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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]

Found recently in stignomatales, the Stig cyclases catalyze the Cope rearrangement and intramolecular cyclization to produce complex indole alkaloids. Five crystal structures were solved of subfamily 1 and 2 Stig cyclases, which adopt a β‐sandwich fold like the non‐catalytic carbohydrate‐binding motif. Several complex structures were also determined of indole‐based compounds, which are bound to the hydrophobic terminal cavity, where a conserved Asp residue makes an H‐bond to the indole N and triggers the acid‐catalyzed Cope rearrangement. Through analyzing the enzyme–ligand interactions and mutagenesis experiments, several aromatic residues were found important in catalysis. Apart from a common substrate binding mode and catalytic mechanism, potential subfamily variations that may attribute to the different product specificity are implicated. These results shall expand our scope of enzymology, in particular for further investigation of the biosynthetic Cope rearrangement. Complex structure elucidations of Stig cyclases reveal a common substrate‐binding mode and mechanism of action of these enzymes. Located near the bottom of a terminal cavity in the β‐sandwich, a strictly conserved aspartate plays a vital role in catalysis, while the surrounding active‐site residues including those in the overhanging loop may determine the type of cyclization and the final product. [ABSTRACT FROM AUTHOR]

Abstract: We report the first X‐ray crystallographic structure of the “head‐to‐middle” prenyltransferase, isosesquilavandulyl diphosphate synthase, involved in biosynthesis of the merochlorin class of antibiotics. The protein adopts the ζ or cis‐prenyl transferase fold but remarkably, unlike tuberculosinol adenosine synthase and other cis‐prenyl transferases (e.g. cis‐farnesyl, decaprenyl, undecaprenyl diphosphate synthases), the large, hydrophobic side chain does not occupy a central hydrophobic tunnel. Instead, it occupies a surface pocket oriented at 90° to the hydrophobic tunnel. Product chain‐length control is achieved by squeezing out the ligand from the conventional allylic S1 binding site, with proton abstraction being achieved using a diphosphate‐Asn‐Ser relay. The structures revise and unify our thinking as to the mechanism of action of many other prenyl transferases and may also be of use in engineering new merochlorin‐class antibiotics. [ABSTRACT FROM AUTHOR]

Structures of the iridoid synthase nepetalactol synthase in the presence of NAD+, NADPH or NAD+/10- oxogeranial were solved. The 10-oxogeranial substrate binds in a transoid-O1-C3 conformation and can be reduced by hydride addition to form the byproduct S-10-oxo-citronellal. Tyr178 Oζ is positioned 2.5 &#197 from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1–C2 to form the cisoid isomer, leading to formation of the cis-enolate, together with rotation about C4–C5, which enables cyclization and lactol production. The structure is similar to that of progesterone-5β-reductase, with almost identical positioning of NADP, Lys146- (147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10- oxogeranial structure also serves as a model for β-face hydride attack in progesterone 5β-reductases and is of general interest in the context of asymmetric synthesis. [ABSTRACT FROM AUTHOR]

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease. [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]

Pyridoxal kinase catalyzes the transfer of a phosphate group from ATP to the 5′ alcohol of pyridoxine, pyridoxamine, and pyridoxal. In this work, kinetic studies were conducted to examine monovalent cation dependence of human pyridoxal kinase kinetic parameters. The results show that hPLK affinity for ATP and PL is increased manyfold in the presence of K+ when compared to Na+; however, the maximal activity of the Na+ form of the enzyme is more than double the activity in the presence of K+. Other monovalent cations, Li+, Cs+, and Rb+ do not show significant activity. We have determined the crystal structure of hPLK in the unliganded form, and in complex with MgATP to 2.0 and 2.2 Å resolution, respectively. Overall, the two structures show similar open conformation, and likely represent the catalytically idle state. The crystal structure of the MgATP complex also reveals Mg2+ and Na+ acting in tandem to anchor the ATP at the active site. Interestingly, the active site of hPLK acts as a sink to bind several molecules of MPD. The features of monovalent and divalent metal cation binding, active site structure, and vitamin B6 specificity are discussed in terms of the kinetic and structural studies, and are compared with those of the sheep and Escherichia coli enzymes. [ABSTRACT FROM AUTHOR]

Pyridoxine 5′-phosphate oxidase catalyzes the terminal step in the synthesis of pyridoxal 5′-phosphate. The cDNA for the human enzyme has been cloned and expressed in Escherichia coli. The purified human enzyme is a homodimer that exhibits a low catalytic rate constant of ∼0.2 sec−1 and Km values in the low micromolar range for both pyridoxine 5′phosphate and pyridoxamine 5′-phosphate. Pyridoxal 5′-phosphate is an effective product inhibitor. The three-dimensional fold of the human enzyme is very similar to those of the E. coli and yeast enzymes. The human and E. coli enzymes share 39% sequence identity, but the binding sites for the tightly bound FMN and substrate are highly conserved. As observed with the E. coli enzyme, the human enzyme binds one molecule of pyridoxal 5′-phosphate tightly on each subunit. [ABSTRACT FROM AUTHOR]

The crystal structure of TM-3, a small snake-venom metallo-proteinase (SVMP) isolated from Taiwan habu (Trimeresurus mucrosquamatus), was determined at 1.35 A resolution with resultant R and Rfree values of 0.181 and 0.204, respectively. The overall structure of TM-3 is an oblate ellipsoid that contains three disulfide crosslinks, Cys118-Cys197, Cys159-Cys181 and Cys161-Cys164. It exhibits the typical structural features of SVMPs and is closely related to the structure of the catalytic proteinase domain of TNFα-converting enzyme (TACE). In the present structure, the essential catalytic zinc ion was found to be replaced by a cadmium ion during crystallization, as revealed by atomic absorption analysis and X-ray data. This cadmium ion is bound to six ligands, including three conserved histidines and three water molecules, displaying the coordination geometry of a distorted octahedron. One of the water molecules is proposed to play the role of stabilizing the tetrahedral intermediate during the catalysis of SVMPs. The putative S' specificity pocket of TM-3 is relatively shallow, in contrast to the deep pockets of adamalysin II, atrolysin C and H2-proteinase, but is similar to those in acutolysin A and TACE. The shallow pocket is a consequence of the presence of the non-conserved disulfide bond Cys159-Cys181 and the residue Gln174 at the bottom of the S'j pocket. The results indicate that the active-site structure of TM-3, among the know structures of SVMPs examined thus far, is most similar to that of TACE owing to their close disulfide configurations and the S'j specificity pocket. [ABSTRACT FROM AUTHOR]

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]

Acute hepatopancreatic necrosis disease (AHPND) is a newly emergent penaeid shrimp disease which can cause 70–100% mortality in Penaeus vannamei and Penaeus monodon, and has resulted in enormous economic losses since its appearance. AHPND is caused by the specific strains of Vibrio parahaemolyticus that harbor the pVA1 plasmid and express PirAvp and PirBvp toxins. These two toxins have been reported to form a binary complex. When both are present, they lead to the death of shrimp epithelial cells in the hepatopancreas and cause the typical histological symptoms of AHPND. However, the binding mode of PirAvp and PirBvp has not yet been determined. Here, we used isothermal titration calorimetry (ITC) to measure the binding affinity of PirAvp and PirBvp. Since the dissociation constant (Kd = 7.33 ± 1.20 μM) was considered too low to form a sufficiently stable complex for X-ray crystallographic analysis, we used alternative methods to investigate PirAvp-PirBvp interaction, first by using gel filtration to evaluate the molecular weight of the PirAvp/PirBvp complex, and then by using cross-linking and hydrogen-deuterium exchange (HDX) mass spectrometry to further understand the interaction interface between PirAvp and PirBvp. Based on these results, we propose a heterotetrameric interaction model of this binary toxin complex. This model provides insight of how conformational changes might activate the PirBvp N-terminal pore-forming domain and should be helpful for devising effective anti-AHPND strategies in the future. [ABSTRACT FROM AUTHOR]

The Cope rearrangement generally occurs under thermal conditions in the absence of catalyst (examples in red half). In their Communication on page 15060 ff., Y. Zhang, W. Liu, R.‐T. Guo, et al. present the crystal structures of two groups of stigonematalean cyclases that catalyze Cope rearrangement under physiological conditions (structure example in blue half). Complex structures and mutagenesis results provide insights into substrate binding, catalytic residues, and mechanisms. [ABSTRACT FROM AUTHOR]

Dehydroascorbate reductase (DHAR) is a key enzyme for glutathione (GSH)-dependent reduction of dehydroascorbate (DHA) to recycled ascorbate (AsA) in plants, and plays a major role against the toxicity of reactive oxygen species (ROS). Previously, we proposed that the increase of AsA regeneration via enhanced DHAR activity modulates the ascorbate-glutathione cycle activity against photooxidative stress in Chlamydomonas reinhardtii . In the present work, we use site-directed mutagenesis and crystal structure analysis to elucidate the molecular basis of how C. reinhardtii DHAR (CrDHAR1) is involved in the detoxification mechanisms. Mutagenesis data show that the D21A, D21N and C22A mutations result in severe loss of the enzyme's function, suggesting crucial roles of Asp-21 and Cys-22 in substrate binding and catalysis. The mutant K11A also exhibits reduced redox activity (∼50%). The crystal structure of apo CrDHAR1 further provides insights into the proposed mechanism centering on the strictly conserved Cys-22, which is suggested to initiate the redox reactions of DHA and GSH. Furthermore, in vitro oxidation of the recombinant CrDHAR1 in the presence of 1 mM H 2 O 2 has minor effects on the K m for the substrates but significantly reduces the k cat . The enzyme's activity and its mRNA abundance in the C. reinhardtii cells are increased by treatment with 0.2–1 mM H 2 O 2 but decreased when H 2 O 2 is ≥ 1.5 mM. The latter decrease is accompanied by oxidative damage and lower AsA concentrations. These biochemical and physiological data provide new insights into the catalytic mechanism of CrDHAR1, which protects the C. reinhardtii cells from oxidative stress-induced toxicity. [ABSTRACT FROM AUTHOR]

Vespid phospholipase A 1 (vPLA 1 ) from the black-bellied hornet ( Vespa basalis ) catalyzes the hydrolysis of emulsified phospholipids and shows potent hemolytic activity that is responsible for its lethal effect. To investigate the mechanism of vPLA 1 towards its function such as hemolysis and emulsification, we isolated vPLA 1 from V. basalis venom and determined its crystal structure at 2.5 Å resolution. vPLA 1 belongs to the α/β hydrolase fold family. It contains a tightly packed β-sheet surrounded by ten α-helices and a Gly-X-Ser-X-Gly motif, characteristic of a serine hydrolyase active site. A bound phospholipid was modeled into the active site adjacent to the catalytic Ser-His-Asp triad indicating that Gln95 is located at hydrogen-bonding distance from the substrate's phosphate group. Moreover, a hydrophobic surface comprised by the side chains of Phe53, Phe62, Met91, Tyr99, Leu197, Ala167 and Pro169 may serve as the acyl chain-binding site. vPLA 1 shows global similarity to the N-terminal domain of human pancreatic lipase (HPL), but with some local differences. The lid domain and the β9 loop responsible for substrate selectivity in vPLA 1 are shorter than in HPL. Thus, solvent-exposed hydrophilic residues can easily accommodate the polar head groups of phospholipids, thereby accounting for the high activity level of vPLA 1 . Our result provides a potential explanation for the ability of vPLA 1 to hydrolyze phospholipids of cell membrane. [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]