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Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus.

de la Peña AH, Suarez A, Duong-Ly KC, Schoeffield AJ, Pizarro-Dupuy MA, Zarr M, Pineiro SA, Amzel LM, Gabelli SB - PLoS ONE (2015)

Bottom Line: Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes.We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively).Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Structural Enzymology and Thermodynamics Group, Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.

No MeSH data available.


Overall Structure Bd-NDPSase.A) Structure of E140Q Bd-NDPSase bound to ADPR (PDB ID 5C7T). B) Sequence alignment of the Ec-ADPRase (PDB ID 1KHZ) and Ec-NDPSase (PDB ID 3O61) to Bd-NDPSase. The N-terminal domain (residues 1–44) consists of an antiparallel beta sheet (β1- β3) and is denoted by green. The Nudix fold consists of a mixed beta sheet (β1- β10, yellow) flanked by helix α1 (cyan) on one side and helices α2 and α3 (residues 148–182, gray) on the other side. The location of the E140Q mutation on loop L9 (magenta) is denoted by an asterisk. The prime symbol (‘) denotes residues of the opposite monomer (lighter color shade).
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pone.0141716.g003: Overall Structure Bd-NDPSase.A) Structure of E140Q Bd-NDPSase bound to ADPR (PDB ID 5C7T). B) Sequence alignment of the Ec-ADPRase (PDB ID 1KHZ) and Ec-NDPSase (PDB ID 3O61) to Bd-NDPSase. The N-terminal domain (residues 1–44) consists of an antiparallel beta sheet (β1- β3) and is denoted by green. The Nudix fold consists of a mixed beta sheet (β1- β10, yellow) flanked by helix α1 (cyan) on one side and helices α2 and α3 (residues 148–182, gray) on the other side. The location of the E140Q mutation on loop L9 (magenta) is denoted by an asterisk. The prime symbol (‘) denotes residues of the opposite monomer (lighter color shade).

Mentions: Bd-NDPSase is a homodimer whose monomers are related by a non-crystallographic 2-fold axis. Each monomer comprises an N-terminal and a Nudix domain (Fig 3). The N-terminal domain contains a beta sheet composed of three anti-parallel strands (residues 1–40) connected by loops. Loop L1, which connects strands β1 and β2, contributes to a π-stacking interaction with the substrate via Y19. The C-terminal domain, comprising the Nudix fold, is composed of an α-β-α motif (α1, β4-β10, α2). The specificity loop L8 joining strands β8 and β9 of the mixed beta sheet interacts with the substrate in the catalytic site of the opposite monomer via hydrogen bonding. The swapped N-terminal domains and the Nudix fold form two catalytic cavities with an exposed surface of ~689 Å2 each. The signature Nudix sequence is folded as loop-helix-loop (β7-L6-α1-L7). Its conserved glutamates , together with E140 of loop L9, are positioned to coordinate Mg2+ binding.


Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus.

de la Peña AH, Suarez A, Duong-Ly KC, Schoeffield AJ, Pizarro-Dupuy MA, Zarr M, Pineiro SA, Amzel LM, Gabelli SB - PLoS ONE (2015)

Overall Structure Bd-NDPSase.A) Structure of E140Q Bd-NDPSase bound to ADPR (PDB ID 5C7T). B) Sequence alignment of the Ec-ADPRase (PDB ID 1KHZ) and Ec-NDPSase (PDB ID 3O61) to Bd-NDPSase. The N-terminal domain (residues 1–44) consists of an antiparallel beta sheet (β1- β3) and is denoted by green. The Nudix fold consists of a mixed beta sheet (β1- β10, yellow) flanked by helix α1 (cyan) on one side and helices α2 and α3 (residues 148–182, gray) on the other side. The location of the E140Q mutation on loop L9 (magenta) is denoted by an asterisk. The prime symbol (‘) denotes residues of the opposite monomer (lighter color shade).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4629899&req=5

pone.0141716.g003: Overall Structure Bd-NDPSase.A) Structure of E140Q Bd-NDPSase bound to ADPR (PDB ID 5C7T). B) Sequence alignment of the Ec-ADPRase (PDB ID 1KHZ) and Ec-NDPSase (PDB ID 3O61) to Bd-NDPSase. The N-terminal domain (residues 1–44) consists of an antiparallel beta sheet (β1- β3) and is denoted by green. The Nudix fold consists of a mixed beta sheet (β1- β10, yellow) flanked by helix α1 (cyan) on one side and helices α2 and α3 (residues 148–182, gray) on the other side. The location of the E140Q mutation on loop L9 (magenta) is denoted by an asterisk. The prime symbol (‘) denotes residues of the opposite monomer (lighter color shade).
Mentions: Bd-NDPSase is a homodimer whose monomers are related by a non-crystallographic 2-fold axis. Each monomer comprises an N-terminal and a Nudix domain (Fig 3). The N-terminal domain contains a beta sheet composed of three anti-parallel strands (residues 1–40) connected by loops. Loop L1, which connects strands β1 and β2, contributes to a π-stacking interaction with the substrate via Y19. The C-terminal domain, comprising the Nudix fold, is composed of an α-β-α motif (α1, β4-β10, α2). The specificity loop L8 joining strands β8 and β9 of the mixed beta sheet interacts with the substrate in the catalytic site of the opposite monomer via hydrogen bonding. The swapped N-terminal domains and the Nudix fold form two catalytic cavities with an exposed surface of ~689 Å2 each. The signature Nudix sequence is folded as loop-helix-loop (β7-L6-α1-L7). Its conserved glutamates , together with E140 of loop L9, are positioned to coordinate Mg2+ binding.

Bottom Line: Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes.We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively).Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Structural Enzymology and Thermodynamics Group, Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.

No MeSH data available.