Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants

Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (ΔG = −33 kcal mol⁻¹) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (K_D = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (K_D) and the regulation of cysteine sensitivity of SAT within the CSC.     

Escherichia coli phosphoenolpyruvate carboxylase: studies on the mechanism of multiple allosteric interactions.

Some kinetic studies of the interactions between Escherichia coli phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylase (phosphorylating) EC 4.1.1.31) acetyl coenzyme A, fructose 1,6-bisphosphate, and aspartate were performed. Activation of the enzyme by fructose 1,6-bisphosphate is anomalous by comparison with acetyl coenzyme A in that it confers hysteretic properties on the enzyme. In the presence of both activators and aspartate, hysteresis is observed also, but the approach to optimum catalytic activity can be fit to an equation for a second-order reaction with respect to enzyme concentration. Since, however, hysteresis is not a result of any apparent association-dissociation reaction, the apparent fit to a second-order kinetic equation is probably not real but is the result of a multistep activation mechanism. Hysteresis is not eliminated by preincubation of the enzyme with fructose 1,6-bisphosphate, acetyl coenzyme A, or phosphoenolpyruvate singly or in any pair of combinations. Hysteresis is associated, therefore, with the slow conformation change from the inactive species to the active species under the influence of all three of those reactants. The enzyme complex resulting from the binding of each activator, including phosphoenolpyruvate, has an increased affinity for the other activators. A kinetic method for estimating the relative changes in affinity of these complexes for some of the other reactants is presented. At concentrations of the activators below their K_a, synergistic effects are evident, particularly in their ability to relieve aspartate inhibition. Aspartate inhibition is competitive with acetyl coenzyme A both in the absence and in the presence of low concentrations of fructose 1,6-bisphosphate. Increasing the concentrations of fructose 1,6-bisphosphate results in an increase in the apparent K_l for aspartate, suggesting that synergistic activation by fructose 1,6-bisphosphate is a result of the increased affinity of the fructose 1,6-bisphosphate-enzyme complex for acetyl coenzyme A, and a shift in the concentration of enzyme species away from the one(s) to which aspartate can bind most easily. In the presence of fructose 1,6-bisphosphate alone optimal activation can be achieved, but the concentrations required in vitro are high and suggest that fructose 1,6-bisphosphate alone does not function in that capacity physiologically, but primes the enzyme for more effective activation by acetyl coenzyme A and/or phosphoenolpyruvate.     

Divergence of cofactor recognition across evolution: coenzyme A binding in a prokaryotic arylamine N-acetyltransferase

Arylamine N-acetyltransferase (NAT) enzymes are widespread in nature. They serve to acetylate xenobiotics and/or endogenous substrates using acetyl coenzyme A (CoA) as a cofactor. Conservation of the architecture of the NAT enzyme family from mammals to bacteria has been demonstrated by a series of prokaryotic NAT structures, together with the recently reported structure of human NAT1. We report here the cloning, purification, kinetic characterisation and crystallographic structure determination of NAT from Mycobacterium marinum, a close relative of the pathogenic Mycobacterium tuberculosis. We have also determined the structure of M. marinum NAT in complex with CoA, shedding the first light on cofactor recognition in prokaryotic NATs. Surprisingly, the principal CoA recognition site in M. marinum NAT is located some 30 Å from the site of CoA recognition in the recently deposited structure of human NAT2 bound to CoA. The structure explains the Ping-Pong Bi-Bi reaction mechanism of NAT enzymes and suggests mechanisms by which the acetylated enzyme intermediate may be protected. Recognition of CoA in a much wider groove in prokaryotic NATs suggests that this subfamily may accommodate larger substrates than is the case for human NATs and may assist in the identification of potential endogenous substrates. It also suggests the cofactor-binding site as a unique subsite to target in drug design directed against NAT in mycobacteria.     

The structure and mechanism of serine acetyltransferase from Escherichia coli

Serine acetyltransferase (SAT) catalyzes the first step of cysteine synthesis in microorganisms and higher plants. Here we present the 2.2 A crystal structure of SAT from Escherichia coli, which is a dimer of trimers, in complex with cysteine. The SAT monomer consists of an amino-terminal alpha-helical domain and a carboxyl-terminal left-handed beta-helix. We identify His(158) and Asp(143) as essential residues that form a catalytic triad with the substrate for acetyl transfer. This structure shows the mechanism by which cysteine inhibits SAT activity and thus controls its own synthesis. Cysteine is found to bind at the serine substrate site and not the acetyl-CoA site that had been reported previously. On the basis of the geometry around the cysteine binding site, we are able to suggest a mechanism for the O-acetylation of serine by SAT. We also compare the structure of SAT with other left-handed beta-helical structures.     

Kinetic mechanism of the serine acetyltransferase from Haemophilus influenzae

The kinetic mechanism of serine acetyltransferase from Haemophilus influenzae was studied in both reaction directions. The enzyme catalyzes the conversion of acetyl CoA and L-serine to O-acetyl-L-serine (OAS) and coenzyme A (CoASH). In the direction of L-serine acetylation, an equilibrium ordered mechanism is assigned at pH 6.5. The initial velocity pattern in the absence of added inhibitors is best described by a series of lines converging on the ordinate when L-serine is varied at different fixed levels of acetyl CoA. The initial velocity pattern at pH 7.5 is also intersecting, but the lines are nearly parallel. Product inhibition by OAS is noncompetitive against acetyl CoA, while it is uncompetitive against L-serine. Product inhibition by L-serine in the reverse reaction direction is noncompetitive with respect to both OAS and CoASH. Glycine and S-methyl-L-cysteine (SMC) were used as dead-end analogs of L-serine and OAS, respectively. Glycine is competitive versus L-serine and uncompetitive versus acetyl CoA, while SMC is competitive against OAS and uncompetitive against CoASH. Desulfo-CoA was used as a dead-end analog of both acetyl CoA and CoASH, and is competitive versus both substrates in the direction of L-serine acetylation; while it is competitive against CoASH and noncompetitive against OAS in the direction of CoASH acetylation. All of the above kinetic parameters are consistent with those predicted for an ordered mechanism at pH 6.5 with the exception of the uncompetitive inhibition by OAS vs. serine. The latter inhibition pattern suggests combination of OAS with the central E:acetyl CoA:serine complex. Cysteine is known to regulate its own biosynthesis at the level of SAT. As a dead-end inhibitor, L-cysteine is competitive against both substrates in both reaction directions. These results are discussed in terms of the mechanism of regulation.     

Structure of Soybean Serine Acetyltransferase and Formation of the Cysteine Regulatory Complex as a Molecular Chaperone

Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase. Formation of the cysteine regulatory complex (CRC) is a critical biochemical control feature in plant sulfur metabolism. Here we present the 1.75–3.0 Å resolution x-ray crystal structures of soybean (Glycine max) SAT (GmSAT) in apoenzyme, serine-bound, and CoA-bound forms. The GmSAT-serine and GmSAT-CoA structures provide new details on substrate interactions in the active site. The crystal structures and analysis of site-directed mutants suggest that His¹⁶⁹ and Asp¹⁵⁴ form a catalytic dyad for general base catalysis and that His¹⁸⁹ may stabilize the oxyanion reaction intermediate. Glu¹⁷⁷ helps to position Arg²⁰³ and His²⁰⁴ and the β1c-β2c loop for serine binding. A similar role for ionic interactions formed by Lys²³⁰ is required for CoA binding. The GmSAT structures also identify Arg²⁵³ as important for the enhanced catalytic efficiency of SAT in the CRC and suggest that movement of the residue may stabilize CoA binding in the macromolecular complex. Differences in the effect of cold on GmSAT activity in the isolated enzyme versus the enzyme in the CRC were also observed. A role for CRC formation as a molecular chaperone to maintain SAT activity in response to an environmental stress is proposed for this multienzyme complex in plants.     

The Molecular Structure of Ornithine Acetyltransferase from Mycobacterium tuberculosis Bound to Ornithine, a Competitive Inhibitor

Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT; E.C. 2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to l-glutamate. Mtb OAT is a member of the N-terminal nucleophile fold family of enzymes. The crystal structures of Mtb OAT in native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 Å resolutions, respectively. ORN is a competitive inhibitor of this enzyme against l-glutamate as substrate. Although the acyl-enzyme complex of Streptomyces clavuligerus ornithine acetyltransferase has been determined, ours is the first crystal structure to be reported of an ornithine acetyltransferase in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, stabilization of the C-terminal residues by ORN reduces the size of the active-site pocket volume in the structure of the ORN complex. The interactions of ORN and the protein residues of Mtb OAT unambiguously delineate the active-site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN-Mtb OAT complex reveal important interactions of the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly128 and with the side-chain oxygen of Thr127. These interactions likely help in the stabilization of oxyanion formation during enzymatic reaction and also will polarize the carbonyl carbon-oxygen bond, thereby enabling the side-chain atom O^γ1 of Thr200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN.     

Detection of broad-spectrum aminoglycoside antibiotics through fluorescence-labeling aminoglycoside acetyltransferase(6')-Ii

Aminoglycosides are among the most commonly used antibiotics. The intensive use of aminoglycoside antibiotics has led to the problem of food contamination and the development of antibiotic-resistant bacteria. In the present study, we developed an effective method for easy sensitive detection of broad-spectrum aminoglycoside antibiotics. Aminoglycoside 6′-N-acetyltransferase family catalyzes the transfer of an acetyl group from acetyl coenzyme A (acetyl-CoA) to the 6′ amino group of the aminoglycoside, which is one of the most widespread determinants of aminoglycoside resistance. Because acetyl-CoA is naturally present only in living organisms, it is expected that the enzyme can bind with aminoglycoside antibiotics without catalysis in vitro. The enzyme was mutated for the introduction of a cysteine residue to flexible loops close to the binding site, which was then labeled with thio-labeling reagent fluorescein-5-maleimide. The labeled enzymes were characterized with kinetic and binding studies of various known aminoglycoside antibiotics. The binding of the labeled enzyme with aminoglycoside antibiotics causes a conformational change of the enzyme, which subsequently changes the hydrophobicity and hydrophilicity environment of fluorescent labeling reagent resulting in emission of fluorescence. This study provides a sensitive detection method for residual aminoglycoside antibiotics and strategies to screen and discover new effective aminoglycoside antibiotics.     

Crystal Structure of Glyceraldehyde-3-Phosphate Dehydrogenase 1 from Methicillin-Resistant Staphylococcus aureus MRSA252 Provides Novel Insights into Substrate Binding and Catalytic Mechanism

The dreaded pathogen Staphylococcus aureus is one of the causes of morbidity and mortality worldwide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the key glycolytic enzymes, is irreversibly oxidized under oxidative stress and is responsible for sustenance of the pathogen inside the host. With an aim to elucidate the catalytic mechanism and identification of intermediates involved, we describe in this study different crystal structures of GAPDH1 from methicillin-resistant S. aureus MRSA252 (SaGAPDH1) in apo and holo forms of wild type, thioacyl intermediate, and ternary complexes of active-site mutants with physiological substrate d-glyceraldehyde-3-phosphate (G3P) and coenzyme NAD⁺. A new phosphate recognition site, “new P_i” site, similar to that observed in GAPDH from Thermotoga maritima, is reported here, which is 3.40 Å away from the “classical P_i” site. Ternary complexes discussed are representatives of noncovalent Michaelis complexes in the ground state. d-G3P is bound to all the four subunits of C151S.NAD and C151G.NAD in more reactive hydrate (gem-di-ol) form. However, in C151S + H178N.NAD, the substrate is bound to two chains in aldehyde form and in gem-di-ol form to the other two. This work reports binding of d-G3P to the C151G mutant in an inverted manner for the very first time. The structure of the thiaocyl complex presented here is formed after the hydride transfer. The C3 phosphate of d-G3P is positioned at the “P_s” site in the ternary complexes but at the “new P_i” site in the thioacyl complex and C1-O1 bond points opposite to His178 disrupting the alignment between itself and NE2 of His178. A new conformation (Conformation I) of the 209-215 loop has also been identified, where the interaction between phosphate ion at the “new P_i” site and conserved Gly212 is lost. Altogether, inferences drawn from the kinetic analyses and crystal structures suggest the “flip-flop” model proposed for the enzyme mechanism.     

Kinetic and X-ray structural evidence for negative cooperativity in substrate binding to nicotinate mononucleotide adenylyltransferase (NMAT) from Bacillus anthracis

Biosynthesis of NAD(P) in bacteria occurs either de novo or through one of the salvage pathways that converge at the point where the reaction of nicotinate mononucleotide (NaMN) with ATP is coupled to the formation of nicotinate adenine dinucleotide (NaAD) and inorganic pyrophosphate. This reaction is catalyzed by nicotinate mononucleotide adenylyltransferase (NMAT), which is essential for bacterial growth, making it an attractive drug target for the development of new antibiotics. Steady-state kinetic and direct binding studies on NMAT from Bacillus anthracis suggest a random sequential Bi-Bi kinetic mechanism. Interestingly, the interactions of NaMN and ATP with NMAT were observed to exhibit negative cooperativity, i.e. Hill coefficients < 1.0. Negative cooperativity in binding is supported by the results of X-ray crystallographic studies. X-ray structures of the B. anthracis NMAT apoenzyme, and the NaMN- and NaAD-bound complexes were determined to resolutions of 2.50 Å, 2.60 Å and 1.75 Å, respectively. The X-ray structure of the NMAT-NaMN complex revealed only one NaMN molecule bound in the biological dimer, supporting negative cooperativity in substrate binding. The kinetic, direct-binding, and X-ray structural studies support a model in which the binding affinity of substrates to the first monomer of NMAT is stronger than that to the second, and analysis of the three X-ray structures reveals significant conformational changes of NMAT along the enzymatic reaction coordinate. The negative cooperativity observed in B. anthracis NMAT substrate binding is a unique property that has not been observed in other prokaryotic NMAT enzymes. We propose that regulation of the NAD(P) biosynthetic pathway may occur, in part, at the reaction catalyzed by NMAT.     

Molecular mechanism underlying promiscuous polyamine recognition by spermidine acetyltransferase

Spermidine acetyltransferase (SAT) from Escherichia coli, which catalyses the transfer of acetyl groups from acetyl-CoA to spermidine, is a key enzyme in controlling polyamine levels in prokaryotic cells. In this study, we determined the crystal structure of SAT in complex with spermidine (SPD) and CoA at 2.5 Å resolution. SAT is a dodecamer organized as a hexamer of dimers. The secondary structural element and folding topology of the SAT dimer resemble those of spermidine/spermine N¹-acetyltransferase (SSAT), suggesting an evolutionary link between SAT and SSAT. However, the polyamine specificity of SAT is distinct from that of SSAT and is promiscuous. The SPD molecule is also located at the inter-dimer interface. The distance between SPD and CoA molecules is 13 Å. A deep, highly acidic, water-filled cavity encompasses the SPD and CoA binding sites. Structure-based mutagenesis and in-vitro assays identified SPD-bound residues, and the acidic residues lining the walls of the cavity are mostly essential for enzymatic activities. Based on mutagenesis and structural data, we propose an acetylation mechanism underlying promiscuous polyamine recognition for SAT.     

Plant serine acetyltransferase: new insights for regulation of sulphur metabolism in plant cells

Plant serine acetyltransferase (SAT, E.C. 2.3.1.30) catalyses the first connecting reaction between nitrogen/carbon and sulphate metabolism. SAT is associated with the second committed enzyme, O-acetylserine(thiol)lyase (OASTL, E.C. 4.2.99.8), in a bi-enzyme complex called cysteine synthase (CS). Metabolic regulation of SAT-bound OASTL in the presence of cysteine (Cys) is analysed with the extracts from the leaf cell compartments of Pisum sativum. To this end, a high performance liquid chromatography (HPLC) technique is developed to measure the rate of O-acetylserine (OAS) formation by SAT. Under physiological experimental conditions, L-Cys specifically inhibits chloroplast-SAT activity, which is linked to the sulphate assimilation network. This metabolic feedback control does not apply to the SAT activity located in the cytosol. The non-physiological range of L-Cys inhibits the mitochondrial isoform. L-Cys in a non-competitive manner in presence of L-serine or acetyl-CoA (K_i of 12–20 μM) inhibits partially purified chloroplast SAT, free of bound OASTL. The K_i values are in the range of Cys concentrations estimated in this compartment. Furthermore, we report for the first time that the multi-enzyme complex, CS dissociates in the presence of Cys as previously described with OAS. From this study, and with the integration of data previously reported in the literature, we hypothesize a new model for the regulation of Cys synthesis in plant cells containing a chloroplastic Cys-sensitive SAT.     

Structural and Functional Characterization of Plant Aminoaldehyde Dehydrogenase from Pisum sativum with a Broad Specificity for Natural and Synthetic Aminoaldehydes

Aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the large aldehyde dehydrogenase (ALDH) superfamily, namely, the ALDH9 family. They oxidize polyamine-derived ω-aminoaldehydes to the corresponding ω-amino acids. Here, we report the first X-ray structures of plant AMADHs: two isoenzymes, PsAMADH1 and PsAMADH2, from Pisum sativum in complex with β-nicotinamide adenine dinucleotide (NAD⁺) at 2.4 and 2.15 Å resolution, respectively. Both recombinant proteins are dimeric and, similarly to other ALDHs, each monomer is composed of an oligomerization domain, a coenzyme binding domain and a catalytic domain. Each subunit binds NAD⁺ as a coenzyme, contains a solvent-accessible C-terminal peroxisomal targeting signal (type 1) and a cation bound in the cavity close to the NAD⁺ binding site. While the NAD⁺ binding mode is classical for PsAMADH2, that for PsAMADH1 is unusual among ALDHs. A glycerol molecule occupies the substrate binding site and mimics a bound substrate. Structural analysis and substrate specificity study of both isoenzymes in combination with data published previously on other ALDH9 family members show that the established categorization of such enzymes into distinct groups based on substrate specificity is no more appropriate, because many of them seem capable of oxidizing a large spectrum of aminoaldehyde substrates. PsAMADH1 and PsAMADH2 can oxidize N,N,N-trimethyl-4-aminobutyraldehyde into γ-butyrobetaine, which is the carnitine precursor in animal cells. This activity highly suggests that in addition to their contribution to the formation of compatible osmolytes such as glycine betaine, β-alanine betaine and γ-aminobutyric acid, AMADHs might participate in carnitine biosynthesis in plants.     

Molecular basis of ligand recognition by OASS from E. histolytica: Insights from structural and molecular dynamics simulation studies

Background

O-acetyl serine sulfhydrylase (OASS) is a pyridoxal phosphate (PLP) dependent enzyme catalyzing the last step of the cysteine biosynthetic pathway. Here we analyze and investigate the factors responsible for recognition and different conformational changes accompanying the binding of various ligands to OASS.

Methods

X ray crystallography was used to determine the structures of OASS from Entamoeba histolytica in complex with methionine (substrate analog), isoleucine (inhibitor) and an inhibitory tetra-peptide to 2.00 Å, 2.03 Å and 1.87 Å resolutions, respectively. Molecular dynamics simulations were used to investigate the reasons responsible for the extent of domain movement and cleft closure of the enzyme in presence of different ligands.

Results

Here we report for the first time an OASS-methionine structure with an unmutated catalytic lysine at the active site. This is also the first OASS structure with a closed active site lacking external aldimine formation. The OASS-isoleucine structure shows the active site cleft in open state. Molecular dynamics studies indicate that cofactor PLP, N88 and G192 form a triad of energy contributors to close the active site upon ligand binding and orientation of the Schiff base forming nitrogen of the ligand is critical for this interaction.

Conclusions

Methionine proves to be a better binder to OASS than isoleucine. The β branching of isoleucine does not allow it to reorient itself in suitable conformation near PLP to cause active site closure.

General significance

Our findings have important implications in designing better inhibitors against OASS across all pathogenic microbial species.     

A Random Sequential Mechanism of Aminoglycoside Acetylation by Mycobacterium tuberculosis Eis Protein

An important cause of bacterial resistance to aminoglycoside antibiotics is the enzymatic acetylation of their amino groups by acetyltransferases, which abolishes their binding to and inhibition of the bacterial ribosome. Enhanced intracellular survival (Eis) protein from Mycobacterium tuberculosis (Mt) is one of such acetyltransferases, whose upregulation was recently established as a cause of resistance to aminoglycosides in clinical cases of drug-resistant tuberculosis. The mechanism of aminoglycoside acetylation by MtEis is not completely understood. A systematic analysis of steady-state kinetics of acetylation of kanamycin A and neomycin B by Eis as a function of concentrations of these aminoglycosides and the acetyl donor, acetyl coenzyme A, reveals that MtEis employs a random-sequential bisubstrate mechanism of acetylation and yields the values of the kinetic parameters of this mechanism. The implications of these mechanistic properties for the design of inhibitors of Eis and other aminoglycoside acetyltransferases are discussed.     

Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein from Escherichia coli: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes

The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme from Escherichia coli was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. The K_m values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.     

Mitochondrial cysteine synthase complex regulates o-acetylserine biosynthesis in plants

Cysteine synthesis is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) in the cytosol, plastids, and mitochondria of plants. Biochemical analyses of recombinant plant SAT and OAS-TL indicate that the reversible association of the proteins in the cysteine synthase complex (CSC) controls cellular sulfur homeostasis. However, the relevance of CSC formation in each compartment for flux control of cysteine synthesis remains controversial. Here, we demonstrate the interaction between mitochondrial SAT3 and OAS-TL C in planta by FRET and establish the role of the mitochondrial CSC in the regulation of cysteine synthesis. NMR spectroscopy of isolated mitochondria from WT, serat2;2, and oastl-C plants showed the SAT-dependent export of OAS. The presence of cysteine resulted in reduced OAS export in mitochondria of oastl-C mutants but not in WT mitochondria. This is in agreement with the stronger in vitro feedback inhibition of free SAT by cysteine compared with CSC-bound SAT and explains the high OAS export rate of WT mitochondria in the presence of cysteine. The predominant role of mitochondrial OAS synthesis was validated in planta by feeding [(3)H]serine to the WT and loss-of-function mutants for OAS-TLs in the cytosol, plastids, and mitochondria. On the basis of these results, we propose a new model in which the mitochondrial CSC acts as a sensor that regulates the level of SAT activity in response to sulfur supply and cysteine demand.     

Molecular determinants of substrate specificity in plant 5'-methylthioadenosine nucleosidases

5′-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5′-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5′-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 Å resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5′-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5′-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK_a of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.     

A mechanistic model of the cysteine synthase complex

Plants and bacteria assimilate and incorporate inorganic sulfur into organic compounds such as the amino acid cysteine. Cysteine biosynthesis involves a bienzyme complex, the cysteine synthase (CS) complex. The CS complex is composed of the enzymes serine acetyl transferase (SAT) and O-acetyl-serine-(thiol)-lyase (OAS-TL). Although it is experimentally known that formation of the CS complex influences cysteine production, the exact biological function of the CS complex, the mechanism of reciprocal regulation of the constituent enzymes and the structure of the complex are still poorly understood. Here, we used docking techniques to construct a model of the CS complex from mitochondrial Arabidopsis thaliana. The three-dimensional structures of the enzymes were modeled by comparative techniques. The C-termini of SAT, missing in the template structures but crucial for CS formation, were modeled de novo. Diffusional encounter complexes of SAT and OAS-TL were generated by rigid-body Brownian dynamics simulation. By incorporating experimental constraints during Brownian dynamics simulation, we identified complexes consistent with experiments. Selected encounter complexes were refined by molecular dynamics simulation to generate structures of bound complexes. We found that although a stoichiometric ratio of six OAS-TL dimers to one SAT hexamer in the CS complex is geometrically possible, binding energy calculations suggest that, consistent with experiments, a ratio of only two OAS-TL dimers to one SAT hexamer is more likely. Computational mutagenesis of residues in OAS-TL that are experimentally significant for CS formation hindered the association of the enzymes due to a less-favorable electrostatic binding free energy. Since the enzymes from A. thaliana were expressed in Escherichia coli, the cross-species binding of SAT and OAS-TL from E. coli and A. thaliana was explored. The results showed that reduced cysteine production might be due to a cross-binding of A. thaliana OAS-TL with E. coli SAT. The proposed models of the enzymes and their complexes provide mechanistic insights into CS complexation.     

Crystal Structure of the Mycoplasma arthritidis-Derived Mitogen in Apo Form Reveals a 3D Domain-Swapped Dimer

Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular Vβ elements of T cell receptor. Here, we report the crystal structure of a MAM mutant K201A in apo form (unliganded) at 2.8-Å resolutions. We also partially refined the crystal structures of the MAM wild type and another MAM mutant L50A in apo forms at low resolutions. Unexpectedly, the structures of these apo MAM molecules display a three-dimensional domain-swapped dimer. The entire C-terminal domains of these MAM molecules are involved in the domain swapping. Functional analyses demonstrated that the K201A and L50A mutants do not show altered ability to bind to their host receptors and that they stimulate the activation of T cells as efficiently as does the wild type. Structural comparisons indicated that the “reconstituted” MAM monomer from the domain-swapped dimer displays large differences at the hinge regions from the MAM_wt molecule in the receptor-bound form. Further comparison indicated that MAM has a flexible N-terminal loop, implying that conformational changes could occur upon receptor binding.