Structural Analysis of N-acetylglucosamine-6-phosphate Deacetylase Apoenzyme from Escherichia coli

Ferreira, Frederico Moraes; Mendoza‐Hernández, Guillermo; Castañeda‐Bueno, María; Aparício, Ricardo; Fischer, Hannes; Calcagno, Mario L.; Oliva, Glaucius

doi:10.1016/j.jmb.2006.03.024

Cited by 24 publications

(42 citation statements)

References 55 publications

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“…The Ni enzyme has the highest K m while the Zn enzyme has the lowest. Similar trends in the range of the catalytic constants were reported previously for the Co, Zn, Mn and Fe reconstitutions of apo-NagA from E. coli (7).…”

Section: Kinetic Constants For Nagasupporting

confidence: 87%

N-Acetyl-d-glucosamine-6-phosphate Deacetylase: Substrate Activation via a Single Divalent Metal Ion

Hall

Xiang

et al. 2007

Biochemistry

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NagA is a member of the amidohydrolase superfamily and catalyzes the deacetylation of N-acetyl-D-glucosamine-6-phosphate. The catalytic mechanism of this enzyme was addressed by the characterization of the catalytic properties of metal-substituted derivatives of NagA from Escherichia coli with a variety of substrate analogs. The reaction mechanism is of interest since NagA from bacterial sources is found with either one or two divalent metal ions in the active site. This observation indicates that there has been a divergence in the evolution of NagA and suggests that there are fundamental differences in the mechanistic details for substrate activation and hydrolysis. NagA from E. coli was inactivated by the removal of the zinc bound to the active site and the apo-enzyme reactivated upon incubation with one equivalent of Zn 2+ , Cd 2+ , Co 2+ , Mn 2+ , Ni 2+ or Fe 2+ . In the proposed catalytic mechanism the reaction is initiated by the polarization of the carbonyl group of the substrate via a direct interaction with the divalent metal ion and His-143. The invariant aspartate found at the end of β-strand 8 in all members of the amidohydrolase superfamily abstracts a proton from the metal-bound water molecule (or hydroxide) to promote the hydrolytic attack on the carbonyl group of the substrate. A tetrahedral intermediate is formed and then collapses with cleavage of the C-N bond after proton transfer to the leaving group amine by Asp-273. The lack of a solvent isotope effect by D 2 O and the absence of any changes to the kinetic constants with increases in solvent viscosity indicate that net product formation is not limited to any significant extent by proton transfer steps or the release of products. N-trifluoroacetyl-D-glucosamine-6-phosphate is hydrolyzed by NagA 26-fold faster than the corresponding N-acetyl derivative. This result is consistent with the formation or collapse of the tetrahedral intermediate as the rate limiting step in the catalytic mechanism of NagA.NagA 1 catalyzes the hydrolytic cleavage of N-acetyl-D-glucosamine-6-phosphate as illustrated in Scheme 1. The deacetylation of this compound provides a source of carbon and nitrogen by preparing this substrate for entry into the glycolytic pathway. This reaction is a key step in the catabolism of N-acetyl-D-glucosamine, derived from the degradation of chitin, and is an essential component for the biosynthesis of lipopolysaccharides and peptidoglycans. More recently, the reaction catalyzed by NagA has been shown to be an important step in the recycling of cell wall murein (1-3).The purification of NagA from Escherichia coli was originally reported by White and Pasternak (4). The enzyme oligomerizes as a tetramer and each subunit contains a reactive sulfhydryl group near the active site (5). In the forward reaction, inhibition occurs at high substrate † This work was supported in part by the NIH (GM 71790) and the Robert A. Welch Foundation (A-840). RSH was supported by a Chemical Biology Interface Training Grant (GM 08523). * To whom cor...

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Section: Kinetic Constants For Nagasupporting

confidence: 87%

N-Acetyl-d-glucosamine-6-phosphate Deacetylase: Substrate Activation via a Single Divalent Metal Ion

Hall

Xiang

et al. 2007

Biochemistry

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“…TatD and the PHP domain of DNA polymerase III exhibit metal-dependent nuclease activity (6,36) catalyzing the hydrolysis of a phosphoester bond. The amidohydrolases catalyze the hydrolysis of carbon-nitrogen bonds in amido or amino groups and phosphoester bonds (29,31,37,38). Holm and Sander (8) first demonstrated the existence of the amidohydrolase superfamily, which have a distorted ( R) 8 barrel fold bearing metal sites consisting of conserved histidine and aspartate residues.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of Monofunctional Histidinol Phosphate Phosphatase from Thermus thermophilus HB8

et al. 2007

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Monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8, which catalyzes the dephosphorylation of l-histidinol phosphate, belongs to the PHP family, together with the PHP domain of bacterial DNA polymerase III and family X DNA polymerase. We have determined the structures of the complex with a sulfate ion, the complex with a phosphate ion, and the unliganded form at 1.6, 2.1, and 1.8 A resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of a distorted (betaalpha)7 barrel with one linker and one C-terminal tail. Three metal sites located on the C-terminal side of the barrel are occupied by Fe1, Fe2, and Zn ions, respectively, forming a trinuclear metal center liganded by seven histidines, one aspartate, one glutamate, and one hydroxide with two Fe ions bridged by the hydroxide. In the complexes, the sulfate or phosphate ion is coordinated to three metal ions, resulting in octahedral, trigonal bipyramidal, and tetrahedral geometries around the Fe1, Fe2, and Zn ions, respectively. The ligand residues are derived from the four motifs that characterize the PHP family and from two motifs conserved in histidinol phosphate phosphatases. The (betaalpha)7 barrel and the metal cluster are closely related in nature and architecture to the (betaalpha)8 barrel and the mononuclear or dinuclear metal center in the amidohydrolase superfamily, respectively. The coordination behavior of the phosphate ion toward the metal center supports the mechanism in which the bridging hydroxide makes a direct attack on the substrate phosphate tridentately bound to the two Fe ions and Zn ion to hydrolyze the phosphoester bond.

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“…NagA from E. coli and B. subtilis have been purified, both enzymes are dimeric and their crystallographic structures have been determined [Ferreira et al, 2006;Souza et al, 1997;Vincent et al, 2004]. Both enzymes require a metal cofactor for activity.…”

Section: The Enzymes Of the Glcn And Glcnac Degradative Pathway Nagbmentioning

confidence: 99%

“…Whereas Fe was found in the B. subtilis enzyme, Zn was found in the E. coli enzyme. Various divalent metal ions were shown to function with the E. coli enzyme [Ferreira et al, 2006;Hall et al, 2007].…”

Section: The Enzymes Of the Glcn And Glcnac Degradative Pathway Nagbmentioning

confidence: 99%

Regulation of the Utilization of Amino Sugars by Escherichia coli and Bacillus subtilis: Same Genes, Different Control

Plumbridge

2015

Microb Physiol

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Amino sugars are dual-purpose compounds in bacteria: they are essential components of the outer wall peptidoglycan (PG) and the outer membrane of Gram-negative bacteria and, in addition, when supplied exogenously their catabolism contributes valuable supplies of energy, carbon and nitrogen to the cell. The enzymes for both the synthesis and degradation of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) are highly conserved but during evolution have become subject to different regulatory regimes. Escherichia coli grows more rapidly using GlcNAc as a carbon source than with GlcN. On the other hand, Bacillus subtilis, but not other Bacilli tested, grows more efficiently on GlcN than GlcNAc. The more rapid growth on this sugar is associated with the presence of a second, GlcN-specific operon, which is unique to this species. A single locus is associated with the genes for catabolism of GlcNAc and GlcN in E. coli, although they enter the cell via different transporters. In E. coli the amino sugar transport and catabolic genes have also been requisitioned as part of the PG recycling process. Although PG recycling likely occurs in B. subtilis, it appears to have different characteristics.

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Structural Analysis of N-acetylglucosamine-6-phosphate Deacetylase Apoenzyme from Escherichia coli

Cited by 24 publications

References 55 publications

N-Acetyl-d-glucosamine-6-phosphate Deacetylase: Substrate Activation via a Single Divalent Metal Ion

N-Acetyl-d-glucosamine-6-phosphate Deacetylase: Substrate Activation via a Single Divalent Metal Ion

Crystal Structure of Monofunctional Histidinol Phosphate Phosphatase from Thermus thermophilus HB8

Regulation of the Utilization of Amino Sugars by <b><i>Escherichia coli</i></b> and <b><i>Bacillus subtilis</i></b>: Same Genes, Different Control

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