Figure 4 - available from: Extremophiles
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Relationship between the substrate concentration and the specific activity of cysteine synthesis toward a OPS and b Na2S. A reaction mixture of 100 mM KPi buffer (pH 7.5), 1 mM DTT, 0.2 mM PLP, and a 20–120 mM OPS in the presence of 4 mM Na2S, or b 0.5–6 mM Na2S in the presence of 80 mM OPS was incubated at 80 °C. Enzyme activity (1 U) was defined as the amount of enzyme required to produce 1 mmol of l-cysteine per 1 min. Black and gray circles represent the specific activity of the wt ApOPSS and the F225A mutants, respectively. The average values of three independent experiments are plotted, and the error bars show the standard deviation
Source publication
O-Phosphoserine sulfhydrylase (OPSS) synthesizes cysteine from O-phospho-l-serine (OPS) and sulfide. We have determined the three-dimensional structures of OPSS from hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) in complex with aminoacrylate intermediate (AA) formed from pyridoxal 5′-phosphate with OPS or in complex with cysteine and comp...
Citations
... Cysteine synthesis begins with the formation of either O-acetyl-L-serine (OAS) or O-phospho-L-serine (OPS) by serine acetyltransferase (CysE, Cj0763) or phosphoserine aminotransferase (SerC, Cj0326), respectively [9,10]. The resultant metabolites act as sulfur acceptors in the generation of cysteine by either OAS sulfhydrylase (OASS) or OPS sulfhydrylase (OPSS), broadly referred to as cysteine synthase. ...
Campylobacter jejuni is a highly successful enteric pathogen with a small, host-adapted genome (1.64 Mbp, ~1650 coding genes). As a result, C. jejuni has limited capacity in numerous metabolic pathways, including sulfur metabolism. Unable to utilise ionic sulfur, C. jejuni relies on the uptake of exogenous cysteine and its derivatives for its supply of this essential amino acid. Cysteine can also be synthesized de novo by the sole cysteine synthase, CysM. In this study, we explored the substrate specificity of purified C. jejuni CysM and define it as an O-acetyl-L-serine sulfhydrylase with an almost absolute preference for sulfide as sulfur donor. Sulfide is produced in abundance in the intestinal niche C. jejuni colonises, yet sulfide is generally viewed as highly toxic to bacteria. We conducted a series of growth experiments in sulfur-limited media and demonstrate that sulfide is an excellent sulfur source for C. jejuni at physiologically relevant concentrations, combating the view of sulfide as a purely deleterious compound to bacteria. Nonetheless, C. jejuni is indeed inhibited by elevated concentrations of sulfide and we sought to understand the targets involved. Surprisingly, we found that inactivation of the sulfide-sensitive primary terminal oxidase, the cbb3-type cytochrome c oxidase CcoNOPQ, did not explain the majority of growth inhibition by sulfide. Therefore, further work is required to reveal the cellular targets responsible for sulfide toxicity in C. jejuni.
Pyridoxal-5′-phosphate-dependent cysteine synthases synthesize l-cysteine from their primary substrates, O-acetyl-l-serine (OAS) and O-phospho-l-serine (OPS), and their secondary substrate, sulfide. The mechanism by which cysteine synthases recognize OPS remains unclear; hence, we investigated the OPS recognition mechanism of the OPS sulfhydrylase obtained from Aeropyrum pernix K1 (ApOPSS) and the OAS sulfhydrylase-B obtained from Escherichia coli (EcOASS-B), using protein engineering methods. From the amino acid sequence alignment data, we found that some OPS sulfhydrylases (OPSSs) had a Tyr corresponding to the Phe225 and Phe141 residues in ApOPSS and EcOASS-B, respectively, and that the Tyr residue could facilitate OPS recognition. The enzymatic activity of the ApOPSS F225Y mutant toward OPS decreased compared with that of the wild-type; the kcat value decreased 2.3-fold during cysteine synthesis. X-ray crystallography results of the complex of ApOPSS F225Y and F225Y/R297A mutants bound to OPS and l-cysteine showed that kcat might have decreased because of the stronger interactions of the reaction product phosphate with Tyr225, Thr203, and Arg297, and that of the l-cysteine with Tyr225. The specific activity of the EcOASS-B F141Y mutant toward OPS increased by 50-fold compared with that of the wild-type. Thus, a Tyr within a cysteine synthase corresponding to the Phe225 in ApOPSS and Phe141 in EcOASS-B could act as a key residue for classifying an unknown cysteine synthase as an OPSS. The elucidation of the substrate recognition system of cysteine synthases would enable us to effectively classify cysteine synthases and develop pathogen-specific drug targets, as OPSS is absent in mammalian hosts.
The structures of aminoacrylate intermediates of wild-type, F448A mutant, and perdeuterated tyrosine phenol-lyase (TPL) formed from L-tyrosine, 3-F-L-tyrosine, S-ethyl-L-cysteine, and L-serine, with 4-hydroxpyridine bound, were determined by X-ray crystallography. All the aminoacrylate Schiff’s base structures in chain A are identical regardless of the substrate used to form them. The 4-hydroxypyridine is also in an identical location, except for F448A TPL, where it is displaced about 1 Å due to the increased size of the active site. In chain B, we have found different complexes depending on the substrate. With wild-type TPL, L-tyrosine gave no density, 3-F-L-tyrosine gave a gem-diamine, and L-serine gave a gem-diamine, in chain B. S-Ethyl-L-cysteine formed an aminoacrylate in chain B with both wild-type and F448A TPL, but perdeuterated TPL with S-ethyl-L-cysteine formed a gem-diamine of aminoacrylate. The kinetics of aminoacrylate intermediate formation from L-tyrosine and S-ethyl-L-cysteine were followed by stopped-flow spectrophotometry at temperatures from 281 to 320 K, and hydrostatic pressures ranging from 1 bar to 1.5 kbar at 293 K. There are large negative values of ΔS‡, ΔCp‡, ΔV‡, and Δβ‡ for aminoacrylate intermediate formation for L-tyrosine, but not for S-ethyl-L-cysteine. Formation of the aminoacrylate intermediates from L-tyrosine and S-ethyl-L-cysteine show heavy enzyme deuterium kinetic isotope effects with perdeuterated TPL that are strongly temperature and pressure dependent, and may be normal or inverse depending on conditions. These results suggest that conformational dynamics as well as vibrational coupling play a key role in the mechanism of the elimination reaction of L-tyrosine catalyzed by TPL.
Background
O-phospho-L-serine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) is thermostable and tolerant to organic solvents. It can produce non-natural amino acids in addition to L-cysteine.
Objective
We aimed to obtain higher amounts of ApOPSS compared to those reported with previous methods for convenience of research and for industrial production of L-cysteine and non-natural amino acids.
Method
We performed codon optimization of cysO that encodes ApOPSS, for optimal expression in Escherichia coli. We then examined combinations of conditions such as the host strain, plasmid, culture medium, and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration to improve ApOPSS yield.
Results and Discussion
E. coli strain Rosetta (DE3) harboring the expression plasmid pQE-80L with the codon-optimized cysO was cultured in Terrific broth with 0.01 mM IPTG at 37°C for 48 h to yield a 10-times higher amount of purified ApOPSS (650 mg·L-1) compared to that obtained by the conventional method (64 mg·L-1). We found that the optimal culture conditions along with codon optimization were essential for the increased ApOPSS production.The expressed ApOPSS had a 6-histidine tag at the N-terminal, which did not affect its activity. This method may facilitate the industrial production of cysteine and non-natural amino acids using ApOPSS.
Conclusion
We conclude that these results could be used in applied research on enzymatic production of L-cysteine in E. coli, large scale production of non-natural amino acids, enzymatic reaction in organic solvent, and environmental remediation by sulfur removal.
O-Acetyl-L-serine sulfhydrylase (OASS) from plants and bacteria synthesizes cysteine and unnatural amino acids that are important building blocks for active pharmaceuticals and agrochemicals. A thermostable O-phospho-L-serine sulfhydrylase from hyperthermophilic archaeon Aeropyrum pernix K1 (OPSSAp) exhibits a function similar to OASS. In the present study, we examined the synthesis of various unnatural amino acids using OPSSAp and demonstrated OPSSAp could efficiently synthesize various sulfur-containing amino acids. OPSSAp would be useful for industrial production of biologically important unnatural amino acids.
