Brucellosis also known as ’undulant fever’, ‘Mediterranean fever’ or ‘malta fever’ is zoonosis, an infectious disease caused by bacteria called brucella. It can spread from animals to humans, when person comes into contact with an animal or animal products of infected with brucella. Brucellosis is considered a significant health treat in many parts of the world especially in Mediterranean countries of Europe, north and east Africa, the Middle East, South and Central Asia and Central and South America. Activation of NADPH oxidase and low ROS production needed for the bacterium to induce premature cell death of neutrophils without inducing pro inflammatory phenotypic changes. This event majorly achieved by important antioxidative property of Cystein, involved in survival and protection of pathogenic microbes from oxidative stress and is the precursors for biosynthesis of many metabolites like glutathione, tripanthione and taurine etc., involved in maintaining cellular homeostasis, as cysteine biosynthesis pathway is crucial for inhibitor designing. In bacteria, plants and most of the pathogenic protozoans, cysteine biosynthesis is a two-step pathway. Serine acetyl transferase (SAT) catalyzes the first step of the pathway in which it transfers the acetyl group from acetyl Coenzyme A to serine to form O-acetyl serine (OAS) and in second step O-acetyl serine sulfydralase (OASS) catalyzes the condensation of O-acetyl serine with sulfide to produce cysteine.
Cysteine biosynthetic pathway is regulated by formation of a decameric complex called cysteine synthase complex (CSC), plays an important role in maintaining intracellular cysteine level. The SAT C-terminal tail binds at the active site of OASS to form CSC, which was reported earlier in E.coli, Salmonella, Hemophilus, leishmania, and Arabidopsis. And there are few structural models for CSC, although no structural evidence has been given so far.
¦nbsp;Here in Brucella abortus OASS, does not interacts with its cognate SAT C-terminal tail. We recently determined the crystal structure of native BaOASS at 2.2Å resolution. The detailed comparison of BaOASS crystal structure with OASS complexes with SAT C-terminal peptides from other organisms showed the two residues 96Q and 125Y of BaOASS present instead of M and G from all other OASS respectively, occupying the active site pocket and interfering the entry of SAT C-terminal tail into the active site pocket of OASS. Thus blacking the active site pocket for entry of SAT C-terminal peptide. Point mutation of these residues (Q96 and Y125) was done to generate three mutations (Q96A, Y125A and Q96A Y125A). The binding affinity of SAT C-terminal peptides were measured by fluorescence spectrometer for all three mutants, it was found that the binding affinity (Kd) of double mutant BaOASS (Q96A Y125A) is much stronger (1000 fold) and single mutants of BaOASS (Q96A), BaOASS (Y125A) about (10 fold stronger) up on titration with SAT C-terminal mimicking peptides compared to Native BaOASS.
The Surface Plasmon Resonance (SPR) has been done for both native BAOASS and mutant BaOASS (Q96A Y125A) with BaSAT protein, where double mutant can bind to BaSAT with about 7uM binding affinity while native protein can’t bind to BaSAT. These observations clearly conforms the reason for not forming CSC in BaOASS and we have validated these observations by mutational studies.