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Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames).

Couñago RM, Davlieva M, Strych U, Hill RE, Krause KL - BMC Struct. Biol. (2009)

Bottom Line: Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure.The chloride ion in AlrBax is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure.Despite differences in space group and crystal form, the two AlrBax structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Otago, Dunedin, New Zealand. rafael.counago@otago.ac.nz

ABSTRACT

Background: Bacillus anthracis is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of B. anthracis (Ames) alanine racemase (AlrBax), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native AlrBax structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation.

Results: B. anthracis has two open reading frames encoding for putative alanine racemases. We show that only one, dal1, is able to complement a D-alanine auxotrophic strain of E. coli. Purified Dal1, which we term AlrBax, is shown to be a dimer in solution by dynamic light scattering and has a Vmax for racemization (L- to D-alanine) of 101 U/mg. The crystal structure of unmodified AlrBax is reported here to 1.95 A resolution. Despite the overall similarity of the fold to other alanine racemases, AlrBax makes use of a chloride ion to position key active site residues for catalysis, a feature not yet observed for this enzyme in other species. Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure.

Conclusion: The chloride ion in AlrBax is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure. Despite differences in space group and crystal form, the two AlrBax structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.

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Organization of the active site residues in B. anthracis Alr is facilitated by a chloride ion. (A) Electron density map (contoured at 1.5σ in the final refined 2Fo-Fc map) showing details of the active site for AlrBax. (B) Structural alignment of residues making the active site of various Alrs (TB structure was not included). For all available Alr structures, Arg138 makes polar contacts to the PLP and, possibly, to the substrate. In AlrBax this arginine residue is positioned in the active site by a chloride ion (Cl-). Polar contacts between the chloride ion and Asn-131 and Arg-138 are shown in Panel A by dashes. For all other alanine racemase structures available to date the equivalent interactions are mediated by a carbamylated lysine (shown in Panel B). Residues in the active site of various Alrs are shown as a stick model. In Panel A, the acetate molecule and the modified lysine residue (LLP) are depicted as ball and stick models; carbons are colored in green, nitrogen in blue, oxygen in red, phosphate in orange, sulfur in yellow and the chloride ion is depicted as a light green sphere. In Panel B residues are shown as stick model and are colored according to the legend on figure 3; the PLP cofactors are shown as ball and stick models. In both panels, primed numbers denote residues from the second monomer.
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Figure 6: Organization of the active site residues in B. anthracis Alr is facilitated by a chloride ion. (A) Electron density map (contoured at 1.5σ in the final refined 2Fo-Fc map) showing details of the active site for AlrBax. (B) Structural alignment of residues making the active site of various Alrs (TB structure was not included). For all available Alr structures, Arg138 makes polar contacts to the PLP and, possibly, to the substrate. In AlrBax this arginine residue is positioned in the active site by a chloride ion (Cl-). Polar contacts between the chloride ion and Asn-131 and Arg-138 are shown in Panel A by dashes. For all other alanine racemase structures available to date the equivalent interactions are mediated by a carbamylated lysine (shown in Panel B). Residues in the active site of various Alrs are shown as a stick model. In Panel A, the acetate molecule and the modified lysine residue (LLP) are depicted as ball and stick models; carbons are colored in green, nitrogen in blue, oxygen in red, phosphate in orange, sulfur in yellow and the chloride ion is depicted as a light green sphere. In Panel B residues are shown as stick model and are colored according to the legend on figure 3; the PLP cofactors are shown as ball and stick models. In both panels, primed numbers denote residues from the second monomer.

Mentions: As observed for other Alrs, the active site of AlrBax is formed by residues from both monomers, with the two catalytic bases Lys41 and Tyr270' found in different monomers. In the AlrBax structure, Lys41 is seen covalently linked to the PLP cofactor. As was observed for one of the AlrGst structures (1sft) [22], we have identified extra density in the active site of AlrBax, which we have modeled as a molecule of acetate. Acetate, which was present in our crystallization solution, is an inhibitor of Alr [22] and its carboxylate group is thought to bind the enzyme active site in the same way the carboxylate group from alanine is expected to do [22]. The oxygen atoms from the acetate molecule in our model are within hydrogen bonding distance to the side chain oxygen from Tyr289', the main chain nitrogen from Met317' and, perhaps more importantly, to the side chain nitrogen atom from the catalytic Lys41 residue (Figure 6).


Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames).

Couñago RM, Davlieva M, Strych U, Hill RE, Krause KL - BMC Struct. Biol. (2009)

Organization of the active site residues in B. anthracis Alr is facilitated by a chloride ion. (A) Electron density map (contoured at 1.5σ in the final refined 2Fo-Fc map) showing details of the active site for AlrBax. (B) Structural alignment of residues making the active site of various Alrs (TB structure was not included). For all available Alr structures, Arg138 makes polar contacts to the PLP and, possibly, to the substrate. In AlrBax this arginine residue is positioned in the active site by a chloride ion (Cl-). Polar contacts between the chloride ion and Asn-131 and Arg-138 are shown in Panel A by dashes. For all other alanine racemase structures available to date the equivalent interactions are mediated by a carbamylated lysine (shown in Panel B). Residues in the active site of various Alrs are shown as a stick model. In Panel A, the acetate molecule and the modified lysine residue (LLP) are depicted as ball and stick models; carbons are colored in green, nitrogen in blue, oxygen in red, phosphate in orange, sulfur in yellow and the chloride ion is depicted as a light green sphere. In Panel B residues are shown as stick model and are colored according to the legend on figure 3; the PLP cofactors are shown as ball and stick models. In both panels, primed numbers denote residues from the second monomer.
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Related In: Results  -  Collection

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Figure 6: Organization of the active site residues in B. anthracis Alr is facilitated by a chloride ion. (A) Electron density map (contoured at 1.5σ in the final refined 2Fo-Fc map) showing details of the active site for AlrBax. (B) Structural alignment of residues making the active site of various Alrs (TB structure was not included). For all available Alr structures, Arg138 makes polar contacts to the PLP and, possibly, to the substrate. In AlrBax this arginine residue is positioned in the active site by a chloride ion (Cl-). Polar contacts between the chloride ion and Asn-131 and Arg-138 are shown in Panel A by dashes. For all other alanine racemase structures available to date the equivalent interactions are mediated by a carbamylated lysine (shown in Panel B). Residues in the active site of various Alrs are shown as a stick model. In Panel A, the acetate molecule and the modified lysine residue (LLP) are depicted as ball and stick models; carbons are colored in green, nitrogen in blue, oxygen in red, phosphate in orange, sulfur in yellow and the chloride ion is depicted as a light green sphere. In Panel B residues are shown as stick model and are colored according to the legend on figure 3; the PLP cofactors are shown as ball and stick models. In both panels, primed numbers denote residues from the second monomer.
Mentions: As observed for other Alrs, the active site of AlrBax is formed by residues from both monomers, with the two catalytic bases Lys41 and Tyr270' found in different monomers. In the AlrBax structure, Lys41 is seen covalently linked to the PLP cofactor. As was observed for one of the AlrGst structures (1sft) [22], we have identified extra density in the active site of AlrBax, which we have modeled as a molecule of acetate. Acetate, which was present in our crystallization solution, is an inhibitor of Alr [22] and its carboxylate group is thought to bind the enzyme active site in the same way the carboxylate group from alanine is expected to do [22]. The oxygen atoms from the acetate molecule in our model are within hydrogen bonding distance to the side chain oxygen from Tyr289', the main chain nitrogen from Met317' and, perhaps more importantly, to the side chain nitrogen atom from the catalytic Lys41 residue (Figure 6).

Bottom Line: Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure.The chloride ion in AlrBax is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure.Despite differences in space group and crystal form, the two AlrBax structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Otago, Dunedin, New Zealand. rafael.counago@otago.ac.nz

ABSTRACT

Background: Bacillus anthracis is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of B. anthracis (Ames) alanine racemase (AlrBax), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native AlrBax structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation.

Results: B. anthracis has two open reading frames encoding for putative alanine racemases. We show that only one, dal1, is able to complement a D-alanine auxotrophic strain of E. coli. Purified Dal1, which we term AlrBax, is shown to be a dimer in solution by dynamic light scattering and has a Vmax for racemization (L- to D-alanine) of 101 U/mg. The crystal structure of unmodified AlrBax is reported here to 1.95 A resolution. Despite the overall similarity of the fold to other alanine racemases, AlrBax makes use of a chloride ion to position key active site residues for catalysis, a feature not yet observed for this enzyme in other species. Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure.

Conclusion: The chloride ion in AlrBax is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure. Despite differences in space group and crystal form, the two AlrBax structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.

Show MeSH
Related in: MedlinePlus