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Diazobenzenesulphonate selectively abolishes stimulation of glucuronidation by UDP-N-acetylglucosamine.

ABSTRACT: 1. Basal rates of glucuronidation of oestrone (guinea pig) or of 4-nitrophenol (rat or guinea pig) were not significantly altered in sealed liver microsomal vesicles, treated with the membrane-impermeant protein-modifying agent diazobenzenesulphonate at 0.5-1.0 mM. 2. Contrarily, diazobenzenesulphonate abolished the normal stimulation of glucuronidation by UDP-N-acetylglucosamine. 3. Ultrasonication to increase microsomal permeability activated glucuronidation by 680-750% and permitted significant inhibition by diazobenzenesulphonate. 4. These findings are consistent with a model wherein glucuronyltransferases are embedded in the luminal leaflet of the endoplasmic reticulum and access of UDP-glucuronic acid to the transferases is facilitated by transmembrane carriers, which are stimulated by UDP-N-acetylglucosamine and are available to diazobenzenesulphonate; ultrasonication serves to permit access of diazobenzenesulphonate to glucuronyltransferases themselves, resulting in inhibition of their activity.

SUBMITTER: Haeger B

PROVIDER: S-EPMC1162428 | biostudies-other | 1980 Dec

REPOSITORIES: biostudies-other

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Mechanism of stimulation of microsomal UDP-glucuronosyltransferase by UDP-N-acetylglucosamine.

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Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide.

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Activity and crystal structure of Arabidopsis thaliana UDP-N-acetylglucosamine acyltransferase.

Project description:The UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase, encoded by lpxA, catalyzes the first step of lipid A biosynthesis in Gram-negative bacteria, the (R)-3-hydroxyacyl-ACP-dependent acylation of the 3-OH group of UDP-GlcNAc. Recently, we demonstrated that the Arabidopsis thaliana orthologs of six enzymes of the bacterial lipid A pathway produce lipid A precursors with structures similar to those of Escherichia coli lipid A precursors [Li, C., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 11387-11392]. To build upon this finding, we have cloned, purified, and determined the crystal structure of the A. thaliana LpxA ortholog (AtLpxA) to 2.1 Å resolution. The overall structure of AtLpxA is very similar to that of E. coli LpxA (EcLpxA) with an α-helical-rich C-terminus and characteristic N-terminal left-handed parallel β-helix (LβH). All key catalytic and chain length-determining residues of EcLpxA are conserved in AtLpxA; however, AtLpxA has an additional coil and loop added to the LβH not seen in EcLpxA. Consistent with the similarities between the two structures, purified AtLpxA catalyzes the same reaction as EcLpxA. In addition, A. thaliana lpxA complements an E. coli mutant lacking the chromosomal lpxA and promotes the synthesis of lipid A in vivo similar to the lipid A produced in the presence of E. coli lpxA. This work shows that AtLpxA is a functional UDP-GlcNAc acyltransferase that is able to catalyze the same reaction as EcLpxA and supports the hypothesis that lipid A molecules are biosynthesized in Arabidopsis and other plants.

| S-EPMC3383772 | biostudies-literature

Crystal structure and activity of Francisella novicida UDP-N-acetylglucosamine acyltransferase.

Project description:The first step of lipid A biosynthesis in Escherichia coli (E. coli) is catalyzed by LpxA (EcLpxA), an acyltransferase selective for UDP-N-acetylglucosamine (UDP-GlcNAc) and R-3-hydroxymyristoyl-acyl carrier protein (3-OH-C14-ACP), and is an essential step in majority of Gram-negative bacteria. Since the majority of lipid A species isolated from F. novicida contains 3-OH-C16 or 3-OH-C18 at its C3 and C3' positions, FnLpxA was thought to be selective for longer acyl chain (3-OH-C16 and 3-OH-C18) over short acyl chain (3-OH-C14, 3-OH-C12, and 3-OH-C10). Here we demonstrate that Francisella novicida (F. novicida) lpxA functionally complements an E. coli lpxA knockout mutant and efficiently transfers 3-OH-C14 as well as 3-OH-C16 in E. coli. Our results implicate that the acyl chain length of lipid A is determined by several factors including acyl chain selectivity of LpxA and downstream enzymes, as well as the composition of the acyl-ACP pool in vivo. We also report the crystal structure of F. novicida LpxA (FnLpxA) at 2.06 Å. The N-terminal parallel beta-helix (LβH) and C-terminal alpha-helical domain are similar to other reported structures of LpxAs. However, our structure indicates that the supposed ruler residues for hydrocarbon length, 171L in one monomer and 168H in the adjacent monomer in a functional trimer of FnLpxA, are located just 3.8 Å apart that renders not enough space for binding of 3-OH-C12 or longer acyl chains. This implicates that FnLpxA may have an alternative hydrophobic pocket, or the acyl chain may bend while binding to FnLpxA. In addition, the FnLpxA structure suggests a potential inhibitor binding site for development of antibiotics.

| S-EPMC5021990 | biostudies-literature

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