BMB reports Biochemical characterization of Alanine racemase- a spore protein produced by Bacillus anthracis Shivani Kanodia1,*, Shivangi Agarwal1,*, Priyanka Singh1,*, Shivani Agarwal2, Preeti Singh1 & Rakesh Bhatnagar1,* 1Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi-110067, India, 2Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi-110067, India Alanine racemase catalyzes the interconversion of L-alanine and racemization proceeds through a two-base mechanism that in- D-alanine and plays a crucial role in spore germination and cell volves lysine (pyridoxal 5'-phosphate binding residue) and ty- wall biosynthesis. In this study, alanine racemase produced by rosine (plays a role in binding/catalysis). Crystallographic analysis Bacillus anthracis was expressed and purified as a monomer in of B. stearothermophilus alanine racemase has shown that the resi- Escherichia coli and the importance of lysine 41 in the cofactor dues at the active site play a role in its racemization (5). Based on binding octapeptide and tyrosine 270 in catalysis was evaluated. its known structure, it is plausible that B. anthracis produces ala- The native enzyme exhibited an apparent Km of 3 mM for L-ala- nine racemase via an analogous catalytic site, wherein Lys41 and nine, and a Vmax of 295 μmoles/min/mg, with the optimum activ- Tyr270 remove α-hydrogen from the alanyl-PLP aldimine and pro- ity occurring at 37oC and a pH of 8-9. The activity observed in the tonate the carbanion to induce racemization. absence of exogenous pyridoxal 5 -phosphaté suggested that the Immunoproteome analysis of the spore antigens produced cofactor is bound to the enzyme. Additionally, the UV-visible ab- by Bacillus anthracis has led to the identification of several im- sorption spectra indicated that the activity was pH independece, munogenic proteins including exosporium associated alanine of VV-visible absorption spectra suggests that the bound PLP ex- racemase (6, 7). However, several studies have demonstrated ists as a protonated Schiff’s base. Furthermore, the loss of activity that few antigens produced by anthrax spores can augment the observed in the apoenzyme suggested that bound PLP is required protective efficacy of currently used protective antigen based for catalysis. Finally, the enzyme followed non-competitive and vaccines (7). B. anthracis spores being a potential biological mixed inhibition kinetics for hydroxylamine and propionate with warfare agent, development of vaccines or inhibitors against a Ki of 160 μM and 30 mM, respectively. [BMB reports 2009; proteins/enzymes crucial for B. anthracis germination and out- 42(1): 47-52] growth-associated factors or combining PA with one of the im- munodominant antigens in vaccine formulation may lead to anefficacious safer vaccine. INTRODUCTION The development of mechanism-based inhibitors of alanine racemase requires understanding the mechanism by which ala- Alanine racemase (EC 5.1.1.1) (Alr) is a pyridoxal 5'-phosphate nine racemase enforces substrate specificity and maintains (PLP) dependent enzyme that catalyzes the racemization between enantioselectivity. Understanding these mechanisms requires L- and D- alanine. This enzyme is indispensable to prokaryotes be- enzyme-substrate complex analysis to determine if it requires a cause D- alanine is essential for peptidoglycan biosynthesis in the PLP cofactor. Therefore, the present study was conducted to cell wall of gram-positive and gram-negative bacteria (1, 2). characterize alanine racemase (YP_026523), a spore-specific Escherichia coli and other gram-negative bacteria possess two in- enzyme produced by B. anthracis (8). dependent alanine racemases with distinct functions (3). Alanine racemase is ubiquitous in ubiquitous, which makes it an attractive RESULTS AND DISCUSSION target for the development of antimicrobial drugs. As a result, L-flu- oroalanine, a structural analog of L-alanine, is currently being eval- Biochemical characterization of Alanine racemase uated to determine its potential as an antibacterial drug (4). Alanine PCR amplification of alr resulted in amplification of an 1170 bp fragment, which was subsequently cloned into an expression *Corresponding author. Tel: 91-11-26704079; Fax: 91-11-26742040; vector. Blast P analysis of the deduced amino acid sequence of B. E-mail: [email protected] anthracis Alr revealed that it was 96-99% homologous with the #Authors contributed equally to this work. Alr produced by closely related bacillus species. The PLP binding octapeptide of B. anthracis Alr, KGNAYGHD, was also found to Received 4 June 2008, Accepted 18 July 2008 be conserved among species. SDS-PAGE of E. coli BL21 (λDE3) cells harboring pPSAlr fol- Keywords: Alanine racemase, Bacillus anthracis, Enantiomers, Exospo- rium, Spore germination lowing IPTG induction revealed that a high level of an≈45 http://bmbreports.org BMB reports 47 Alanine racemase of Bacillus anthracis Shivani Kanodia, et al. Fig. 1. (A) Expression and purification of wild type rAlr. Lanes 1 and 2: unin- duced and induced cell lysates; 3: puri- fied rAlr; M: molecular mass standards. (B) UV-Visible spectrum of rAlr. (C) Kinetics of wild type rAlr. Lineweaver- Burk plot of 1/(S) vs. 1/Vo. Inset: tem- perature and pH optimum for rAlr. (D) Determination of Tm for rAlr. Denatura- tion transition of rAlr obtained using DSC. Inset: percentage of residual activ- ity for rAlr at each temperature indi- cated. (E) Determination of the molec- ular weight of rAlr. kDa rAlr was expressed (Fig. 1A, Lane 2), and that this protein which resulted in irreversible unfolding. As a result, the post was purified to≈98% homogeneity (Fig. 1A, Lane 3). A shake transitional baseline could not be evaluated. Additionally, the flask culture of the transformed organisms yielded approx- residual activity of rAlr (functional stability) resulted in a 50% imately 180 mg of the purified protein per liter of culture. The reduction in the enzyme activity between 60 and 70oC (Fig. protein produced using the technique described here had a 1D, inset). Additionally, the Tm (structural stability) suggests specific activity of 295 U/mg. The specific activity of the rAlr that 50% of the protein molecules are completely denatured at produced B. anthracis was found to be lower than that re- 85oC. This finding indicates that the remainder of the protein ported for dimeric alanine racemases (2000-7700 U/mg) (9-13); molecules in the solution may have already undergone con- however, it was comparable to that of monomeric alanine formational perturbations at this temperature, thereby reducing racemases (36-220 U/mg) (2, 14-16). its catalytic efficiency (4% residual activity). The high degree The UV-Visible spectrum of rAlr revealed a small shoulder at of thermostability associated with rAlr could be attributed to its 420 nm, which is characteristic of a Schiff’s base complex be- presence on the spore surface. The optimum activity for the di- tween PLP and the ε-amino group of the lysine residue (Fig. 1B). meric enzyme from Acidiphilium organovorum and B. stear- The absorption maximum for free PLP was observed at 291 nm. othermophilus also falls within the range of 50-60oC (10, 16). The rAlr produced here was active within a temperature range However, even though rAlr produced by B. anthracis is mono- of 25oC to 45oC, with the optimum activity being observed at meric; it is thermostable, which indicates that it is a potential 37oC. In addition, rAlr was active over a wide pH range, with the candidate for the development of a recombinant vaccine. optimum activity being observed between pH 8-9. These find- ings are in accordance with those of all other alanine racemases Determination of the molecular mass of rAlr that have been evaluated to date (14-18) (Fig. 1C, Inset). Bacterial alanine racemases are classified as monomeric (13, The wild type rAlr followed classical Michaelis-Menten’s ki- 15, 18) or homodimeric (3, 11, 12) based on the structure of netics; therefore, the kinetic coefficients were calculated using their subunits. SDS-PAGE revealed that the molecular mass of a Lineweaver-Burk plot (Fig. 1C). The Km was calculated to be the purified rAlr of B. anthracis was≈45 kDa. In addition, gel 3 mM for L-alanine with a Vmax of 295 U/mg. The kcat for B. an- filtration analysis of purified rAlr (1 and 10 mg/ml) in its native thracis rAlr was determined to be 22 s-1, which differs greatly form revealed that it was a monomer (Fig. 1E). No multi- from the values reported for H. pylori (632 s-1) (1), P. monodon merization was observed at this concentration. The eluted frac- (2568 s-1) (19) and crayfish (7504 s-1) (9). These findings in- tion of rAlr (peak 2) exhibited a specific activity of 260 U/mg, dicate that rAlr has a high catalytic efficiency. which demonstrates that the rAlr monomer is catalytically The thermal stability and Tm determination for rAlr were as- active. Peak 1 shown in the inset, which was eluted just before sessed by Differential Scanning Calorimetry (DSC). The results rAlr, possessed no activity. of this analysis revealed that rAlr is an extremely stable en- o zyme with an apparent Tm of 85 C (Fig. 1D). However, rAlr Substrate specificity of rAlr was prone to aggregation at temperatures higher than 90oC, rAlr acted exclusively on L- alanine, and the formation of D-ala- 48 BMB reports http://bmbreports.org Alanine racemase of Bacillus anthracis Shivani Kanodia, et al. nine was demonstrated by a decrease in the CD signal following when compared to Ki' (7 mM). This finding indicates that pro- racemization (Data not shown). Other amino acids were in- pionate influences both Km (affinity for substrate) and Vmax active as substrates, which suggests that B. anthracis rAlr is high- (enzyme catalysis), which has also been proposed for the ala- ly specific for L-alanine. However, the 20% racemization that nine racemase of B.