Main content area

Two Distinct Assembly States of the Cysteine Regulatory Complex of Salmonella typhimurium Are Regulated by Enzyme–Substrate Cognate Pairs

Kaushik, Abhishek, Ekka, Mary Krishna, Kumaran, Sangaralingam
Biochemistry 2017 v.56 no.18 pp. 2385-2399
Salmonella Typhimurium, binding sites, biosynthesis, calorimetry, cysteine, cysteine synthase, enzyme substrates, gel chromatography, models, serine O-acetyltransferase, sulfates, sulfides, surface plasmon resonance, titration, ultracentrifugation
Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), which catalyze the last two steps of cysteine biosynthesis, interact and form the cysteine regulatory complex (CRC). The current model of Salmonella typhimurium predicts that CRC is composed of one [SAT]ₕₑₓₐₘₑᵣ unit and two molecules of [OASS]dᵢₘₑᵣ. However, it is not clear why [SAT]ₕₑₓₐₘₑᵣ cannot engage all of its six high-affinity binding sites. We examined the assembly state(s) of CRC by size exclusion chromatography, analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) approaches. We show that CRC exists in two major assembly states, low-molecular weight (CRC₁; 1[SAT]ₕₑₓₐₘₑᵣ + 2[OASS]dᵢₘₑᵣ) and high-molecular weight (CRC₂; 1[SAT]ₕₑₓₐₘₑᵣ + 4[OASS]dᵢₘₑᵣ) states. Along with AUC results, ITC and SPR studies show that [OASS]dᵢₘₑᵣ binds to [SAT]ₕₑₓₐₘₑᵣ in a stepwise manner but the formation of fully saturated CRC₃ (1[SAT]ₕₑₓₐₘₑᵣ + 6[OASS]dᵢₘₑᵣ) is not favorable. The fraction of CRC₂ increases as the [OASS]dᵢₘₑᵣ/[SAT]ₕₑₓₐₘₑᵣ ratio increases to >4-fold, but CRC₂ can be selectively dissociated into either CRC₁ or free enzymes, in the presence of OAS and sulfide, in a concentration-dependent manner. Together, we show that CRC is a regulatable multienzyme assembly, sensitive to OASS–substrate(s) levels but subject to negative cooperativity and steric hindrance. Our results constitute the first report of the dual-assembly-state nature of CRC and suggest that physiological conditions, which limit sulfate uptake, would favor CRC₁ over CRC₂.