Structure of the Hexapeptide Xenobiotic Acetyltransferase from Pseudomonas aeruginosa,

Beaman, Todd W.; Sugantino, Michele; Roderick, Steven L.

doi:10.1021/bi980106v

Cited by 103 publications

(131 citation statements)

References 35 publications

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“…common with other CoA-dependent acyltransferases containing an L␤H domain (33)(34)(35) and as demonstrated previously for VatD (11), both substrates are bound at the interfaces between subunits. The structure of the VatD apoenzyme reveals a short tunnel formed, at the N-terminal end, by residues of the extended loop region of one monomer and the L␤H domain of the symmetry-related subunit and, at the C-terminal end, by residues provided by two adjacent L␤H domains.…”

Section: Location Of the Dalfopristin And Accoa Binding Sites-insupporting

confidence: 74%

Structural Basis of Synercid® (Quinupristin-Dalfopristin) Resistance in Gram-positive Bacterial Pathogens

Kehoe

Snidwongse

Courvalin

et al. 2003

Journal of Biological Chemistry

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Synercid®, a new semisynthetic streptogramin-derived antibiotic containing dalfopristin and quinupristin, is used in treatment of life-threatening infections caused by glycopeptide-resistant Enterococcus faecium and other bacterial pathogens. However, dissemination of genes encoding virginiamycin acetyltransferases, enzymes that confer resistance to streptogramins, threatens to limit the medical utility of the quinupristin-dalfopristin combination. Here we present structures of virginiamycin acetyltransferase D (VatD) determined at 1.8 Å resolution in the absence of ligands, at 2.8 Å resolution bound to dalfopristin, and at 3.0 Å resolution in the presence of acetyl-coenzyme A. Dalfopristin is bound by VatD in a similar conformation to that described previously for the streptogramin virginiamycin M1. However, specific interactions with the substrate are altered as a consequence of a conformational change in the pyrollidine ring that is propagated to adjacent constituents of the dalfopristin macrocycle. Inactivation of dalfopristin involves acetyl transfer from acetylcoenzyme A to the sole (O-18) hydroxy group of the antibiotic that lies close to the side chain of the strictly conserved residue, His-82. Replacement of residue 82 by alanine is accompanied by a fall in specific activity of >10 5 -fold, indicating that the imidazole moiety of His-82 is a major determinant of catalytic rate enhancement by VatD. The structure of the VatD-dalfopristin complex can be used to predict positions where further structural modification of the drug might preclude enzyme binding and thereby circumvent Synercid® resistance.

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Section: Location Of the Dalfopristin And Accoa Binding Sites-insupporting

confidence: 74%

Structural Basis of Synercid® (Quinupristin-Dalfopristin) Resistance in Gram-positive Bacterial Pathogens

Kehoe

Snidwongse

Courvalin

et al. 2003

Journal of Biological Chemistry

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“…In each case, a loop extending from the left-handed ␤-helix interacts with the neighboring subunit and covers the active site. These enzymes are presumed to have similar mechanisms, with a histidine acting as a general base catalyst for acetyl transfer (34). All of these structures have varying amounts of ␣-helical content, which is at either the amino-or carboxyl-terminal end of the left-handed ␤-helix.…”

Section: Discussionmentioning

confidence: 99%

The Structure and Mechanism of Serine Acetyltransferase from Escherichia coli

Pye

Tingey

Robson

et al. 2004

Journal of Biological Chemistry

104

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Serine acetyltransferase (SAT) catalyzes the first step of cysteine synthesis in microorganisms and higher plants. Here we present the 2.2 Å crystal structure of SAT from Escherichia coli, which is a dimer of trimers, in complex with cysteine. The SAT monomer consists of an amino-terminal ␣-helical domain and a carboxyl-terminal left-handed ␤-helix. We identify His 158 and Asp 143as essential residues that form a catalytic triad with the substrate for acetyl transfer. This structure shows the mechanism by which cysteine inhibits SAT activity and thus controls its own synthesis. Cysteine is found to bind at the serine substrate site and not the acetyl-CoA site that had been reported previously. On the basis of the geometry around the cysteine binding site, we are able to suggest a mechanism for the O-acetylation of serine by SAT. We also compare the structure of SAT with other left-handed ␤-helical structures.Serine acetyltransferase (SAT) 1 participates in the dual-step process of sulfur assimilation of microorganisms (1-3) and higher plants (4, 5). First, SAT catalyzes the production of O-acetyl-L-serine from acetyl-CoA and L-serine, and then Oacetyl serine (thiol) lyase (OAS-TL) converts O-acetyl-L-serine into L-cysteine in the presence of sulfide. Kredich and co-workers (2, 6) reported that SAT (from Salmonella typhimurium) represents the rate-limiting component and is reversibly associated with ϳ5% of the total cellular OAS-TL to form the multi-enzyme complex referred to as "cysteine synthase." Cysteine constitutes the almost exclusive metabolic entrance for reduced sulfur into cell metabolism, where it is required for biosynthesis of essential compounds including methionine, several vitamins, and metal clusters (7). The production of cysteine is therefore of biotechnological interest for pharmacological processes and as a nutritional supplement for food and feed.SAT is known to be a member of the bacterial O-acetyltransferases subfamily of O-acyltransferases (8, 9), where amino acid sequence, tertiary structures, and mechanisms are known. The folding pattern that dominates the O-acetyltransferase family is the left-handed ␤-helix, which is recognized by a hexapeptide repeating signature in which residue i is aliphatic, i ϩ 1 is usually glycine, and i ϩ 4 is a small residue, thusStructures are triangular in crosssection and are formed by parallel ␤-strands folding into a helix with three strands per turn. The first of such proteins to be studied by x-ray crystallography was the lpxA gene product that was shown to be a trimeric protein with this left-handed ␤-helical fold (10). A folding pattern arises from a hexapeptide repeat, which occurs, albeit to varying degrees, in other members of the O-acyltransferase family. SAT has such hexapeptide repeats at its carboxyl-terminal region. The three clefts between the three subunits form the catalytic centers in which a histidine residue is essential for transfer of the acetyl or succinyl moiety from CoA to the second substrate. From the sequence alignment in Fig. 1...

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“…2 A and B). Many other bacterial acyl and acetyl transferases later were shown to possess this fold (17)(18)(19)(20)(21)(22).Structures of LpxA have not been solved in the presence of substrates or inhibitors. Mutagenesis suggests that the active site is located in a large cleft between adjacent subunits (8, 23).…”

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confidence: 99%

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

Williams

Immormino

Gewirth

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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UDP-GlcNAc acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein (ACP) to the glucosamine 3-OH group of UDP-GlcNAc. LpxA is essential for the growth of Escherichia coli and related Gram-negative bacteria. The crystal structure of the E. coli LpxA homotrimer, determined previously at 2.6 Å in the absence of substrates or inhibitors, revealed that LpxA contains an unusual, left-handed parallel ␤-helix fold. We now present the crystal structure at 1.8 Å resolution of E. coli LpxA in a complex with a pentadecapeptide, peptide 920. Three peptides, each of which adopts a ␤-hairpin conformation, are bound per LpxA trimer. The peptides are located at the interfaces of adjacent subunits in the vicinity of the three active sites. Each peptide interacts with residues from both adjacent subunits. Peptide 920 is a potent inhibitor of E. coli LpxA (Ki ‫؍‬ 50 nM). It is competitive with respect to acyl-ACP but not UDP-GlcNAc. The compact ␤-turn structure of peptide 920 bound to LpxA may open previously uncharacterized approaches to the rational design of LpxA inhibitors with antibiotic activity.antibiotic ͉ crystal structure ͉ Escherichia coli ͉ inhibitor ͉ outer membrane L ipopolysaccharide constitutes the outer monolayer of the outer membrane in Gram-negative bacteria (1-3). Lipopolysaccharide maintains the integrity of the outer membrane, which functions as a barrier to molecules Ͼ500 Da (4). Lipid A (endotoxin) is the hydrophobic moiety that anchors lipopolysaccharide into the outer membrane (1, 2). It is a potent activator of the human innate immune system via Toll-like receptor 4 (TLR4) (5-7). The minimal lipopolysaccharide required for growth usually consists of two 3-deoxy-D-manno-octulosonic acid (Kdo) residues attached to lipid A ( Fig. 1; ref. 2). Because lipid A biosynthetic enzymes have no mammalian counterparts, they are attractive targets for the design of new antibiotics (10, 11).Kdo 2 -lipid A is synthesized by a conserved pathway consisting of nine enzymes (1, 2). LpxA catalyzes the first step (Fig. 1), the thermodynamically unfavorable 3-O-acylation of UDP-GlcNAc (K eq ϭ 0.01) (12-14). The crystal structure of Escherichia coli LpxA, previously determined at 2.6 Å (15), revealed that the enzyme is a homotrimer. It adopts a distinctive, left-handed parallel ␤-helix fold ( Fig. 2 A and B), which is specified by the presence of 24 complete and six partial hexad repeats (15). Three contiguous hexad repeats (18-aa residues) fold into one coil of the ␤-helix (Fig. 2 A and B). Many other bacterial acyl and acetyl transferases later were shown to possess this fold (17)(18)(19)(20)(21)(22).Structures of LpxA have not been solved in the presence of substrates or inhibitors. Mutagenesis suggests that the active site is located in a large cleft between adjacent subunits (8, 23). This region contains several conserved basic residues (K76, H122, H125, H144, H160, and R204) (23). H125 (Fig. 2 C and D) may be the catalytic ...

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Structure of the Hexapeptide Xenobiotic Acetyltransferase from Pseudomonas aeruginosa^,

Cited by 103 publications

References 35 publications

Structural Basis of Synercid® (Quinupristin-Dalfopristin) Resistance in Gram-positive Bacterial Pathogens

Structural Basis of Synercid® (Quinupristin-Dalfopristin) Resistance in Gram-positive Bacterial Pathogens

The Structure and Mechanism of Serine Acetyltransferase from Escherichia coli

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

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