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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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Related in: MedlinePlus

Ribbon representation of the dimer of the alanine racemase from B. anthracis. The PLP co-factor is shown as a ball and stick model. Monomers are shown in different colors. N and C indicate the position of the C- and N-termini of one monomer; primed letters denote the termini for the second monomer.
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Figure 2: Ribbon representation of the dimer of the alanine racemase from B. anthracis. The PLP co-factor is shown as a ball and stick model. Monomers are shown in different colors. N and C indicate the position of the C- and N-termini of one monomer; primed letters denote the termini for the second monomer.

Mentions: Consistent with other alanine racemases, the tertiary structure of AlrBax is a homodimer formed by head-to-tail-association of two monomers (Figure 2). Each monomer is crystallographically distinct in this crystal form (Table 1), but the two monomers have very similar folds. The rms difference obtained for their Cα atoms after least-squares superposition is 0.22 Å. AlrBax monomers consist of two structurally distinct domains. Residues in the N-terminus (16–245) fold into an eight-stranded α/β barrel, while the C-terminal residues (246–389) and the first 15 N-terminal amino acids are part of a predominantly β-structure. The homodimer displays two active sites, formed by residues from the N-terminus of one monomer and residues from the C-terminus of the other monomer. The PLP cofactor forms a covalent bond to Lys41 and points at the center of the α/β barrel. As previously observed for AlrGst [22], extra density was present in the active of AlrBax, which we model here as a molecule of acetate.


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)

Ribbon representation of the dimer of the alanine racemase from B. anthracis. The PLP co-factor is shown as a ball and stick model. Monomers are shown in different colors. N and C indicate the position of the C- and N-termini of one monomer; primed letters denote the termini for the second monomer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Ribbon representation of the dimer of the alanine racemase from B. anthracis. The PLP co-factor is shown as a ball and stick model. Monomers are shown in different colors. N and C indicate the position of the C- and N-termini of one monomer; primed letters denote the termini for the second monomer.
Mentions: Consistent with other alanine racemases, the tertiary structure of AlrBax is a homodimer formed by head-to-tail-association of two monomers (Figure 2). Each monomer is crystallographically distinct in this crystal form (Table 1), but the two monomers have very similar folds. The rms difference obtained for their Cα atoms after least-squares superposition is 0.22 Å. AlrBax monomers consist of two structurally distinct domains. Residues in the N-terminus (16–245) fold into an eight-stranded α/β barrel, while the C-terminal residues (246–389) and the first 15 N-terminal amino acids are part of a predominantly β-structure. The homodimer displays two active sites, formed by residues from the N-terminus of one monomer and residues from the C-terminus of the other monomer. The PLP cofactor forms a covalent bond to Lys41 and points at the center of the α/β barrel. As previously observed for AlrGst [22], extra density was present in the active of AlrBax, which we model here as a molecule of acetate.

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