligosaccharide deacetylase with additional subdomains that could enhance substrate binding, and it acts on diltiazem just by accident. • Structure of diltiazem deacetylase BT4096 from a human gut microbe is solved. • The catalytic domain of BT4096 features GDSL/SGNH-family hydrolase. • The roles of the unique C-terminal domains are proposed by structural analyses. [ABSTRACT FROM AUTHOR]

The epimerase MoeE5 from Streptomyces viridosporus converts UDP-glucuronic acid (UDP-GlcA) to UDP-galacturonic acid (UDP-GalA) to provide the first sugar in synthesizing moenomycin, a potent inhibitor against bacterial peptidoglycan glycosyltransferases. The enzyme belongs to the UDP-hexose 4-epimerase family, and uses NAD+ as its cofactor. Here we present the complex crystal structures of MoeE5/NAD+/UDP-GlcA and MoeE5/NAD+/UDP-glucose, determined at 1.48 Å and 1.66 Å resolution. The cofactor NAD+ is bound to the N-terminal Rossmann-fold domain and the substrate is bound to the smaller C-terminal domain. In both crystals the C4 atom of the sugar moiety of the substrate is in close proximity to the C4 atom of the nicotinamide of NAD+, and the O4 atom of the sugar is also hydrogen bonded to the side chain of Tyr154, suggesting a productive binding mode. As the first complex structure of this protein family with a bound UDP-GlcA in the active site, it shows an extensive hydrogen-bond network between the enzyme and the substrate. We further built a model with the product UDP-GalA, and found that the unique Arg192 of MoeE5 might play an important role in the catalytic pathway. Consequently, MoeE5 is likely a specific epimerase for UDP-GlcA to UDP-GalA conversion, rather than a promiscuous enzyme as some other family members. Image 1 • The crystal structures of MoeE5 in complex with NAD+ and sugars were determined. • Substrate disposition in the active site suggests a productive binding mode. • Extensive hydrogen-bond network to UDP-GlcA suggests highly specificity of MoeE5. • The catalytic pathway is elucidated by constructing a product model of UDP-GalA. [ABSTRACT FROM AUTHOR]

The polyprenoid glycan carriers are produced by cis -prenyltransferases (cis -PTs), which function as heterodimers in metazoa and fungi or homodimers in bacteria, but both are found in plants, protista and archaea. Heterodimeric cis -PTs comprise catalytic and non-catalytic subunits while homodimeric enzymes contain two catalytic subunits. The non-catalytic subunits of cis -PT shows low sequence similarity to known cis -PTs and their structure information is of great interests. Here we report the crystal structure of Nus1, the non-catalytic subunit of cis -PT from Saccharomyces cerevisiae. We also investigate the heterodimer formation and active site conformation by constructing a homology model of Nus1 and its catalytic subunit. Nus1 does not contain an active site, but its C-terminus may participate in catalysis by interacting with the substrates bound to the catalytic subunit. These results provide important basis for further investigation of heterodimeric cis -PTs. • The structure of N-terminally truncated Nus1 was determined at 2.0 Å resolution. • Significant differences from other cis -prenyltransferase structures were observed. • A model of Nus1/Rer2 complex suggests conserved subunit interface and active site. • The C-terminus of Nus1 interacts with Rer2 but does not contain an RXG motif. • A majority of disease-causing mutation sites in human NgBR can now be located. [ABSTRACT FROM AUTHOR]

Abstract The antibiotic moenomycin A is a phosphoglycerate derivative with a C 25 -moenocinyl chain and a branched oligosaccharide. Formation of the C 25 -chain is catalyzed by the enzyme MoeN5 with geranyl pyrophosphate (GPP) and the sugar-linked 2- Z,E -farnesyl-3-phosphoglycerate (FPG) as its substrates. Previous complex crystal structures with GPP and long-chain alkyl glycosides suggested that GPP binds to the S1 site in a similar way as in most other α-helical prenyltransferases (PTs), and FPG is likely to assume a bent conformation in the S2 site. However, two FPG derivatives synthesized in the current study were found in the S1 site rather than S2 in their complex crystal structures with MoeN5. Apparently S1 is the preferred site for prenyl-containing ligand, and S2 binding may proceed only after S1 is occupied. Thus, like most trans -type PTs, MoeN5 may employ a sequential ionization-condensation-elimination mechanism that involves a carbocation intermediate. Highlights • The complex crystal structures of MoeN5/FQ1 and MoeN5/FQ2 were determined. • FQ1 and FQ2 were found in the other binding site for geranyl pyrophosphate (GPP). • The ordered binding suggests a sequential mechanism starting with GPP ionization. • Docking of the Sso7d tag on MoeN5 helped with crystallization. [ABSTRACT FROM AUTHOR]

Two-component system SaeRS of Staphylococcus regulates virulence factor expression through phosphorylation of the DNA-binding regulator SaeR by the sensor histidine kinase SaeS. Here crystal structures of the DNA-binding domain (DBD) of SaeR from two Staphylococcal species Staphylococcus epidermidis and Staphylococcus aureus were determined and showed similar folds. Analyzing the DNA binding activity of three mutants of Se SaeR, we observed that Thr217 is important in binding to the phosphate group of DNA and Trp219 may interact with the base pairs. Additionally, the tandem arrangement of DBD may represent a possible way for SaeR oligomerization on DNA. [ABSTRACT FROM AUTHOR]

Oxidized polyvinyl alcohol hydrolase (OPH) catalyzes the cleavage of C–C bond in β-diketone. It belongs to the α/β-hydrolase family and contains a unique lid region that covers the active site. The lid is the most variable region when pOPH from Pseudomonas sp. VM15C and sOPH from Sphingopyxis sp. 113P3 are compared. The wild-type enzymes and the pOPH mutants W255A, W255Y and W255F were analyzed for lipase activity by using p-nitrophenyl (pNP) esters as the substrates. The wild-type enzymes showed increased K m and decreased k cat / K m with the acyl chain length, and the mutants showed reduced k cat / K m for pNP acetate, indicating the importance of Trp255 in sequestering the active site from solvent. The significantly lower activity for pNP butyrate can be a result of product inhibition, as suggested by the complex crystal structures, in which butyric acid, DMSO or PEG occupied the same substrate-binding cleft. The mutant activity was retained with pNP caprylate and pNP laurate as the substrates, reflecting the amphipathic nature of the cleft. Moreover, the disulfide bond formation of Cys257/267 is important for the activity of pOPH, but it is not essential for sOPH, which has a shorter lid structure. [ABSTRACT FROM AUTHOR]

Abstract: Cationic modification of lipid A with 4-amino-4-deoxy-l-arabinopyranose (l-Ara4N) allows the pathogen Klebsiella pneumoniae to resist the antibiotic polymyxin and other cationic antimicrobial peptides. UDP-glucose dehydrogenase (Ugd) catalyzes the NAD+-dependent twofold oxidation of UDP-glucose (UPG) to produce UDP-glucuronic acid (UGA), a requisite precursor in the biosynthesis of l-Ara4N and bacterial exopolysaccharides. Here we report five crystal structures of K. pneumoniae Ugd (KpUgd) in its apo form, in complex with UPG, UPG/NADH, two UGA molecules, and finally with a C-terminal His6-tag. The UGA-complex structure differs from the others by a 14° rotation of the N-terminal domain toward the C-terminal domain, and represents a closed enzyme conformation. It also reveals that the second UGA molecule binds to a pre-existing positively charged surface patch away from the active site. The enzyme is thus inactivated by moving the catalytically important residues C253, K256 and D257 from their original positions. Kinetic data also suggest that KpUgd has multiple binding sites for UPG, and that UGA is a competitive inhibitor. The conformational changes triggered by UGA binding to the allosteric site can be exploited in designing potent inhibitors. [Copyright &y& Elsevier]

Abstract: Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general. [Copyright &y& Elsevier]

Abstract: 6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway, catalyzes the oxidative decarboxylation of 6-phosphogluconate, making ribulose 5-phosphate, along with the reduction of NADP+ to NADPH and the release of CO2. Here, we report the first apo-form crystal structure of the pathogenic Klebsiella pneumoniae 6PGDH (Kp6PGDH) and the structures of the highly homologous Escherichia coli K12 6PGDH (Ec6PGDH) complexed with substrate, substrate/NADPH and glucose at high resolution. The binding of NADPH to one subunit of the homodimeric structure triggered a 10° rotation and resulting in a 7Å movement of the coenzyme-binding domain. The coenzyme was thus trapped in a closed enzyme conformation, in contrast to the open conformation of the neighboring subunit. Comparison of our Ec/Kp6PGDH structures with those of other species illustrated how the domain conformation can be affected upon binding of the coenzyme, which in turn gives rise to concomitant movements of two important NADP+-interacting amino acids, M14 and N102. We propose that the catalysis follows an ordered binding mechanism with alternating conformational changes in the corresponding subunits, involving several related amino acid residues. [Copyright &y& Elsevier]

Abstract: l-Aspartate 4-decarboxylase catalyzes the conversion of aspartate to alanine and CO2. The wild-type enzyme was observed as dodecamers at pH 5.0. The mutation of Ser298 into Arg resulted in an almost complete loss of the enzyme activity, and caused regional structural distortion and defects in the enzyme assembly, as shown in circular dichroism spectra and gel filtration profiles. Mutating Tyr207 and Pro257 into His also resulted in inactivation of the enzyme, but did not affect the overall structure. Computer modeling suggests that Ser298 is located on the surface, and its mutation may result in enzyme disassembly, whereas Tyr207 and Pro257 are near the active site, and their mutations may cause local structure perturbation. [Copyright &y& Elsevier]

Abstract: The structural protein VP2 of infectious bursal disease virus (IBDV) spontaneously forms a dodecahedral T=1 subviral particle (SVP), and is a primary immunogen of the virus. In this study, the structure of IBDV SVP was determined in a cubic crystal and refined to 2.6Å resolution. It contains 20 independent VP2 subunits in a crystallographic asymmetric unit. Each subunit is folded mainly into a shell domain and a protrusion domain, both with the Swiss-roll topology, plus a small helical base domain. Three VP2 subunits constitute a tight trimer, which is the building block of IBDV (sub)viral particles. The structure revealed a calcium ion bound to three pairs of symmetry-related Asp31 and Asp174 to stabilize the VP2 trimer. Our results of treatment of SVP with EGTA, a Ca2+-chelating reagent, indicated that the metal-ion may be important not only in maintaining highly stable quaternary structure but also in regulating the swelling and dissociation of the icosahedral particles. A Ca2+-dependent assembly pathway was thus proposed, which involves further interactions between the trimers. The 20 independent subunits showed conformational variations, with the surface loops of the protrusion domain being the most diverse. These loops are targets of the neutralizing antibodies. Several common interactions between the surface loops were clearly observed, suggesting a possible major conformation of the immunogenic epitopes. [Copyright &y& Elsevier]

The SARS-CoV spike protein, a glycoprotein essential for viral entry, is a primary target for vaccine and drug development. Two peptides denoted HR-N(SN50) and HR-C(SC40), corresponding to the Leu/Ile/Val-rich heptad-repeat regions from the N-terminal and C-terminal segments of the SARS-CoV spike S2 sequence, respectively, were synthesized and predicted to form trimeric assembly of hairpin-like structures. The polyclonal antibodies produced by recombinant S2 protein were tested for antigenicity of the two heptad repeats. We report here the first crystallographic study of the SARS spike HR-N/HR-C complex. The crystal belongs to the triclinic space group P1 and the data-set collected to 2.98Å resolution showed noncrystallographic pseudo-222 and 3-fold symmetries. Based on these data, comparative modeling of the SARS-CoV fusion core was performed. The immunological and structural information presented herein may provide a more detailed understanding of the viral fusion mechanism as well as the development of effective therapy against SARS-CoV infection. [Copyright &y& Elsevier]

Pyridoxine-5′-phosphate oxidase catalyzes the oxidation of either the C4′ alcohol group or amino group of the two substrates pyridoxine 5′-phosphate and pyridoxamine 5′-phosphate to an aldehyde, forming pyridoxal 5′-phosphate. A hydrogen atom is removed from C4′ during the oxidation and a pair of electrons is transferred to tightly bound FMN. A new crystal form of the enzyme in complex with pyridoxal 5′-phosphate shows that the N-terminal segment of the protein folds over the active site to sequester the ligand from solvent during the catalytic cycle. Using (4′R)-[3H]PMP as substrate, nearly 100 % of the radiolabel appears in water after oxidation to pyridoxal 5′-phosphate. Thus, the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4′ carbon atom of pyridoxamine 5′-phosphate. Site mutants were made of all residues at the active site that interact with the oxygen atom or amine group on C4′ of the substrates. Other residues that make interactions with the phosphate moiety of the substrate were mutated. The mutants showed a decrease in affinity, but exhibited considerable catalytic activity, showing that these residues are important for binding, but play a lesser role in catalysis. The exception is Arg197, which is important for both binding and catalysis. The R197 M mutant enzyme catalyzed removal of the proS hydrogen atom from (4′R)-[3H]PMP, showing that the guanidinium side-chain plays an important role in determining stereospecificity. The crystal structure and the stereospecificity studies suggests that the pair of electrons on C4′ of the substrate are transferred to FMN as a hydride ion. [ABSTRACT FROM AUTHOR]

Bacterial wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising the transmembrane and ATPase subunits TagG and TagH. Here the dimeric structure of the N-terminal domain of TagH (TagH-N) was solved by single-wavelength anomalous diffraction using a selenomethionine-containing crystal, which shows an ATP-binding cassette (ABC) architecture with RecA-like and helical subdomains. Besides significant structural differences from other ABC transporters, a prominent patch of positively charged surface is seen in the center of the TagH-N dimer, suggesting a potential binding site for the glycerol phosphate chain of WTA. The ATPase activity of TagH-N was inhibited by clodronate, a bisphosphonate, in a non-competitive manner, consistent with the proposed WTA-binding site for drug targeting. • The dimeric structure of the N-terminal RecA-like domain of TagH was solved. • Positively charged surface in the center suggests a potential binding site for WTA. • The ATPase activity was inhibited by clodronate in a non-competitive manner. [ABSTRACT FROM AUTHOR]

S-adenosylhomocysteine (SAH) hydrolase catalyzes the reversible hydrolysis of SAH into adenosine and homocysteine by using NAD + as a cofactor. The enzyme from Thermotoga maritima (tmSAHH) has great potentials in industrial applications because of its hyperthermophilic properties. Here, two crystal structures of tmSAHH in complex with NAD + show both open and closed conformations despite the absence of bound substrate. Each subunit of the tetrameric enzyme is composed of three domains, namely the catalytic domain, the NAD + -binding domain and the C-terminal domain. The NAD + binding mode is clearly observed and a substrate analogue can also be modeled into the active site, where two cysteine residues in mesophilic enzymes are replaced by serine and threonine in tmSAHH. Notably, the C-terminal domain of tmSAHH lacks the second loop region of mesophilic SAHH, which is important in NAD + binding, and thus exposes the bound cofactor to the solvent. The difference explains the higher NAD + requirement of tmSAHH because of the reduced affinity. Furthermore, the feature of missing loop is consistently observed in thermophilic bacterial and archaeal SAHHs, and may be related to their thermostability. [ABSTRACT FROM AUTHOR]

Abstract: Isoprenoids, most of them synthesized by prenyltransferases (PTSs), are a class of important biologically active compounds with diverse functions. The mint geranyl pyrophosphate synthase (GPPS) is a heterotetramer composed of two LSU·SSU (large/small subunit) dimers. In addition to C10-GPP, the enzyme also produces geranylgeranyl pyrophosphate (C20-GGPP) in vitro, probably because of the conserved active-site structures between the LSU of mint GPPS and the homodimeric GGPP synthase from mustard. By contrast, the SSU lacks the conserved aspartate-rich motifs for catalysis. A major active-site cavity loop in the LSU and other trans-type PTSs is replaced by the regulatory R-loop in the SSU. Only C10-GPP, but not C20-GGPP, was produced when intersubunit interactions of the R-loop were disrupted by either deletion or multiple point mutations. The structure of the deletion mutant, determined in two different crystal forms, shows an intact (LSU·SSU)2 heterotetramer, as previously observed in the wild-type enzyme. The active-site of LSU remains largely unaltered, except being slightly more open to the bulk solvent. The R-loop of SSU acts by regulating the product release from LSU, just as does its equivalent loop in a homodimeric PTS, which prevents the early reaction intermediates from escaping the active site of the other subunit. In this way, the product-retaining function of R-loop provides a more stringent control for chain-length determination, complementary to the well-established molecular ruler mechanism. We conclude that the R-loop may be used not only to conserve the GPPS activity but also to produce portions of C20-GGPP in mint. [ABSTRACT FROM AUTHOR]

Abstract: Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the formation of pyroglutamate (pGlu) at the N-terminus of many proteins and peptides, a critical step for the maturation of these bioactive molecules. Proteins having QC activity have been identified in animals and plants, but not in bacteria. Here, we report the first bacterial QC from the plant pathogen Xanthomonas campestris (Xc). The crystal structure of the enzyme was solved and refined to 1.44-Å resolution. The structure shows a five-bladed β-propeller and exhibits a scaffold similar to that of papaya QC (pQC), but with some sequence deletions and conformational changes. In contrast to the pQC structure, the active site of XcQC has a wider substrate-binding pocket, but its accessibility is modulated by a protruding loop acting as a flap. Enzyme activity analyses showed that the wild-type XcQC possesses only 3% QC activity compared to that of pQC. Superposition of those two structures revealed that an active-site glutamine residue in pQC is substituted by a glutamate (Glu45) in XcQC, although position 45 is a glutamine in most bacterial QC sequences. The E45Q mutation increased the QC activity by an order of magnitude, but the mutation E45A led to a drop in the enzyme activity, indicating the critical catalytic role of this residue. Further mutagenesis studies support the catalytic role of Glu89 as proposed previously and confirm the importance of several conserved amino acids around the substrate-binding pocket. XcQC was shown to be weakly resistant to guanidine hydrochloride, extreme pH, and heat denaturations, in contrast to the extremely high stability of pQC, despite their similar scaffold. On the basis of structure comparison, the low stability of XcQC may be attributed to the absence of both a disulfide linkage and some hydrogen bonds in the closure of β-propeller structure. These results significantly improve our understanding of the catalytic mechanism and extreme stability of type I QCs, which will be useful in further applications of QC enzymes. [Copyright &y& Elsevier]

Abstract: Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 Å resolution. This enzyme has optimal activity in the temperature range of 70–90 °C and pH 10–11. AFL consists of an N-terminal α/β-hydrolase fold domain, a small lid domain, and a C-terminal β-barrel domain. The N-terminal catalytic domain consists of a 6-stranded β-sheet flanked by seven α-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a β-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL–substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications. [Copyright &y& Elsevier]

Borneol is a bicyclic plant monoterpene. It can be degraded by soil microorganisms through the conversion of borneol dehydrogenase (BDH) and a known camphor degradation pathway. Recombinant BDH from Pseudomonas sp. TCU-HL1 was produced in the form of inclusion body. The refolded BDH1 tends to precipitate. Insoluble recombinant BDH1 was converted into a soluble form by adding glycerol in LB medium. The k cat and k cat / K m values of soluble form BDH1 for (+)-borneol turned out to be about 34-fold and 45-fold higher, respectively, than those of the refolded enzyme. On the other hand, a gene knockout mutant, TCU-HL1Δ bdh , was constructed to investigate the possible presence of a second copy of the bdh gene in TCU-HL1 genome. A new gene, bdh2 , encoding a BDH isozyme, was identified, and the recombinant BDH2 protein was produced in a soluble form. Both bdh1 and bdh2 genes are expressed in the crude extract of wild type TCU-HL1, as shown by RT-qPCR results. Both BDH isozymes prefer to degrade (+)-borneol, rather than (−)-borneol, probably because (+)-camphor is the main form present in nature. • Two borneol dehydrogenases (BDH) from Pseudomonas sp. TCU-HL1 were characterized. • Insoluble BDH1 was converted into a soluble form by adding glycerol in LB medium. • The k cat for (+)-borneol of soluble BDH1 is 34-fold higher than that of the refolded. • A new gene, bdh2 , encoding a BDH isozyme, was identified in strain TCU-HL1. • Both BDH1 and BDH2 prefer to degrade (+)-borneol, rather than (−)-borneol. [ABSTRACT FROM AUTHOR]

Thebaine synthase 2 (THS2) that can transform (7 S)-salutaridinol 7- O -acetate to thebaine catalyzes the final step of thebaine biosynthesis in Papaver somniferum. Here, the crystal structures of THS2 and its complex with thebaine are reported, revealing the interaction network in the substrate-binding pocket. Subsequent docking and QM/MM studies was performed to further explore the catalytic mechanism of THS2. Our results suggest that T105 may abstract the proton of C4–OH from the substrate under the assistance of H89. The resulting C4–O- phenolate anion then attacks the nearby C5, and triggers intramolecular S N 2′ syn displacement with the elimination of O -acetyl group. Moreover, the latter S N 2′ reaction is the rate-determining step of the whole enzymatic reaction with an overall energy barrier of 18.8 kcal/mol. These findings are of pivotal importance to understand the mechanism of action of thebaine biosynthesis, and would guide enzyme engineering to enhance the production of opiate alkaloids via metabolic engineering. Image 1 • The crystal structure of thebaine synthase 2 (THS2) from opium poppy was solved. • The crystal structure of complex of THS2 and thebaine was reported. • A catalytic mechanism THS2 was proposed on the basis of QM/MM calculation. [ABSTRACT FROM AUTHOR]

[Display omitted] • A high-quality cryo-EM structure of malate synthase G (MSG) at 2.89 Å. • A complete structural comparison of a 723-residue MSG determined by cryo-EM and X-ray crystallography. • Cryo-EM revealed the structural dynamics of MSG. • The structural dynamics unraveled by cryo-EM, crystallography and NMR are highly correlated. Determination of sub-100 kDa (kDa) structures by cryo-electron microscopy (EM) is a longstanding but not straightforward goal. Here, we present a 2.9-Å cryo-EM structure of a 723-amino acid apo-form malate synthase G (MSG) from Escherichia coli. The cryo-EM structure of the 82-kDa MSG exhibits the same global folding as structures resolved by crystallography and nuclear magnetic resonance (NMR) spectroscopy, and the crystal and cryo-EM structures are indistinguishable. Analyses of MSG dynamics reveal consistent conformational flexibilities among the three experimental approaches, most notably that the α/β domain exhibits structural heterogeneity. We observed that sidechains of F453, L454, M629, and E630 residues involved in hosting the cofactor acetyl-CoA and substrate rotate differently between the cryo-EM apo-form and complex crystal structures. Our work demonstrates that the cryo-EM technique can be used to determine structures and conformational heterogeneity of sub-100 kDa biomolecules to a quality as high as that obtained from X-ray crystallography and NMR spectroscopy. [ABSTRACT FROM AUTHOR]

The cold-adapted xylanases can catalyze at low temperature and hold great potential in food industry applications. Here we describe the first crystal structure of a cold-adapted glycoside hydrolase (GH) family 10 xylanase XynGR40 and its complex with xylobiose at 2.15 and 2.50 Å resolution. The enzyme folds into a typical GH10 (β/α) 8 TIM-barrel, with E132 and E243 serving as the catalytic residues. The xylobiose was observed to occupy the −1 and −2 subsites. Structural comparison with a thermophilic GH10 xylanase highlighting various parameters that may explain the cold adaptation features were analyzed. Synergistic effects of the increased exposure of hydrophobic residues, the higher flexibility of substrate-binding residues, more flexible loops, and the ratios of special amino acid residues, may result in the cold adaptation of XynGR40. [ABSTRACT FROM AUTHOR]

Highlights: [•] This is the structure of GH131 family enzyme in complex with polysaccharide. [•] The broadened substrate binding cleft may explain the substrate diversity. [•] The hydrolysis of GH131 family enzyme may perform via an inverting mechanism. [ABSTRACT FROM AUTHOR]

Abstract: The pectate lyase (Bsp165PelA) from Bacillus sp. N16-5 has great potential in industrial applications because it shows high specific activity under extremely alkaline conditions. Besides, activity measurement of Bsp165PelA does not require addition of calcium, in a way different from the other pectate lyases. Here we report crystal structures of Bsp165PelA in apo-form and in complex with trigalacturonate. The parallel β-helix, active site residues and substrate binding cleft are similar to those in the other pectate lyases from Polysaccharide Lyase family 1. However, some of the highly conserved Ca2+ binding residues and secondary structures are altered in Bsp165PelA, making it difficult to coordinate with Ca2+ as in the other pectate lyases. We found Bsp165PelA forms some direct enzyme–substrate interactions instead of using Ca2+ ions bridging in the extremely alkaline environment. [Copyright &y& Elsevier]

[Display omitted] • Mammalian QCs remain indistinguishable from M28-family peptidases at sequence level. • A unique carboxylic-acid hydrogen-bond network (CAHBN) is required for QC activity. • CAHBN can discriminate QCs from other M28-family enzymes. • Mammalian-type QCs are indeed widely distributed in the three domains of life. • Interplay between CAHBN and the binuclear metal-binding center of QCs is clarified. Proteins with sequence or structure similar to those of di-Zn exopeptidases are usually classified as the M28-family enzymes, including the mammalian-type glutaminyl cyclases (QCs). QC catalyzes protein N-terminal pyroglutamate formation, a posttranslational modification important under many physiological and pathological conditions, and is a drug target for treating neurodegenerative diseases, cancers and inflammatory disorders. Without functional characterization, mammalian QCs and their orthologs remain indistinguishable at the sequence and structure levels from other M28-family proteins, leading to few reported QCs. Here, we show that a low-barrier carboxylic-acid hydrogen-bond network (CAHBN) is required for QC activity and discriminates QCs from M28-family peptidases. We demonstrate that the CAHBN-containing M28 peptidases deposited in the PDB are indeed QCs. Our analyses identify several thousands of QCs from the three domains of life, and we enzymatically and structurally characterize several. For the first time, the interplay between a CAHBN and the binuclear metal-binding center of mammalian QCs is made clear. We found that the presence or absence of CAHBN is a key discriminator for the formation of either the mono-Zn QCs or the di-Zn exopeptidases. Our study helps explain the possible roles of QCs in life. [ABSTRACT FROM AUTHOR]