Structural Basis for Interaction of O-Acetylserine Sulfhydrylase and Serine Acetyltransferase in the Arabidopsis Cysteine Synthase Complex

In plants, association of O-acetylserine sulfhydrylase (OASS) and Ser acetyltransferase (SAT) into the Cys synthase complex plays a regulatory role in sulfur assimilation and Cys biosynthesis. We determined the crystal structure of Arabidopsis thaliana OASS (At-OASS) bound with a peptide corresponding to the C-terminal 10 residues of Arabidopsis SAT (C10 peptide) at 2.9-Å resolution. Hydrogen bonding interactions with key active site residues (Thr-74, Ser-75, and Gln-147) lock the C10 peptide in the binding site. C10 peptide binding blocks access to OASS catalytic residues, explaining how complex formation downregulates OASS activity. Comparison with bacterial OASS suggests that structural plasticity in the active site allows binding of SAT C termini with dissimilar sequences at structurally similar OASS active sites. Calorimetric analysis of the effect of active site mutations (T74S, S75A, S75T, and Q147A) demonstrates that these residues are important for C10 peptide binding and that changes at these positions disrupt communication between active sites in the homodimeric enzyme. We also demonstrate that the C-terminal Ile of the C10 peptide is required for molecular recognition by At-OASS. These results provide new insights into the molecular mechanism underlying formation of the Cys synthase complex and provide a structural basis for the biochemical regulation of Cys biosynthesis in plants

Amino-terminal extension present in the methionine aminopeptidase type 1c of <it>Mycobacterium tuberculosis </it>is indispensible for its activity

<p>Abstract</p> <p>Background</p> <p>Methionine aminopeptidase (MetAP) is a ubiquitous enzyme in both prokaryotes and eukaryotes, which catalyzes co-translational removal of N-terminal methionine from elongating polypeptide chains during protein synthesis. It specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged. The MetAP action for exclusion of N-terminal methionine is mandatory in 50-70% of nascent proteins. Such an activity is required for proper sub cellular localization, additional processing and eventually for the degradation of proteins.</p> <p>Results</p> <p>We cloned genes encoding two such metalloproteases (<it>Mt</it>MetAP1a and <it>Mt</it>MetAP1c) present in <it>Mycobacterium tuberculosis </it>and expressed them as histidine-tagged proteins in <it>Escherichia coli</it>. Although they have different substrate preferences, for Met-Ala-Ser, we found, <it>Mt</it>MetAP1c had significantly high enzyme turnover rate as opposed to <it>Mt</it>MetAP1a. Circular dichroism spectroscopic studies as well as monitoring of enzyme activity indicated high temperature stability (up to 50°C) of <it>Mt</it>MetAP1a compared to that of the <it>Mt</it>MetAP1c. Modelling of <it>Mt</it>MetAP1a based on <it>Mt</it>MetAP1c crystal structure revealed the distinct spatial arrangements of identical active site amino acid residues and their mutations affected the enzymatic activities of both the proteins. Strikingly, we observed that 40 amino acid long N-terminal extension of <it>Mt</it>MetAP1c, compared to its other family members, contributes towards the activity and stability of this enzyme, which has never been reported for any methionine aminopeptidase. Furthermore, mutational analysis revealed that Val-18 and Pro-19 of <it>Mt</it>MetAP1c are crucial for its enzymatic activity. Consistent with this observation, molecular dynamic simulation studies of wild-type and these variants strongly suggest their involvement in maintaining active site conformation of <it>Mt</it>MetAP1c.</p> <p>Conclusion</p> <p>Our findings unequivocally emphasized that N-terminal extension of <it>Mt</it>MetAP1c contributes towards the functionality of the enzyme presumably by regulating active site residues through "action-at-a-distance" mechanism and we for the first time are reporting this unique function of the enzyme.</p

Assembly of the Cysteine Synthase Complex and the Regulatory Role of Protein-Protein Interactions*

Macromolecular assemblies play critical roles in regulating cellular functions. The cysteine synthase complex (CSC), which is formed by association of serine O-acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), acts as a sensor and modulator of thiol metabolism by responding to changes in nutrient conditions. Here we examine the oligomerization and energetics of formation of the soybean CSC. Biophysical examination of the CSC by size exclusion chromatography and sedimentation ultracentrifugation indicates that this assembly (complex Mr ∼ 330,000) consists of a single SAT trimer (trimer Mr ∼ 110,000) and three OASS dimers (dimer Mr ∼ 70,000). Analysis of the SAT-OASS interaction by isothermal titration calorimetry reveals negative cooperativity with three distinct binding events during CSC formation with Kd values of 0.3, 7.5, and 78 nm. The three binding events are also observed using surface plasmon resonance with comparable affinities. The stability of the CSC derives from rapid association and extremely slow dissociation of OASS with SAT and requires the C terminus of SAT for the interaction. Steady-state kinetic analysis shows that CSC formation enhances SAT activity and releases SAT from substrate inhibition and feedback inhibition by cysteine, the final product of the biosynthesis pathway. Cysteine inhibits SAT and the CSC with Ki values of 2 and 70 μm, respectively. These results suggest a new model for the architecture of this regulatory complex and additional control mechanisms for biochemically controlling plant cysteine biosynthesis. Based on previous work and our results, we suggest that OASS acts as an enzyme chaperone of SAT in the CSC