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

Highlights

• CysK catalyzes the last step of cysteine biosynthesis in bacteria and plants.

• CysK interacts with CysE to form the cysteine synthase complex.

• Recently, additional non-biosynthetic functions have been discovered for CysK.

• CysK moonlighting activities require interactions with binding partners.

• Moonlighting partners invariably mimic CysE to form complexes with CysK.

Abstract

O-acetylserine sulfhydrylase A (CysK) is the pyridoxal 5′-phosphate-dependent enzyme that catalyzes the final reaction of cysteine biosynthesis in bacteria. CysK was initially identified in a complex with serine acetyltransferase (CysE), which catalyzes the penultimate reaction in the synthetic pathway. This “cysteine synthase” complex is stabilized by insertion of the CysE C-terminus into the active-site of CysK. Remarkably, the CysK/CysE binding interaction is conserved in most bacterial and plant systems. For the past 40 years, CysK was thought to function exclusively in cysteine biosynthesis, but recent studies have revealed a repertoire of additional “moonlighting” activities for this enzyme. CysK and its paralogs influence transcription in both Gram-positive bacteria and the nematode Caenorhabditis elegans. CysK also activates an antibacterial nuclease toxin produced by uropathogenic Escherichia coli. Intriguingly, each moonlighting activity requires a binding partner that invariably mimics the C-terminus of CysE to interact with the CysK active site. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Introduction

Cysteine biosynthesis in bacteria occurs through two interconnected pathways. Sulfate is reduced to bisulfide (HS⁻) through a multistep, energy-dependent process, and l-serine is activated via O-acetylation to produce O-acetylserine (OAS). The two branches converge on the enzyme O-acetylserine sulfhydrylase (OASS) (EC 2.5.1.47), a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the replacement of the β-acetotoxy group of OAS with bisulfide (Fig. 1). CysK was the first OASS isoform to be identified and was originally isolated and characterized in Salmonella typhimurium [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. Those early studies showed that CysK can physically associate with serine acetyltransferase (CysE), which catalyzes the preceding step in the biosynthetic pathway. CysE is a hexameric protein that catalyzes an acetyl-transfer reaction from acetyl-CoA to serine via a random-order ternary complex reaction mechanism [12]. Together, the two enzymes form a heteromeric complex known as the cysteine synthase complex (CSC) [2], [13]. Subsequent studies identified CysK and CysE homologs also in plants [14], [15] and mycobacteria [16]. CysE shows the typical left-handed β-helix domain of acetyltransferases, but its C-terminus is flexible and not resolved in available crystal structures [17], [18]. It was proposed [19], and later demonstrated [20], [21], [22], that the C-terminal tail of CysE inserts into the active site of CysK. The three-dimensional structure of the CSC has not yet been determined. However, the structure of Haemophilus influenzae CysK (HiCysK) in complex with a decapeptide corresponding to the C-terminus of HiCysE supports the notion that the CysK active site serves as the anchor point for complex formation [20]. Interestingly, CysE proteins from different organisms show a high degree of sequence variability at the C-terminus (Fig. 2), yet carry an invariant C-terminal Ile residue. Ile plays a pivotal role in complex formation and its removal completely prevents CSC association [23]. From the beginning, the functional significance of the CSC was unclear because no substrate channeling was envisaged for the complex [2], [24], [25]. Additionally, only a fraction (~ 5–25%) of total CysK was reported to be in the complex [13], [26]. Subsequently, it has been proposed that CysK-binding provides a mechanism to protect the bacterial and plant CysE from cold-inactivation and proteolysis [27], [28].

A second OASS isozyme, CysM, whose biological role remains unclear [11], has been identified in several bacteria. CysM is less abundant than CysK [26] and can use alternative sulfur donors like thiosulfate [26], [29], [30]. Some reports suggest that CysM is differentially expressed during anaerobic growth conditions [26], [31], but these findings have not been supported by more recent studies (SalCom, “Salmonella enterica serovar Typhimurium Gene Expression Compendium” [32]). In contrast with CysK, CysM does not interact with CysE [23], [33], suggesting that the two isozymes play distinct roles in the regulation of cysteine biosynthesis.

The interaction between CysK and CysE appears quite unique, yet very similar binding interactions have evolved between CysK and other partner proteins. These various interactions account for the different moonlighting functions of CysK (Table 1). In Bacillus subtilis, CysK modulates the affinity of an Rrf2-type transcription factor for its operator sequences, thereby regulating expression of the cysteine regulon. In E. coli CysK acts as a so-called “permissive” factor to activate an antibacterial contact-dependent growth inhibition (CDI) toxin [34]. Although alternative partners bind CysK for new functions, these interactions could also directly influence cysteine biosynthesis by inhibiting CysK activity. Here, we discuss the biological roles of known CysK complexes in the context of cysteine metabolism.