A Two-step Process Controls the Formation of the Bienzyme Cysteine Synthase Complex

Abstract

The regulation of enzyme activity through the transient formation of multiprotein assemblies plays an important role in the control of biosynthetic pathways. One of the first regulatory complexes to be discovered was cysteine synthase (CS), formed by the pyridoxal 5-phosphate-dependent enzyme O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). These enzymes are at the branch point of the sulfur, carbon, and nitrogen assimilation pathways. Understanding the mechanism of complex formation helps to clarify the role played by CS in the regulation of sulfur assimilation in bacteria and plants. To this goal, stopped-flow fluorescence spectroscopy was used to characterize the interaction of SAT with OASS, at different temperatures and pH values, and in the presence of the physiological regulators cysteine and bisulfide. Results shed light on the mechanism of complex formation and regulation, so far poorly understood. Cysteine synthase assembly occurs via a two-step mechanism involving rapid formation of an encounter complex between the two enzymes, followed by a slow conformational change. The conformational change likely results from the closure of the active site of OASS upon binding of the SAT C-terminal peptide. Bisulfide, the second substrate and a feedback inhibitor of OASS, stabilizes the CS complex mainly by decreasing the back rate of the isomerization step. Cysteine, the product of the OASS reaction and a SAT inhibitor, slightly affects the kinetics of CS formation leading to destabilization of the complex.

Three-stage Assembly of the Cysteine Synthase Complex from Escherichia coli

“…Two such intermediates were identified using this strategy. A single intermediate has been suggested for the CSC from Haemophilus influenza (36). Comparison of the kinetics and energetics of the binding of SAT with its 10-mer, C-terminal peptide supports a mechanism in which the C terminus of SAT attaches to OASS through a non-allosteric interaction that tethers the CSC proteins.…”

Cyclopropane-1,2-dicarboxylic acids as new tools for the biophysical investigation ofO-acetylserine sulfhydrylases by fluorimetric methods and saturation transfer difference (STD) NMR

“…Over the years, along with other groups 20,21 , we have investigated structural and functional properties of OASS isozymes with the aim of further characterizing the biological features of these proteins and to explore the possibility of their inhibition by small molecules. The rational design of the first inhibitors was based on the structure of SAT, that physiologically inhibits OASS activity upon formation of the cysteine synthase bioenzyme complex 22,23 ; in particular, it was considered that the carboxylic moiety of Ile267 of SAT is essential for the interaction between the two proteins [22][23][24] . Therefore, combining a structure-based with a ligand-based drug design approach, we planned and synthesized a series of cyclopropanecarboxylic acid derivatives that bind to both OASS isoforms at nanomolar concentrations [25][26][27] .…”

Moonlighting O-acetylserine sulfhydrylase: New functions for an old protein

“…In fact, the Glu-Tyr-Tyr-Ile C-terminal sequence of EGL-1 resembles the Asp-Tyr-Val-Ile C-terminal sequence of A. thaliana CysE (Table 1) and deletion or substitution of Ile with Ala disrupts binding to CYSL-1. An interesting feature of the EGL-1/CYSL-1 complex is the stabilizing effect of H 2 S, which is very similar to the effects observed with the bacterial and plant CSCs [2, 59] [48]. Here, H 2 S-dependent complex stabilization is exploited to fine-tune the inhibitory activity of EGL-1 on HIF and to coordinate the response to hypoxia with increased H 2 S inside the neurons.…”

“…Here, H 2 S-dependent complex stabilization is exploited to fine-tune the inhibitory activity of EGL-1 on HIF and to coordinate the response to hypoxia with increased H 2 S inside the neurons. It has been speculated that CYSL-1 has an allosteric bisulfide-binding site, similar to that observed in StCysK, whose occupation by bisulfide stabilizes a partially closed conformation of the enzyme that also shows increased affinity for CysE [2, 10, 59]. Notably, both in the case of CymR and EGL-1 the interaction with CysK (or CysK paralog) is modulated by ligands, i.e.…”

“…In the absence of a comprehensive interaction network, the most remarkable finding is the observation that substitution of the C-terminal Ile of CysE with Ala completely prevents CSC formation. Upon binding the active-site pocket, the C-terminal Ile triggers a conformational change that probably closes the CysK active site, thereby promoting high-affinity binding to CysE [59, 63]. This conformational transition could be obligatory for the formation of high-affinity complexes with CysE and other binding partners.…”