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

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 ？ 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.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.Bacillus anthracis is a soil-dwelling, spore-forming, Gram-positive bacterium that is the causative agent of the zoonotic disease anthrax. Although the disease is most common in wild and domestic mammals, it can also occur in humans when exposed to infected animals or living spores [1]. The severity of anthrax in humans depends on the route of infection. Inhalation of B. anthracis spores can lead to the most severe form of the disease, historically associated with a case-fatality rate as high as 85% [2,3]. The high mortality rate, the existence of a respiratory route of infection and the great resistance of its spores has made B. anthracis the subject of biological warfare research programs in many countries for over 60 years [4]. The United States Centers for Disease Control and Prevention (CDC) has classified anthrax