Crystal Structure and Catalytic Mechanism of PglD from Campylobacter jejuni

Olivier, N.B.; Imperiali, Barbara

doi:10.1074/jbc.m801207200

Cited by 40 publications

(83 citation statements)

References 49 publications

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“…PglB-ATD was crystallized in the cubic space group P2 1 3 with a single protomer in the asymmetric unit. Previous work has indicated that bacterial acetyltransferases trimerize in solution (10,35). Whereas the structure of PglB-ATD shows a single molecule in the asymmetric unit, the homotrimer can be observed through crystallographic symmetry centered on a 3-fold axis (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For crystallographic studies, the membrane-bound phosphoglycosyltransferase domain was removed based upon a Clustal Omega alignment with PglD, thus leaving behind the acetyltransferase domain referred to herein as PglB-ATD. The structure of the apo-form of PglB-ATD was solved by molecular replacement utilizing the previously solved acetyltransferase PglD (sequence identity ϭ 34%) (10). Difficulties in crystallization of this protein were addressed by removing the final 10 amino acid residues from the C-terminal tail based upon a sequence alignment with PglD.…”

Section: Resultsmentioning

confidence: 99%

“…Key residues that interact with AcCoA in the PglB-ATD crystal structure are mostly conserved in WeeI. ) may serve to recycle histidine back to its precatalytic state by abstracting a proton from Nʦ2 following substrate turnover (10), this cannot be the case in WeeI due to the alanine moiety at this position.…”

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confidence: 99%

“…There is still an ongoing discussion over the acetyltransferase catalytic mechanism and in particular the protonation state of UDP-4-amino substrate (10,11,15). Although this study does not address this question specifically, the role that Glu 124 (PglD) plays in catalysis was explored.…”

mentioning

confidence: 99%

“…Previous structural characterization of the diNAcBac biosynthetic pathway has focused on the acetyltransferase PglD, an N-linked glycosylation pathway enzyme from C. jejuni (10,11). Additionally, genetic studies have shown that deletion of the pglD gene in C. jejuni results in the loss of the final heptasaccharide and dramatic reduction of colonization in a chick animal model; however, a low level of glycosylation was still detected by lectin blotting and mass spectrometry (12).…”

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confidence: 99%

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Biochemical Analysis and Structure Determination of Bacterial Acetyltransferases Responsible for the Biosynthesis of UDP-N,N′-Diacetylbacillosamine

Morrison

Imperiali

2013

Journal of Biological Chemistry

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Background:A connection between glycoproteins containing N,NЈ-diacetylbacillosamine and pathogenicity has previously been shown in Campylobacter jejuni. Results: Structural and kinetic studies of two bacterial acetyltransferases show the diversity within the binding pockets responsible for UDP-N,NЈ-diacetylbacillosamine production. Conclusion: Carbohydrate acetyltransferases from O-linked glycosylation pathways exhibit significant divergence from their N-linked counterparts. Significance: Acetyltransferase characterization increases our understanding of the diverse nature of bacterial glycosylation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Biochemical Analysis and Structure Determination of Bacterial Acetyltransferases Responsible for the Biosynthesis of UDP-N,N′-Diacetylbacillosamine

Morrison

Imperiali

2013

Journal of Biological Chemistry

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Biochemical investigation of an N‐acetyltransferase from Helicobacter pullorum

et al. 2021

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N-acetylated sugars are often found, for example, on the lipopolysaccharides of Gram-negative bacteria, on the S-layers of Gram-positive bacteria, and on the capsular polysaccharides. Key enzymes involved in their biosynthesis are the sugar N-acetyltransferases. Here, we describe a structural and functional analysis of one such enzyme from Helicobacter pullorum, an emerging pathogen that may be associated with gastroenteritis and gallbladder and liver diseases. For this analysis, the gene BA919-RS02330 putatively encoding an N-acetyltransferase was cloned, and the corresponding protein was expressed and purified. A kinetic analysis demonstrated that the enzyme utilizes dTDP-3-amino-3,6-dideoxy-D-glucose as a substrate as well as dTDP-3-amino-3,6-dideoxy-D-galactose, albeit at a reduced rate. In addition to this kinetic analysis, a similar enzyme from Helicobacter bilis was cloned and expressed, and its kinetic parameters were determined. Seven X-ray crystallographic structures of various complexes of the H. pullorum wild-type enzyme (or the C80T variant) were determined to resolutions of 1.7 Å or higher. The overall molecular architecture of the H. pullorum N-acetyltransferase places it into the Class II left-handedβ-helix superfamily (LβH). Taken together, the data presented herein suggest that 3-acetamido-3,6-dideoxy-D-glucose (or the galactose derivative) is found on either the H. pullorum O-antigen or in another of its complex glycoconjugates. A BLAST search suggests that more than 50 non-pylori Helicobacter spp. have genes encoding N-acetyltransferases. Given that there is little information concerning the complex glycans in non-pylori Helicobacter spp. and considering their zoonotic potential, our results provide new biochemical insight into these pathogens

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Investigation of the enzymes required for the biosynthesis of 2,3‐diacetamido‐2,3‐dideoxy‐d‐glucuronic acid in Psychrobacter cryohalolentisK5^T

et al. 2022

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Psychrobacter cryohalolentis K5 T is a Gram-negative bacterium first isolated from Siberian permafrost in 2006. It has a complex O-antigen containing L-rhamnose, D-galactose, two diacetamido-sugars, and one triacetamido-sugar. The biosynthetic pathway for one of the diacetamido-sugars, namely 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid, is presently unknown. Utilizing the published genome sequence of P. cryohalolentis K5 T , we hypothesized that the genes designated Pcryo_0613, Pcryo_0614, Pcryo_0616, and Pcryo_0615 encode for a uridine dinucleotide (UDP)-N-acetyl-D-glucosamine 6-dehydrogenase, an nicotinamide adenine dinucleotide (oxidized) (NAD + )-dependent dehydrogenase, a pyridoxal 5 0 -phosphate (PLP)-dependent aminotransferase, and an N-acetyltransferase, respectively, activities of which would be required for the biosynthesis of this unusual carbohydrate. Here we present the cloning, overexpression, and purification of these hypothetical proteins. Kinetic data on the enzymes encoded by Pcryo_0613, Pcryo_0614, and Pcryo_0615 confirmed their postulated biochemical activities. In addition, the high-resolution X-ray structures of both the internal and external aldimine forms of the aminotransferase were determined to 1.25 and 1.0 Å, respectively. Finally, the three-dimensional architecture of the N-acetyltransferase in complex with its substrate and coenzyme A was solved to 1.8 Å resolution. Strikingly, the N-acetyltransferase was shown to adopt a new motif for UDP-sugar binding. The data presented herein provide additional insight into sugar biosynthesis in Gram-negative bacteria.Abbreviations: CoA, coenzyme A; HEPES, N-2-hydroxyethylpiperazie-N 0 -2-ethanesulfonic acid; HEPPS, N-2-hydroxyethylpiperazine-N 0 -3-propanesulfonic acid; HPLC, high-performance liquid chromatography; MES, 2-(N-morpholino)ethanesulfonic acid; NAD + , nicotinamide adenine dinucleotide (oxidized); NADH, nicotinamide adenine dinucleotide (reduced); NDP, nucleotide diphosphate; Ni-NTA, nickel-nitrilotriacetic acid; PLP, pyridoxal 5 0 -phosphate; rTEV, recombinant tobacco etch virus; Tris, tris-(hydroxymethyl)aminomethane; UDP, uridine dinucleotide; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine.

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Crystal Structure and Catalytic Mechanism of PglD from Campylobacter jejuni

Cited by 40 publications

References 49 publications

Biochemical Analysis and Structure Determination of Bacterial Acetyltransferases Responsible for the Biosynthesis of UDP-N,N′-Diacetylbacillosamine

Biochemical Analysis and Structure Determination of Bacterial Acetyltransferases Responsible for the Biosynthesis of UDP-N,N′-Diacetylbacillosamine

Biochemical investigation of an N‐acetyltransferase from Helicobacter pullorum

Investigation of the enzymes required for the biosynthesis of 2,3‐diacetamido‐2,3‐dideoxy‐d‐glucuronic acid in Psychrobacter cryohalolentisK5^T

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Crystal Structure and Catalytic Mechanism of PglD from Campylobacter jejuni

Cited by 40 publications

References 49 publications

Biochemical Analysis and Structure Determination of Bacterial Acetyltransferases Responsible for the Biosynthesis of UDP-N,N′-Diacetylbacillosamine

Biochemical Analysis and Structure Determination of Bacterial Acetyltransferases Responsible for the Biosynthesis of UDP-N,N′-Diacetylbacillosamine

Biochemical investigation of an N‐acetyltransferase from Helicobacter pullorum

Investigation of the enzymes required for the biosynthesis of 2,3‐diacetamido‐2,3‐dideoxy‐d‐glucuronic acid in Psychrobacter cryohalolentisK5T

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Investigation of the enzymes required for the biosynthesis of 2,3‐diacetamido‐2,3‐dideoxy‐d‐glucuronic acid in Psychrobacter cryohalolentisK5^T