Structure-based mutational studies of O-acetylserine sulfhydrylase reveal the reason for the loss of cysteine synthase complex formation in Brucella abortus

Abstract

Cysteine biosynthesis takes place via a two-step pathway in bacteria, fungi, plants and protozoan parasites, but not in humans, and hence, the machinery of cysteine biosynthesis is an opportune target for therapeutics. The decameric cysteine synthase complex (CSC) is formed when the C-terminal tail of serine acetyltransferase (SAT) binds in the active site of -acetylserine sulfydrylase (OASS), playing a role in the regulation of this pathway. Here, we show that OASS from (BaOASS) does not interact with its cognate SAT C-terminal tail. Crystal structures of native BaOASS showed that residues Gln96 and Tyr125 occupy the active-site pocket and interfere with the entry of the SAT C-terminal tail. The BaOASS (Q96A-Y125A) mutant showed relatively strong binding ( = 32.4 μM) to BaSAT C-terminal peptides in comparison with native BaOASS. The mutant structure looks similar except that the active-site pocket has enough space to bind the SAT C-terminal end. Surface plasmon resonance results showed a relatively strong (7.3 μM ) interaction between BaSAT and the BaOASS (Q96A-Y125A), but no interaction with native BaOASS. Taken together, our observations suggest that the CSC does not form in.

Structural characterization and functional analysis of cystathionine β‐synthase: an enzyme involved in the reverse transsulfuration pathway of Bacillus anthracis

“…BaCBS crystallized as a dimer, but the interface area between the protomers of BaCBS was measured to be less than those of all other available OASS structures (Table ). Comparisons of several PLP‐bound OASS structures and of their active site cleft openings have been made and showed much smaller active site cleft openings for these OASSs than for BaCBS (Table ). Moreover, a sequence comparison of BaCBS and OASS homologs showed approximately 35–43% sequence identity (Table ).…”

A Novel Mitochondrial Serine O-Acetyltransferase, OpSAT1, Plays a Critical Role in Sulfur Metabolism in the Thermotolerant Methylotrophic Yeast Ogataea parapolymorpha

“…It is known that SAT and cysteine synthase (CS), also called O -acetyl-L-serine sulfhydrylase (OASS), form an enzyme complex in some plants, such as chives, spinach, and A. thaliana 16 , 45 , 46 , but apparently not in all of them, as it was reported for Datura innoxia 47 . Similarly, SAT and OASS (CS) exist as an enzyme complex in S. typhimurium and E. coli 5 , 48 , 49 but not in Brucella abortus due to structural differences in OASS that interfere with the entry of the SAT C-terminal tail into the active-site pocket 50 . CS-SAT complex formation also varies in parasites.…”

A Competitive O-Acetylserine Sulfhydrylase Inhibitor Modulates the Formation of Cysteine Synthase Complex

“…In the following decades, CS complexes were identified in other organisms, including bacteria, protozoa, and plants [16,[56][57][58][59]. In this context, the engagement and the affinity of OASS-A and SAT in bacteria have been the object of several biophysical and kinetic studies, because of the potential pharmaceutical relevance of the cysteine biosynthetic pathway [15,17,24,31,52,[60][61][62][63]. Studies on S. Typhimurium and E. coli are of particular relevance because of the inclusion of these two species in the global priority list of the World Health Organization for the insurgence of new antibiotic resistants [64].…”