Abstract
Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase. Formation of the cysteine regulatory complex (CRC) is a critical biochemical control feature in plant sulfur metabolism. Here we present the 1.75-3.0 Å resolution x-ray crystal structures of soybean (Glycine max) SAT (GmSAT) in apoenzyme, serine-bound, and CoA-bound forms. The GmSAT-serine and GmSAT-CoA structures provide new details on substrate interactions in the active site. The crystal structures and analysis of site-directed mutants suggest that His(169) and Asp(154) form a catalytic dyad for general base catalysis and that His(189) may stabilize the oxyanion reaction intermediate. Glu(177) helps to position Arg(203) and His(204) and the β1c-β2c loop for serine binding. A similar role for ionic interactions formed by Lys(230) is required for CoA binding. The GmSAT structures also identify Arg(253) as important for the enhanced catalytic efficiency of SAT in the CRC and suggest that movement of the residue may stabilize CoA binding in the macromolecular complex. Differences in the effect of cold on GmSAT activity in the isolated enzyme versus the enzyme in the CRC were also observed. A role for CRC formation as a molecular chaperone to maintain SAT activity in response to an environmental stress is proposed for this multienzyme complex in plants.
Highlights
Serine acetyltransferase (SAT) catalyzes the limiting step in cysteine biosynthesis
The crystal structures and analysis of site-directed mutants suggest that His169 and Asp154 form a catalytic dyad for general base catalysis and that His189 may stabilize the oxyanion reaction intermediate
SAT catalyzes the first step in the cysteine biosynthesis pathway by formation of O-acetylserine from acetyl-CoA and serine (Fig. 1) [7,8,9,10,11,12]
Summary
Serine acetyltransferase (SAT) catalyzes the limiting step in cysteine biosynthesis. Results: Analysis of soybean SAT provides insight into catalysis and protein-protein interactions. The GmSAT structures identify Arg253 as important for the enhanced catalytic efficiency of SAT in the CRC and suggest that movement of the residue may stabilize CoA binding in the macromolecular complex. In a nutrient-poor state, depletion of sulfur leads to an accumulation of O-acetylserine, which disassociates the CRC to attenuate SAT activity This multienzyme assembly acts as a sensor to coordinate sulfur assimilation and modulate cysteine production in plants. The effect of CRC formation on SAT activity under low temperature conditions may allow OASS to act as a molecular chaperone of SAT in plants when the demand for sulfur assimilation and cysteine production increases in response to low temperatures
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