Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/11/2009
Publication Date: 4/10/2009
Citation: Kumaran, S., Yi, H., Krishnan, H.B., Jez, J.M. 2009. Assembly of the Cysteine Synthase Complex and the Regulatory Role of Protein-Protein Interactions. Journal of Biological Chemistry. 284:10268-10275. Interpretive Summary: Soybean is a rich source of protein. Unfortunately, soybean proteins contain low amounts of two important amino acids, methionine and cysteine, that are vital for optimal growth of humans and animals. Therefore, attempts are being made to increase the amount of these two amino acids in soybean proteins. Serine O-acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS) comprise the two-step pathway for cysteine synthesis found in plants and bacteria. Both enzymes interact to form the cysteine synthase complex (CSC). Because cysteine is the metabolic source of sulfur for all thiol-containing compounds in plants, the interplay between sulfur assimilation and cysteine biosynthesis modulated by the CSC has a central role in thiol metabolism and can affect a variety of cellular processes. To date, biochemical studies provide only a partial view of assembly of the CSC from either plants or bacteria. Using a combination of biophysical approaches, here we have examined the composition of a soybean CSC and suggest a new model for the architecture of this regulatory macromolecular assembly. The information obtained from this in-depth biochemical study will help biotechnologists to genetically manipulate the sulfur-assimilatory enzyme expression levels so that we can improve the overall quality of soybean seed proteins. Superior quality soy proteins can be utilized to meet the nutritional requirements of the multitude of malnourished people around the world.
Technical Abstract: 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), functions as a multienzyme complex that responds to changes in intracellular sulfur levels resulting from metabolic stresses in plants and bacteria to act as a sensor and modulator of thiol metabolism. Here we examine the oligomerization and energetics of formation of the soybean CSC using analytical ultracentrifugation, isothermal titration calorimetry, and surface plasmon resonance. Biophysical examination of CSC oligomerization indicates that this macromolecular assembly from soybean consists of a single SAT trimer and three OASS dimers. Under physiological conditions, the stability of the CSC derives from tight binding resulting from rapid association and extremely slow dissociation of OASS in the assembly, as determined by isothermal titration calorimetry and surface plasmon resonance. Addition of multiple OASS dimers to the SAT trimer displays negative cooperativity and requires the C-terminus of SAT for interaction with OASS. Steady-state kinetic analysis shows that the functional consequences of CSC formation include elevated SAT activity and the release of substrate inhibition and feedback inhibition by cysteine, the final product of the pathway. These results suggest a new model for the architecture of this regulatory complex and additional physiological functions for this macromolecular assembly in modulating plant cysteine biosynthesis.