Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 å resolution

Oliva, Glaucius; Fontes, Marcos R.M.; Garratt, R.C.; Altamirano, Myriam M.; Calcagno, Mario L.; Horjales, E.

doi:10.1016/s0969-2126(01)00270-2

Cited by 84 publications

(113 citation statements)

References 35 publications

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“…7A). Indeed a comparison of the crystal structure of 12 randomly selected proteins binding a phosphomonoester moiety or moieties shows that multiple hydrogen bonds are present between amino acid side chains and the phosphomonoester, which inevitably carries two negative charges (41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53). Lysine and arginine residues form the positively charged binding pocket and often form the hydrogen bond donors as well, but other residues donating side chain hydrogen bonds also regularly occur.…”

Section: Discussionmentioning

confidence: 99%

An Electrostatic/Hydrogen Bond Switch as the Basis for the Specific Interaction of Phosphatidic Acid with Proteins

Kooijman

Tieleman

Testerink

et al. 2007

Journal of Biological Chemistry

219

255

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Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31 P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains. Phosphatidic acid (PA)7 is a minor but important bioactive lipid involved in at least three essential and likely interrelated processes in a typical eukaryotic cell. PA is a key intermediate in the biosynthetic route of the main membrane phospholipids and triglycerides (1); it is involved in membrane dynamics, i.e. fission and fusion (2-4), and has important signaling functions (5-7). The role of PA in membrane dynamics and signaling is most likely 2-fold, either via an effect on the packing properties of the membrane lipids or via the specific binding of effector proteins (4, 7-9). The origin of the specific binding of effector proteins to PA is not known.The PA binding domains thus far identified (for review, see Ref. 10) and more recently (7)) are diverse and share no apparent sequence homology, in contrast to other lipid binding domains, such as e.g. the PH, PX, FYVE, and C2 domains (11-15). One general feature that the PA binding domains do have in common is the presence of basic amino acids (7). Where examined in detail, these basic amino acids were shown to be essential for the interaction with PA, which underscores the importance of electrostatic interactions. Indeed, the negatively charged phosphomonoester head group of PA would be expected to interact electrostatically with basic amino acids in a lipid binding domain. However, such a simple electrostatic interaction cannot explain the strong preference of PA-binding proteins for PA over other, often more abundant, negatively charged phospholipids.In a recent study we showed that the phosphomonoester head group of PA has remarkable properties (8). The phosphomonoester head group is able to form an intramolecular hydrogen bond upon initial deprotonation (when its charge is Ϫ1), which stabilizes the second proton against dissociation. We provided evidence that competing hydrogen bonds, e.g. from the primary amine of the head group of phosphatidylet...

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Section: Discussionmentioning

confidence: 99%

An Electrostatic/Hydrogen Bond Switch as the Basis for the Specific Interaction of Phosphatidic Acid with Proteins

Kooijman

Tieleman

Testerink

et al. 2007

Journal of Biological Chemistry

219

255

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“…The highly clustered genes for this pathway were transcriptionally induced by (14). Previous studies have indicated that GlcN6P deaminase and the isomerase domain of GlcN6P synthase share some general similarities despite the lack of significant homology between their primary structures (21,31). Both proteins have related nucleotide-binding folds, although GlcN6P deaminase has a dehydrogenase-like six-stranded fold, whereas the fold of GlcN6P synthase is a five-stranded flavodoxin type.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Novel Glucosamine-6-Phosphate Deaminase from a Hyperthermophilic Archaeon

et al. 2005

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A key step in amino sugar metabolism is the interconversion between fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). This conversion is catalyzed in the catabolic and anabolic directions by GlcN6P deaminase and GlcN6P synthase, respectively, two enzymes that show no relationship with one another in terms of primary structure. In this study, we examined the catalytic properties and regulatory features of the glmD gene product (GlmD Tk ) present within a chitin degradation gene cluster in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Although the protein GlmD Tk was predicted as a probable sugar isomerase related to the C-terminal sugar isomerase domain of GlcN6P synthase, the recombinant GlmD Tk clearly exhibited GlcN6P deaminase activity, generating Fru6P and ammonia from GlcN6P. This enzyme also catalyzed the reverse reaction, the ammonia-dependent amination/isomerization of Fru6P to GlcN6P, whereas no GlcN6P synthase activity dependent on glutamine was observed. Kinetic analyses clarified the preference of this enzyme for the deaminase reaction rather than the reverse one, consistent with the catabolic function of GlmD Tk . In T. kodakaraensis cells, glmD Tk was polycistronically transcribed together with upstream genes encoding an ABC transporter and a downstream exo-␤-glucosaminidase gene (glmA Tk ) within the gene cluster, and their expression was induced by the chitin degradation intermediate, diacetylchitobiose. The results presented here indicate that GlmD Tk is actually a GlcN6P deaminase functioning in the entry of chitin-derived monosaccharides to glycolysis in this hyperthermophile. This enzyme is the first example of an archaeal GlcN6P deaminase and is a structurally novel type distinct from any previously known GlcN6P deaminase.Amino sugars, such as N-acetylglucosamine (GlcNAc), Nacetylgalactosamine (GalNAc), and N-acetylmuramic acid, are important building blocks for structural polysaccharides or sugar chains in several organisms. In the metabolism of these sugars, the conversion between fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P) is a key step in both anabolic and catabolic directions. The anabolic reaction is catalyzed by GlcN6P synthase (L-glutamine:D-fructose-6-phosphate amidotransferase), while catabolism is mediated by GlcN6P deaminase (Fig. 1A). GlcN6P synthase catalyzes the irreversible formation of GlcN6P and glutamate from Fru6P and glutamine and is classified in a glutamine-dependent amidotransferase family (18) comprised of an N-terminal glutamine amide transfer (GAT) domain joined to a C-terminal sugar isomerase domain (Fig. 1B). The former domain produces ammonia from glutamine, and the generated ammonia is utilized for amination of Fru6P accompanied by isomerization to GlcN6P in the latter domain. Unlike other glutamine-dependent amidotransferases displaying ammonia-dependent activity, GlcN6P synthase cannot utilize free ammonia as the nitrogen donor in place of glutamine (19).On the other hand, GlcN6P deaminase catalyzes the...

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“…Like most allosteric enzymes, GlcN6P deaminase has two extreme structural states, one displaying high substrate affinity, which is defined as the R state, and the other displaying low affinity for GlcN6P, the T state. The crystallographic structures of both the R and T forms of NagB have been solved, and the binding sites for GlcNAc6P and GlcN6P have been described (15,26). The transition from the T state to the R state, which activates the enzyme, can be driven by substrate binding that produces positive cooperativity (homotropic activation) and also by GlcNAc6P binding to the allosteric site (heterotropic activation).…”

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confidence: 99%

“…Mutants with conservative mutations (Tyr121Thr, Tyr121Ser, Tyr121Trp, Tyr254Phe, and Tyr254Trp) were constructed and were found to have modified kinetics and to be affected both in substrateinduced positive cooperativity (homotropic effect) and in the affinity of the allosteric site for GlcNAc6P (1,23) (Table 4). However, when the crystal structures were obtained, it was clear that neither Tyr121 nor Tyr254 is directly involved in GlcNAc6P binding (15,26) and that these amino acids must contribute indirectly to GlcNAc6P binding at the allosteric site. Mutants with substitutions at both positions still displayed substrate (homotropic) cooperativity (although it was reduced for mutants with mutations at Tyr254) and were also allosterically activated by GlcNAc6P, but the effect was different.…”

mentioning

confidence: 99%

Why Does Escherichia coli Grow More Slowly on Glucosamine than on N -Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

2005

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Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.Escherichia coli is renowned for its ability to use a diverse array of organic compounds as sources of carbon and energy. However, different carbohydrates do not produce the same growth yield. One of the best carbon sources, after glucose, is N-acetylglucosamine (GlcNAc), an amino sugar. The other common amino sugar, glucosamine (GlcN), is also a source of carbon and nitrogen for E. coli, but use of this sugar results in lower growth rates than use of GlcNAc. Both GlcNAc and GlcN are phosphotransferase system (PTS) sugars (38) in E. coli so that their uptake occurs concomitantly with their phosphorylation, which produces intracellular N-acetylglucosamine 6-phosphate (GlcNAc6P) and glucosamine 6-phosphate (GlcN6P). GlcNAc6P is first deacetylated by the nagA gene product, GlcNAc6P deacetylase (NagA; EC 3.5.1.25); this produces GlcN6P, which is then subject to deamination and isomerization by the nagB-encoded GlcN6P deaminase (NagB; EC 3.5.99.6; formerly GlcN6P isomerase), resulting in fructose 6-phosphate, which enters the glycolytic pathway, and ammonia. Thus, the last amino sugar-specific step in the catabolism of both GlcN and GlcNAc depends on the nagB-encoded enzyme (Fig. 1A).GlcNAc6P has many functions during the metabolism of the amino sugars, both as a metabolite and as a regulator. As well ...

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Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 å resolution

Cited by 84 publications

References 35 publications

An Electrostatic/Hydrogen Bond Switch as the Basis for the Specific Interaction of Phosphatidic Acid with Proteins

An Electrostatic/Hydrogen Bond Switch as the Basis for the Specific Interaction of Phosphatidic Acid with Proteins

Characterization of a Novel Glucosamine-6-Phosphate Deaminase from a Hyperthermophilic Archaeon

Why Does Escherichia coli Grow More Slowly on Glucosamine than on N -Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

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