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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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Structure-based alignment of alanine racemasesfrom B. anthracis (AlrBax), G. stearothermophilus (AlrGst), M. tuberculosis (AlrMtb), S. lavendulae (AlrSla) and P. aeruginosa (DadXPao). The initial alignment was performed using EXPRESSO (3DCoffee) [63] and adjusted manually upon inspection of the superimposed structures. An asterisk marks the location of the Lys residue bound to PLP, the diamond marks the location of the Tyr residue that functions as the second base in the racemase reaction, a bullet denotes the location of the carbamylated lysine found in other alanine racemase structures and replaced by a chloride ion on AlrBax. I and M denote residues found in the middle or the inner layer of the active site entryway along with their position in the entryway.
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Figure 1: Structure-based alignment of alanine racemasesfrom B. anthracis (AlrBax), G. stearothermophilus (AlrGst), M. tuberculosis (AlrMtb), S. lavendulae (AlrSla) and P. aeruginosa (DadXPao). The initial alignment was performed using EXPRESSO (3DCoffee) [63] and adjusted manually upon inspection of the superimposed structures. An asterisk marks the location of the Lys residue bound to PLP, the diamond marks the location of the Tyr residue that functions as the second base in the racemase reaction, a bullet denotes the location of the carbamylated lysine found in other alanine racemase structures and replaced by a chloride ion on AlrBax. I and M denote residues found in the middle or the inner layer of the active site entryway along with their position in the entryway.

Mentions: The sequences for both dal1 and dal2 genes amplified in our laboratory from B. anthracis (Ames) genomic DNA are 100% identical to those previously deposited in GeneBank (dal1 GeneID: 1087014 and dal2 GeneID: 30262102) [36]. The protein sequences encoded by dal1 and dal2 both contain the characteristic motifs expected for members of the alanine racemase family, such as a PLP-binding site near the N-terminus, the two key conserved catalytic amino acid residues Lys41 (AlrBax numbering) and Tyr270, and a set of conserved residues making up the entrance corridor to the alanine racemase active site [26] (Figure 1). The gene product of dal1, which we term AlrBax, is identical to the alanine racemase protein previously associated with germination in B. anthracis spores [37] and shares 57% amino acid identity with AlrGst. Dal2, on the other hand, shows 41% sequence identity to AlrBax and 40% identity to AlrGst.


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)

Structure-based alignment of alanine racemasesfrom B. anthracis (AlrBax), G. stearothermophilus (AlrGst), M. tuberculosis (AlrMtb), S. lavendulae (AlrSla) and P. aeruginosa (DadXPao). The initial alignment was performed using EXPRESSO (3DCoffee) [63] and adjusted manually upon inspection of the superimposed structures. An asterisk marks the location of the Lys residue bound to PLP, the diamond marks the location of the Tyr residue that functions as the second base in the racemase reaction, a bullet denotes the location of the carbamylated lysine found in other alanine racemase structures and replaced by a chloride ion on AlrBax. I and M denote residues found in the middle or the inner layer of the active site entryway along with their position in the entryway.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Structure-based alignment of alanine racemasesfrom B. anthracis (AlrBax), G. stearothermophilus (AlrGst), M. tuberculosis (AlrMtb), S. lavendulae (AlrSla) and P. aeruginosa (DadXPao). The initial alignment was performed using EXPRESSO (3DCoffee) [63] and adjusted manually upon inspection of the superimposed structures. An asterisk marks the location of the Lys residue bound to PLP, the diamond marks the location of the Tyr residue that functions as the second base in the racemase reaction, a bullet denotes the location of the carbamylated lysine found in other alanine racemase structures and replaced by a chloride ion on AlrBax. I and M denote residues found in the middle or the inner layer of the active site entryway along with their position in the entryway.
Mentions: The sequences for both dal1 and dal2 genes amplified in our laboratory from B. anthracis (Ames) genomic DNA are 100% identical to those previously deposited in GeneBank (dal1 GeneID: 1087014 and dal2 GeneID: 30262102) [36]. The protein sequences encoded by dal1 and dal2 both contain the characteristic motifs expected for members of the alanine racemase family, such as a PLP-binding site near the N-terminus, the two key conserved catalytic amino acid residues Lys41 (AlrBax numbering) and Tyr270, and a set of conserved residues making up the entrance corridor to the alanine racemase active site [26] (Figure 1). The gene product of dal1, which we term AlrBax, is identical to the alanine racemase protein previously associated with germination in B. anthracis spores [37] and shares 57% amino acid identity with AlrGst. Dal2, on the other hand, shows 41% sequence identity to AlrBax and 40% identity to AlrGst.

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