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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.


Bd-NDPSase wild type and E140Q substrate specificity.A) Wild type enzyme (gray bars) exhibited preference for nucleoside diphosphate sugar (NDPS). E140Q mutant (red bars) were catalytically inactive. B) Initial rates of GDPM hydrolysis for the wild type and E140Q mutant were fit by nonlinear least squares to the Michaelis-Menten equation (solid lines) to determine kcat (5.2 (ms)-1) and Km (0.3 mM). Standard deviations of triplicate measurements are shown by the shaded bars for the wild type (gray shade) and mutant (red shade).
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pone.0141716.g002: Bd-NDPSase wild type and E140Q substrate specificity.A) Wild type enzyme (gray bars) exhibited preference for nucleoside diphosphate sugar (NDPS). E140Q mutant (red bars) were catalytically inactive. B) Initial rates of GDPM hydrolysis for the wild type and E140Q mutant were fit by nonlinear least squares to the Michaelis-Menten equation (solid lines) to determine kcat (5.2 (ms)-1) and Km (0.3 mM). Standard deviations of triplicate measurements are shown by the shaded bars for the wild type (gray shade) and mutant (red shade).

Mentions: Phosphate release assays revealed that among the nucleoside diphosphate sugars hydrolyzed by Bd-NDPSase, its highest relative activity was with GDPM. UDPG, GDPG, and ADPR, were also hydrolyzed, but at a lower extent than GDPM (Fig 2A). Michaelis-Menten kinetics revealed a kcat of 5200 s-1 and Km of 0.3 mM for GDPM. This Km is comparable to those of other Nudix sugar hydrolases with a similar structure and molecular weight [5, 6, 17, 19, 52, 53]. Given its cellular localization it is likely that the physiological substrate of Bd-NDPSase will be determined by which compounds are present at high concentration in the periplasmic space or become available from the host cell when Bdellovibrio degrades it.


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)

Bd-NDPSase wild type and E140Q substrate specificity.A) Wild type enzyme (gray bars) exhibited preference for nucleoside diphosphate sugar (NDPS). E140Q mutant (red bars) were catalytically inactive. B) Initial rates of GDPM hydrolysis for the wild type and E140Q mutant were fit by nonlinear least squares to the Michaelis-Menten equation (solid lines) to determine kcat (5.2 (ms)-1) and Km (0.3 mM). Standard deviations of triplicate measurements are shown by the shaded bars for the wild type (gray shade) and mutant (red shade).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141716.g002: Bd-NDPSase wild type and E140Q substrate specificity.A) Wild type enzyme (gray bars) exhibited preference for nucleoside diphosphate sugar (NDPS). E140Q mutant (red bars) were catalytically inactive. B) Initial rates of GDPM hydrolysis for the wild type and E140Q mutant were fit by nonlinear least squares to the Michaelis-Menten equation (solid lines) to determine kcat (5.2 (ms)-1) and Km (0.3 mM). Standard deviations of triplicate measurements are shown by the shaded bars for the wild type (gray shade) and mutant (red shade).
Mentions: Phosphate release assays revealed that among the nucleoside diphosphate sugars hydrolyzed by Bd-NDPSase, its highest relative activity was with GDPM. UDPG, GDPG, and ADPR, were also hydrolyzed, but at a lower extent than GDPM (Fig 2A). Michaelis-Menten kinetics revealed a kcat of 5200 s-1 and Km of 0.3 mM for GDPM. This Km is comparable to those of other Nudix sugar hydrolases with a similar structure and molecular weight [5, 6, 17, 19, 52, 53]. Given its cellular localization it is likely that the physiological substrate of Bd-NDPSase will be determined by which compounds are present at high concentration in the periplasmic space or become available from the host cell when Bdellovibrio degrades it.

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.