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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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Position of residues taking part in intermonomer contacts is highly conserved among various Alrs, despite differences in hinge angles. Following a structural alignment of the N-terminal domains of various Alrs, the position for the Cα atoms from residues that take part in intermonomer contacts and are in the N-terminal domain (shown as colored spheres) was plotted on the main chain representation of AlrBax (shown in green). Likewise, the position for the Cα atoms from residues that take part in intermonomer contacts and are at the C-terminal domain (shown as colored spheres) were plotted on the main chain representation of AlrBax after a structural alignment of the C-terminal domains of various Alrs. Residues are colored according to the legend on figure 3. The PLP cofactor from AlrBax is shown as a ball and stick model. N and C indicate the position of the C- and N-termini in the monomer.
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Figure 5: Position of residues taking part in intermonomer contacts is highly conserved among various Alrs, despite differences in hinge angles. Following a structural alignment of the N-terminal domains of various Alrs, the position for the Cα atoms from residues that take part in intermonomer contacts and are in the N-terminal domain (shown as colored spheres) was plotted on the main chain representation of AlrBax (shown in green). Likewise, the position for the Cα atoms from residues that take part in intermonomer contacts and are at the C-terminal domain (shown as colored spheres) were plotted on the main chain representation of AlrBax after a structural alignment of the C-terminal domains of various Alrs. Residues are colored according to the legend on figure 3. The PLP cofactor from AlrBax is shown as a ball and stick model. N and C indicate the position of the C- and N-termini in the monomer.

Mentions: Despite large differences in hinge angles, the location of interface residues in various Alrs is very similar (Figure 5). In Figure 5, the Cα atoms for residues taking part in intermonomer contacts in various Alrs are shown as colored spheres. The positions for the various spheres were obtained following two independent structural alignments, one using only atoms from the N-terminal domain and the other using only atoms from the C-terminal domain, and then plotted onto a ribbon diagram of AlrBax. If the position of residues taking part in intermonomer contacts is conserved among various Alrs, we would expect the colored spheres to form tight clusters, containing superimposed red, green, blue, yellow and pink spheres. Indeed, as shown in Figure 5, most of the intermonomer contacts from various Alrs are found in clusters and thus are conserved among various Alrs. It is important to keep in mind that the number of residues taking part in intermonomer contacts varies among the analyzed Alrs. For AlrBax, 94 of its 389 residues take part in intermonomer contacts and both N- and C-terminal domains contribute an almost equal number (44 and 50, respectively) of residues to the interface. The total number of residues in the interface of AlrGst, AlrMtb, AlrSla and DadXPao is slightly smaller than in AlrBax. Nevertheless, for all analyzed structures, both domains contribute almost equally to the monomer-monomer interface.


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)

Position of residues taking part in intermonomer contacts is highly conserved among various Alrs, despite differences in hinge angles. Following a structural alignment of the N-terminal domains of various Alrs, the position for the Cα atoms from residues that take part in intermonomer contacts and are in the N-terminal domain (shown as colored spheres) was plotted on the main chain representation of AlrBax (shown in green). Likewise, the position for the Cα atoms from residues that take part in intermonomer contacts and are at the C-terminal domain (shown as colored spheres) were plotted on the main chain representation of AlrBax after a structural alignment of the C-terminal domains of various Alrs. Residues are colored according to the legend on figure 3. The PLP cofactor from AlrBax is shown as a ball and stick model. N and C indicate the position of the C- and N-termini in the monomer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743695&req=5

Figure 5: Position of residues taking part in intermonomer contacts is highly conserved among various Alrs, despite differences in hinge angles. Following a structural alignment of the N-terminal domains of various Alrs, the position for the Cα atoms from residues that take part in intermonomer contacts and are in the N-terminal domain (shown as colored spheres) was plotted on the main chain representation of AlrBax (shown in green). Likewise, the position for the Cα atoms from residues that take part in intermonomer contacts and are at the C-terminal domain (shown as colored spheres) were plotted on the main chain representation of AlrBax after a structural alignment of the C-terminal domains of various Alrs. Residues are colored according to the legend on figure 3. The PLP cofactor from AlrBax is shown as a ball and stick model. N and C indicate the position of the C- and N-termini in the monomer.
Mentions: Despite large differences in hinge angles, the location of interface residues in various Alrs is very similar (Figure 5). In Figure 5, the Cα atoms for residues taking part in intermonomer contacts in various Alrs are shown as colored spheres. The positions for the various spheres were obtained following two independent structural alignments, one using only atoms from the N-terminal domain and the other using only atoms from the C-terminal domain, and then plotted onto a ribbon diagram of AlrBax. If the position of residues taking part in intermonomer contacts is conserved among various Alrs, we would expect the colored spheres to form tight clusters, containing superimposed red, green, blue, yellow and pink spheres. Indeed, as shown in Figure 5, most of the intermonomer contacts from various Alrs are found in clusters and thus are conserved among various Alrs. It is important to keep in mind that the number of residues taking part in intermonomer contacts varies among the analyzed Alrs. For AlrBax, 94 of its 389 residues take part in intermonomer contacts and both N- and C-terminal domains contribute an almost equal number (44 and 50, respectively) of residues to the interface. The total number of residues in the interface of AlrGst, AlrMtb, AlrSla and DadXPao is slightly smaller than in AlrBax. Nevertheless, for all analyzed structures, both domains contribute almost equally to the monomer-monomer interface.

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