Search

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]

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]

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]

[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]