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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]

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]

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]

[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]