The Active Site of O-Acetylserine Sulfhydrylase Is the Anchor Point for Bienzyme Complex Formation with Serine Acetyltransferase

The biosynthesis of cysteine in bacteria and plants is carriedout by a two-step pathway, catalyzed by serine acetyltransferase(SAT) and O-acetylserine sulfhydrylase (OASS; O-acetylserine[thiol] lyase). The aerobic form of OASS forms a tight bienzymecomplex with SAT in vivo, termed cysteine synthase. We havedetermined the crystal structure of OASS in complex with a C-terminalpeptide of SAT required for bienzyme complex formation. Thebinding site of the peptide is at the active site of OASS, andits C-terminal carboxyl group occupies the same anion bindingpocket as the ${alpha}$ -carboxylate of the O-acetylserine substrate ofOASS. These results explain the partial inhibition of OASS bySAT on complex formation as well as the competitive dissociationof the complex by O-acetylserine.

INTRODUCTION

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Introduction

The biosynthesis of cysteine from serine in bacteria and plantsis carried out by a two-step pathway beginning with the O-acetylationof serine by serine acetyltransferase (SAT; EC 2.3.1.30), followedby ß-replacement of the acetyl group by sulfide bythe pyridoxal-5'-phosphate (PLP)-dependent O-acetylserine sulfhydrylase(OASS; EC 2.5.1.47) (10, 16, 17, 23) (Fig. 1). The inhibitionof SAT by cysteine is the key regulatory feature of sulfur metabolism,as isomerization of the O-acetylserine product of SAT producesthe N-acetylserine inducer of the cys regulon of genes responsiblefor sulfur assimilation (16, 17, 23). The approximately 20-foldabundance of the aerobic form of OASS relative to SAT in vivois thought to lead to the inclusion of nearly all SAT into abienzyme complex (1, 16, 18, 23, 32). The role of this complex,termed cysteine synthase (CS), is not well understood and isnot thought to confer the advantages of substrate channeling(6), but its formation decreases the cold inactivation of SAT,reduces its proteolytic sensitivity (21), and partially inhibitsthe OASS component. The complex is reversibly dissociated toits component enzymes by O-acetylserine at concentrations of0.1 to 1.0 mM (16). The C-terminal portion of SAT is requiredfor both cysteine feedback inhibition of SAT and for the associationof SAT and OASS to form the CS complex, based on the feedbackinsensitivity of mutants in this C-terminal region and the inabilityof truncation mutants to form the bienzyme complex (16, 17,20, 22, 23). A 10-residue peptide corresponding to the C-terminalsequence of Escherichia coli SAT (EcSAT) inhibits E. coli OASS(EcOASS) with a K_i of 130 nM (20).

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FIG. 1. The reactions catalyzed by SAT and OASS. The feedback inhibition of SAT is also depicted.

The three-dimensional structures of hexameric Haemophilus influenzaeSAT (HiSAT) and EcSAT have recently been determined as apoenzymeand in binary complexes with their cysteine inhibitor and withcoenzyme A (8, 25, 28), and a kinetic and chemical mechanismof action for these enzymes has been proposed (11-13, 28). Thebinary complexes reveal conformational changes of residues 241to 257 near the C-terminal end of the 267-residue polypeptide(25). These crystal structures, however, never defined a conformationfor the disordered C-terminal 10 residues of SAT that are requiredfor bienzyme complex formation. OASS has also been studied frombiochemical and structural perspectives (3, 29). The availablecrystal structures of dimeric Salmonella enterica serovar TyphimuriumOASS (StOASS) include the apoenzyme covalently bonded in Schiffbase linkage through Lys 41 to its pyridoxal-5'-phosphate (PLP)cofactor as an internal aldimine (3) and the Lys 41 $->$ Ala mutantenzyme in which PLP covalently bonds instead to a methionineresidue from the medium in an external aldimine linkage (5).The structure of StOASS covalently bonded to methionine hasbeen taken to be a structural analog of the covalent enzyme-O-acetylserineintermediate and has served to identify the location of theamino acid binding site. In addition to the "open" apoenzymeconformation and the "closed" conformation bound to methionine,a form of StOASS bound to chloride and sulfate anions resultsin an "inhibited" conformation (4).

In order to characterize the binding interactions of SAT toOASS in a homologous system and to address questions concerningthe physical organization of enzymes leading to cysteine biosynthesis,we prepared crystals of H. influenzae OASS (HiOASS) in complexwith the C-terminal 10-residue peptide of HiSAT. The crystalstructure reveals the binding location and conformation of theC-terminal four residues of this peptide and provides a structuralbasis for the binding and partial inhibition of OASS by SATin the CS complex.

MATERIALS AND METHODS

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Materials and Methods

The H. influenzae cysK gene corresponding to the aerobic formof OASS (HiOASS) was inserted into a pET28a vector encodingan N-terminal removable six-histidine tag and used to transformE. coli BL21(DE3). The protein was purified from 3 liters ofbacterial culture by Ni-nitrilotriacetic acid affinity chromatography,followed by thrombin cleavage of the histidine tag and subsequentSuperdex 200 pg gel filtration chromatography. The final yieldwas 70 mg. The unblocked peptide corresponding to the C-terminal10 residues of HiSAT (²⁵⁸Gly-Ile-Asp-Asp-Gly-Met-Asn-Leu-Asn-Ile²⁶⁷)was synthesized with a Perkins-Elmer Biosystems 433A peptidesynthesizer and subsequently purified by high-pressure liquidchromatography.

Crystals of the HiOASS-peptide complex were prepared under siliconoil by using the sitting drop method by mixing 2 µl ofHiOASS at a concentration of 15 mg/ml (0.45 mM of active sites)in 20 mM HEPES (pH 7.5), 50 mM NaCl, and 5 mM peptide with 2µl of 3.0 M ammonium sulfate, 2% (vol/vol) polyethyleneglycol 400, and 100 mM HEPES (pH 7.5). These crystals belongedto space group I4₁ (a = b = 112.6 and c = 45.8 Å). Crystalswere passed through solutions containing glycerol (but lackingpeptide) before vitrification in liquid nitrogen. These cooledcrystals diffracted X rays to a resolution of 1.55 Å byusing an in-house Rigaku R-Axis IV⁺⁺ image plate detector andRU-H3R rotating anode X-ray generator equipped with Osmic Blueoptics and operating at 50 kV and 100 mA. The data were reducedwith HKL (26).

The structure was solved by molecular replacement with EPMR(15), by using a single subunit of apoenzyme StOASS (1OAS) asthe search probe. Model building and atomic parameter refinementswere carried out with the programs ARP/wARP (24, 27), O (14),and CNS (2). A Ramachandran plot of main chain torsion anglepairs identified no residues occupying the disallowed or generouslyallowed regions (19, 30).

The atomic coordinates corresponding to the structure of HiOASSin complex with the C-terminal decapeptide of SAT have beendeposited in the Research Collaboratory for Structural BioinformaticsProtein Data Bank (accession code 1Y7L).

RESULTS

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Results

The structure of HiOASS in complex with the C-terminal decapeptideof HiSAT was determined to a resolution of 1.55 Å by usingthe structure of a single subunit of apoenzyme StOASS as themolecular replacement search probe. The atomic model was refinedto a crystallographic R_factor of 19.7% (R_free = 20.7%) (Table1). The asymmetric unit of the atomic model included a singlesubunit of the dimeric enzyme (residues 2 to 311 of the 316-residuepolypeptide), a single molecule of its PLP cofactor covalentlybound to Lys 42 (equivalent to StOASS Lys 41), the C-terminalfour residues of the 10-residue peptide, one sulfate ion, and247 water molecules. The overall conformation of HiOASS nearthe active site is most similar to the apoenzyme, or open andanion-inhibited forms of StOASS (root mean square [rms] discrepanciesof 0.5 and 0.4 Å, respectively) (Fig. 2A).

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TABLE 1. Data measurement and refinement statistics^a

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FIG. 2. (A) Stereoview superposition of HiOASS with peptide (yellow), StOASS with methionine in external aldimine linkage (cyan), and StOASS apoenzyme (gray). The atoms corresponding to PLP at the active site of HiOASS are depicted. (B) Space-filling diagram of dimeric HiOASS in complex with the peptide (yellow) and PLP (green) viewed parallel to the dimeric twofold axis. Figures were prepared with PyMOL (7).

The peptide binds at the active site of OASS near the PLP cofactor(Fig. 2B). The distance between the two peptides bound to theOASS dimer is 36 Å. Interpretable electron density forthe peptide was observed for only its C-terminal four residues(Fig. 3A). According to a residue numbering scheme from thenumbering of the full-length 267-residue HiSAT polypeptide,the visible residues of the peptide correspond to P264 to P267(²⁶⁴Asn-Leu-Asn-Ile²⁶⁷). The observed peptide-OASS interactionsare all formed by the last two residues (Fig. 3B and C). Theside chain O ${delta}$ 1 and N ${delta}$ 2 atoms of Asn-P266 interact with Ser 70N and O ${gamma}$ , respectively. One oxygen of the Ile-P267 C-terminalcarboxyl group interacts with Thr 69 O ${gamma}$ and the side chain N ${varepsilon}$ 2of Gln 143. The other oxygen atom interacts with the peptidenitrogen of Thr 73 and with a water molecule trapped betweenthe peptide carboxylate and the ring hydroxyl or phenolate oxygenof PLP. Interatomic distances less than 4 Å in lengthare observed between the side chain atoms of Ile-P267 and thering of Phe 144 as well as the carbonyl oxygen atoms of Gly228 and Asn 264. A least-squares superposition of the StOASS-methionineand HiOASS-peptide structures revealed that the carboxyl groupsof the methionine and Ile P-267 superimpose near the N-terminalend of a helix ${alpha}$ 2 (residues 71 to 84) as do their side chains.

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FIG. 3. (A) Unbiased F_o – F_c electron density corresponding to the last four residues of the C-terminal peptide of SAT bound to the active site of HiOASS. The map was calculated before fitting the peptide to the electron density. The contour level is 3.0 rms units. (B) Superposition of the active sites of HiOASS in complex with the C-terminal four residues of HiSAT (yellow bonds) and StOASS in external aldimine linkage with methionine (cyan bonds). Hydrophilic interactions are indicated by dotted lines and illustrate the shared interactions of OASS with the C-terminal carboxylate of the peptide and the ${alpha}$ -carboxylate group of the methionine ligand bound to PLP. The locations of PLP, the methionine of StOASS linked to PLP, and the C-terminal residue of the peptide (Ile-P267) are labeled. (C) Stereoview of the active site of the HiOASS peptide complex. The peptide (pink), PLP (green), and water molecules (cyan) are depicted as are the amino-terminal residues of helix ${alpha}$ 2 that interact with the peptide carboxyl.

DISCUSSION

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Discussion

The structure of HiOASS in complex with the C-terminal decapeptideof HiSAT reveals the conformation and interactions of the lastfour residues of SAT with the active site of OASS. A superpositionof the StOASS-methionine and HiOASS-peptide structures overlaysthe carboxyl groups and side chain atoms of the methionine andIle P-267 in an orientation that reverses the direction of theirmain chain atoms. Since the binding location of methionine toStOASS is thought to overlap the O-acetylserine binding pocket(5), it follows that the C-terminal isoleucine residue of SATwould compete with binding of O-acetylserine for the activesite of OASS. Indeed, the C-terminal 10-residue peptide of EcSAT(²⁶⁴Asn-His-Thr-Phe-Glu-Tyr-Gly-Asp-Gly-Ile²⁷³) is a 130 nMcompetitive inhibitor of EcOASS (70% sequence identity withHiOASS) (20). EcSAT and HiSAT share a C-terminal isoleucineresidue in their sequences with many additional bacterial SATsthat may also form the CS complex (e.g., Vibrio cholerae, Neisseriameningitidis, Yersinia pestis, and S. enterica serovar Typhimurium).

In addition, the structure reported here provides an explanationfor the inability of SAT truncation mutants lacking the C-terminal10, 14, 20, and 25 residues to form the CS complex (20) andfor the dissociation of the complex by O-acetylserine, whichwould displace the C-terminal peptide of SAT responsible forcomplex formation. However, full-length EcSAT is a 250-foldtighter binding inhibitor of OASS than its C-terminal 10-residuepeptide (20), and so additional structural features of SAT maybe recognized by OASS apart from the C-terminal peptide butwhich alone are insufficient to direct formation of the complex.Any such additional interactions between SAT and OASS must awaitfurther studies, as the results presented here do not sufficientlyconstrain atomic modeling of the CS complex from its components.

The question of why the CS complex would remain catalyticallycompetent for cysteine biosynthesis in vivo if its formationrequired its OASS active sites to be tethered (and inhibited)in the manner described here may be answered by noting thata dimer of OASS may be tethered to SAT in the CS complex byonly one of its active sites. This is consistent with the measuredturnover number for OASS in the purified CS complex of S. entericaserovar Typhimurium, which is 55% of that measured for the resolvedenzyme (16). The observed mass of the CS complex derived fromequilibrium ultracentrifugation studies is 309 kDa for the S.enterica serovar Typhimurium complex (16) and 293 kDa for thecomplex from E. coli (21). These observations are best explainedby a stoichiometry for the complex of one molecule of hexamericSAT and two dimers of OASS (calculated mass, 314 kDa for bothorganisms) (9, 16). Such a complex could be formed with eachdimer of OASS contributing one active site as an anchor pointfor bienzyme complex formation, leaving the other active siteunblocked.

These results add to the diverse set of roles played by theC-terminal region of SAT, which now include its participationin the high-affinity cysteine binding site of SAT (25, 28),its competitive inhibition of OASS activity with respect toO-acetylserine, and its role in tethering OASS to form the CScomplex. Remaining unanswered, however, are questions concerningthe biological advantages conferred by complex formation sincesubstrates are not thought to channel between the active sitesof SAT and OASS (6). However, EcOASS stimulates the activityof E. coli ATP sulfurylase (31), an enzyme that catalyzes theinitial step of sulfur assimilation into activated organic molecules,suggesting that OASS may form additional bienzyme or higherorder enzyme complexes.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health GrantAI42154 to S.L.R. M.W.V. was supported by grant AI33696 to JohnS. Blanchard. Peptide synthesis was performed by the Laboratoryfor Macromolecular Analysis and Proteomics at the Albert EinsteinCollege of Medicine, which is supported in part by the AlbertEinstein Comprehensive Cancer Center (CA13330) and the DiabetesResearch and Training Center (DK20541).

We acknowledge Paul F. Cook (University of Oklahoma) for helpfuldiscussions.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-2784. Fax: (718) 430-8565. E-mail: roderick{at}aecom.yu.edu.

REFERENCES

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