Crystal Structures of Streptococcus pneumoniaeN-Acetylglucosamine-1-phosphate Uridyltransferase, GlmU, in Apo Form at 2.33 Å Resolution and in Complex with UDP-N-Acetylglucosamine and Mg2+ at 1.96 Å Resolution

doi:10.1006/jmbi.2000.4296

Journal of Molecular Biology

Volume 305, Issue 2, 12 January 2001, Pages 279-289

https://doi.org/10.1006/jmbi.2000.4296 Get rights and content

Abstract

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential bacterial enzyme with both an acetyltransferase and a uridyltransferase activity which have been mapped to the C-terminal and N-terminal domains, respectively. GlmU performs the last two steps in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), which is an essential precursor in both the peptidoglycan and the lipopolysaccharide metabolic pathways. GlmU is therefore an attractive target for potential antibiotics. Knowledge of its three-dimensional structure would provide a basis for rational drug design. We have determined the crystal structures of Streptococcus pneumoniae GlmU (SpGlmU) in apo form at 2.33 Å resolution, and in complex with UDP-N-acetyl glucosamine and the essential co-factor Mg²⁺ at 1.96 Å resolution. The protein structure consists of an N-terminal domain with an α/β-fold, containing the uridyltransferase active site, and a C-terminal domain with a long left-handed β-sheet helix (LβH) domain. An insertion loop containing the highly conserved sequence motif Asn-Tyr-Asp-Gly protrudes from the left-handed β-sheet helix domain. In the crystal, S. pneumoniae GlmU forms exact trimers, mainly through contacts between left-handed β-sheet helix domains. UDP-N-acetylglucosamine and Mg²⁺ are bound at the uridyltransferase active site, which is in a closed form. We propose a uridyltransferase mechanism in which the activation energy of the double negatively charged phosphorane transition state is lowered by charge compensation of Mg²⁺ and the side-chain of Lys22.

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Citation Excerpt :
In the first reaction, GlcN-1P is acetylated using acetyl-CoA and in the second reaction GlcNAc-1P and UTP are used to generate UDP-GlcNAc. Since we found RjoGalU2 was a GlcN-1P specific pyrophosphorylase enzyme with a trimeric quaternary structure typically described for GlmUs [42–45], we turned our attention to the characterization of the RjoGlmU enzyme. UDP-GlcNAc is a critical metabolite in lipopolysaccharide and peptidoglycan synthetic pathways [28]; therefore, it is important to understand possible GlcN-1P partitioning in R. jostii.
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We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120 min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.
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This chapter reviews enzymic reactions involved in starch synthesis in higher plants and algae. Existing information on the properties of various starch biosynthetic enzymes in the plant, algal, and cyanobacterial systems will be described and compared. Alteration of starch structure due to the mutational effects on these biosynthetic enzymes allows postulation for some specific functions for starch synthases and branching enzymes. Finally regulation of starch synthesis at the enzymatic level will be discussed and in relation to this regulation, recent results indicating how starch content has been increased in certain plants will be indicated. A previous chapter (Shannon and Garwood, l984) in the second edition of Starch Chemistry and Technology discusses the various maize endosperm mutants or mutant combinations (26 of them) that shows an effect on the quantity or the nature of the starch formed and is still of interest. This information remains of interest and the reader is referred to that review.

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^☆: Abbreviations used: GlmU, N-acetylglucosamine phosphate uridyltransferase; UDP-GlcNAc, UDP-N-acetylglucosamine; GlcN-1-P, glucosamine 1-phosphate; GlcNAlc-1-P, N-acetylglucosamine-1-phosphate; Uap1p, UDP-N-acetylglucosamine pyrophosphorylase; LβH, left-handed β-helix structure; SpGlmU, Streptococcus pneumoniae GlmU; tr-EcGlmU, truncated form of Escherichia coli GlmU; DapD, tetrahydrodipicolinate-N-succingltransferase; Cam, Methanosarcina thermophila carbonic anhydrase; LpxA, Escherichia coli UDP-N-acetylglucosamine acetyltransferase; EcGlmU, Escherichia coli GlmU; PaXAT, Pseudomonas aeruginosa hexapeptide xenobiotic acetyltransferase

^☆☆: Edited by R. Huber

^f1: Corresponding author

^f2: E-mail address of the corresponding author: [email protected]

^f3: Present address: D. Kostrewa, Paul Scherrer Institute, Life Sciences, OSRA/007, CH-5232 Villigen PSI, Switzerland

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Journal of Molecular Biology

Regular ArticleCrystal Structures of Streptococcus pneumoniaeN-Acetylglucosamine-1-phosphate Uridyltransferase, GlmU, in Apo Form at 2.33 Å Resolution and in Complex with UDP-N-Acetylglucosamine and Mg2+ at 1.96 Å Resolution☆,☆☆

Abstract

Methods Enzymol.

Advan. Protein Chem.

J. Mol. Biol.

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

J. Mol. Biol.

FEMS Microbiol. Letters

Methods used in the structure determination of bovine mitochondrial F1 ATPase

Acta Crystallog. sect. D

Structure of the hexapeptide xenobiotic acetyltransferase from Pseudomonas aeruginosa

Biochemistry

The conformational change and active site structure of the tetrahydrodipicolinate N-succinyltransferase

Biochemistry

Crystal structure of the bifunctional N-acetylglucosamine 1-phosphate uridyltransferase from Escherichia coli: a paradigm for the related pyrophosphorylase superfamily

EMBO J.

Free R value: a novel statistical quantity for assessing the accuracy of crystal structures

Nature

Crystallographic R factor refinement by molecular dynamics

Science

Crystallography & NMR system: a new software suite for macromolecular structure determination

Acta Crystallog. sect. D

Acta Crystallog. sect. D

Accurate bond and angle parameters for X-ray protein structure refinement

Acta Crystallog. sect. A

Acetyltransfer precedes uridylyltransfer in the formation of UDP-N-acetylglucosamine in separable active sites of the bifunctional GlmU protein of Escherichia coli

Biochemistry

Regular Article
Crystal Structures of Streptococcus pneumoniaeN-Acetylglucosamine-1-phosphate Uridyltransferase, GlmU, in Apo Form at 2.33 Å Resolution and in Complex with UDP-N-Acetylglucosamine and Mg²⁺ at 1.96 Å Resolution☆,☆☆

Methods used in the structure determination of bovine mitochondrial F₁ ATPase