A transient post-translational modification of active site cysteine alters binding properties of the parkinsonism protein DJ-1.

Mutations in the human protein DJ-1 cause early onset of Parkinson's disease. A reactive cysteine residue (Cys106) of DJ-1 is crucial for its protective function, although the underlying mechanisms are unclear. Here we show that a fraction of bacterially expressed polyhistidine-tagged human DJ-1 could not be eluted from a Ni-nitrilotriacetate (Ni-NTA) column with 150 mM imidazole. This unusually tight binding was accompanied by the appearance of blue violet color on the Ni-NTA column. We demonstrate by X-ray crystallography that Cys106 is carboxymethylated in a fraction of DJ-1 tightly bound to Ni-NTA and that the replacement of Cys106 by serine abrogates the tight binding and the appearance of blue violet color. However, carboxymethylation of purified DJ-1 is insufficient to confer the tight binding to Ni-NTA. Moreover, when eluted protein was re-applied to the Ni-NTA column, no tight binding was observed, indicating that the formation of high affinity complex with Ni-NTA depends on a transient modification of Cys106 that transforms into a Cys106-carboxymethyl adduct upon elution from Ni-NTA. We conclude that an unknown metabolite reacts with Cys106 of DJ-1 to result in a transient post-translational modification. This modification is distinct from simple oxidation to sulfinic or sulfenic acids and confers altered binding properties to DJ-1 suggesting that it could serve as a signal for sensing oxidant stress.

Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2.

The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 µM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents.

Dissecting the Structural Elements for the Activation of ß-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae.

UNLABELLED: ß-Ketoacyl-(acyl carrier protein) reductase (FabG) catalyzes the key reductive reaction in the elongation cycle of fatty acid synthesis (FAS), which is a vital metabolic pathway in bacteria and a promising target for new antibiotic development. The activation of the enzyme is usually linked to the formation of a catalytic triad and cofactor binding, and crystal structures of FabG from different organisms have been captured in either the active or inactive conformation. However, the structural elements which enable activation of FabG require further exploration. Here we report the findings of structural, enzymatic, and binding studies of the FabG protein found in the causative agent of cholera, Vibrio cholerae (vcFabG). vcFabG exists predominantly as a dimer in solution and is able to self-associate to form tetramers, which is the state seen in the crystal structure. The formation of the tetramer may be promoted by the presence of the cofactor NADP(H). The transition between the dimeric and tetrameric states of vcFabG is related to changes in the conformations of the α4/α5 helices on the dimer-dimer interface. Two glycine residues adjacent to the dimer interface (G92 and G141) are identified to be the hinge for the conformational changes, while the catalytic tyrosine (Y155) and a glutamine residue that forms hydrogen bonds to both loop ß4-α4 and loop ß5-α5 (Q152) stabilize the active conformation. The functions of the aforementioned residues were confirmed by binding and enzymatic assays for the corresponding mutants. IMPORTANCE: This paper describes the results of structural, enzymatic, and binding studies of FabG from Vibrio cholerae (vcFabG). In this work, we dissected the structural elements responsible for the activation of vcFabG. The structural information provided here is essential for the development of antibiotics specifically targeting bacterial FabG, especially for the multidrug-resistant strains of V. cholerae.

The native structure of annexin A2 peptides in hydrophilic environment determines their anti-angiogenic effects.

The progression of aggressive cancer occurs via angiogenesis and metastasis makes these processes important targets for the development of anti-cancer agents. However, recent studies have raised the concern that selective inhibition of angiogenesis results in a switch towards increased tumour growth and metastasis. Since Annexin A2 (AnxA2) is involved in both angiogenesis and metastasis, it may serve as an ideal target for the simultaneous inhibition of both processes. Based on the discovery that domains I (D(I)) and IV (D(IV)) of AnxA2 are potent inhibitors of angiogenesis, we designed seven peptides derived from these domains based on AnxA2 crystal structures. The peptides were expressed as fusion peptides to increase their folding and solubility. Light scattering, far-UV circular dichroism and thermal transition analyses were employed to investigate their aggregation tendencies, α-helical propensity and stability, respectively. 2,2,2-trifluoroethanol (50%) increased the α-helical propensities of all peptides, indicating that they may favour a hydrophobic environment, but did not enhance their thermal stability. D(I)-P2 appears to be the most stable and folded peptide in a hydrophilic environment. The secondary structure of D(I)-P2 was confirmed by nuclear magnetic resonance spectra. The effect of the seven AnxA2 peptides on the formation and integrity of capillary-like networks was studied in a co-culture system mimicking many of the angiogenesis-related processes. Notably, D(I)-P2 inhibited significantly network formation in this system, indicating that the folded D(I)-P2 peptide interferes with vascular endothelial growth factor-dependent pro-angiogenic processes. Thus, this peptide has the potential of being developed further as an anti-angiogenic drug.

Edc3 function in yeast and mammals is modulated by interaction with NAD-related compounds.

The control of mRNA translation and degradation is mediated in part by a set of proteins that can inhibit translation and promote decapping, as well as function in the assembly of cytoplasmic mRNP granules referred to as processing bodies (P-bodies). The conserved enhancer of mRNA decapping 3 (Edc3) protein functions to promote both decapping and P-body assembly. Crystal structures of the YjeF_N domain in hEdc3 identified a putative binding site for a small molecule. Structure modeling of the human Edc3 Yjef_N along with other Yjef_N-containing proteins suggests that this molecule is related to NAD(H). We now show human Edc3 directly binds NADH. We also show that human and yeast Edc3 chemically modify NAD in vitro. Mutations that are predicted to disrupt the binding and/or hydrolysis of an NAD-related molecule by yeast and human Edc3 affect the control of mRNA degradation and/or P-body composition in vivo. This suggests that the interaction of Edc3 with an NAD-related molecule affects its function in the regulation of mRNA translation and degradation and provides a possible mechanism to couple the energetics of the cell to posttranscriptional control. Moreover, this provides a unique example of and lends strength to the postulated connection of metabolites, enzymes, and RNA.

Biophysical analysis of the putative acetyltransferase SACOL2570 from methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of a myriad of insidious and intractable infections in humans, especially in patients with compromised immune systems and children. Here, we report the apo- and CoA-bound crystal structures of a member of the galactoside acetyltransferase superfamily from methicillin-resistant S. aureus SACOL2570 which was recently shown to be down regulated in S. aureus grown in the presence of fusidic acid, an antibiotic used to treat MRSA infections. SACOL2570 forms a homotrimer in solution, as confirmed by small-angle X-ray scattering and dynamic light scattering. The protein subunit consists of an N-terminal alpha-helical domain connected to a C-terminal LßH domain. CoA binds in the active site formed by the residues from adjacent LßH domains. After determination of CoA-bound structure, molecular dynamics simulations were performed to model the binding of AcCoA. Binding of both AcCoA and CoA to SACOL2570 was verified by isothermal titration calorimetry. SACOL2570 most likely acts as an acetyltransferase, using AcCoA as an acetyl group donor and an as-yet-undetermined chemical moiety as an acceptor. SACOL2570 was recently used as a scaffold for mutations that lead the generation of cage-like assemblies, and has the potential to be used for the generation of more complex nanostructures.

Structural and functional insight into the universal stress protein family.

We present the crystal structures of two universal stress proteins (USP) from Archaeoglobus fulgidus and Nitrosomonas europaea in both apo- and ligand-bound forms. This work is the first complete synthesis of the structural properties of 26 USP available in the Protein Data Bank, over 75% of which were determined by structure genomics centers with no additional information provided. The results of bioinformatic analyses of all available USP structures and their sequence homologs revealed that these two new USP structures share overall structural similarity with structures of USPs previously determined. Clustering and cladogram analyses, however, show how they diverge from other members of the USP superfamily and show greater similarity to USPs from organisms inhabiting extreme environments. We compared them with other archaeal and bacterial USPs and discuss their similarities and differences in context of structure, sequential motifs, and potential function. We also attempted to group all analyzed USPs into families, so that assignment of the potential function to those with no experimental data available would be possible by extrapolation.

Disulfide bond oxidoreductase DsbA2 of Legionella pneumophila exhibits protein disulfide isomerase activity.

The extracytoplasmic assembly of the Dot/Icm type IVb secretion system (T4SS) of Legionella pneumophila is dependent on correct disulfide bond (DSB) formation catalyzed by a novel and essential disulfide bond oxidoreductase DsbA2 and not by DsbA1, a second nonessential DSB oxidoreductase. DsbA2, which is widely distributed in the microbial world, is phylogenetically distinct from the canonical DsbA oxidase and the DsbC protein disulfide isomerase (PDI)/reductase of Escherichia coli. Here we show that the extended N-terminal amino acid sequence of DsbA2 (relative to DsbA proteins) contains a highly conserved 27-amino-acid dimerization domain enabling the protein to form a homodimer. Complementation tests with E. coli mutants established that L. pneumophila dsbA1, but not the dsbA2 strain, restored motility to a dsbA mutant. In a protein-folding PDI detector assay, the dsbA2 strain, but not the dsbA1 strain, complemented a dsbC mutant of E. coli. Deletion of the dimerization domain sequences from DsbA2 produced the monomer (DsbA2N), which no longer exhibited PDI activity but complemented the E. coli dsbA mutant. PDI activity was demonstrated in vitro for DsbA2 but not DsbA1 in a nitrocefin-based mutant TEM ß-lactamase folding assay. In an insulin reduction assay, DsbA2N activity was intermediate between those of DsbA2 and DsbA1. In L. pneumophila, DsbA2 was maintained as a mixture of thiol and disulfide forms, while in E. coli, DsbA2 was present as the reduced thiol. Our studies suggest that DsbA2 is a naturally occurring bifunctional disulfide bond oxidoreductase that may be uniquely suited to the majority of intracellular bacterial pathogens expressing T4SSs as well as in many slow-growing soil and aquatic bacteria.

Identification of unknown protein function using metabolite cocktail screening.

Proteins of unknown function comprise a significant fraction of sequenced genomes. Defining the roles of these proteins is vital to understanding cellular processes. Here, we describe a method to determine a protein function based on the identification of its natural ligand(s) by the crystallographic screening of the binding of a metabolite library, followed by a focused search in the metabolic space. The method was applied to two protein families with unknown function, PF01256 and YjeF_N. The PF01256 proteins, represented by YxkO from Bacillus subtilis and the C-terminal domain of Tm0922 from Thermotoga maritima, were shown to catalyze ADP/ATP-dependent NAD(P)H-hydrate dehydratation, a previously described orphan activity. The YjeF_N proteins, represented by mouse apolipoprotein A-I binding protein and the N-terminal domain of Tm0922, were found to interact with an adenosine diphosphoribose-related substrate and likely serve as ADP-ribosyltransferases. Crystallographic screening of metabolites serves as an efficient tool in functional analyses of uncharacterized proteins.

Substrate specificity of mammalian N-terminal α-amino methyltransferase NRMT.

N-Terminal methylation of free α-amino groups is a post-translational modification of proteins that was first described 30 years ago but has been studied very little. In this modification, the initiating M residue is cleaved and the exposed α-amino group is mono-, di-, or trimethylated by NRMT, a recently identified N-terminal methyltransferase. Currently, all known eukaryotic α-amino-methylated proteins have a unique N-terminal motif, M-X-P-K, where X is A, P, or S. NRMT can also methylate artificial substrates in vitro in which X is G, F, Y, C, M, K, R, N, Q, or H. Methylation efficiencies of N-terminal amino acids are variable with respect to the identity of X. Here we use in vitro peptide methylation assays and substrate immunoprecipitations to show that the canonical M-X-P-K methylation motif is not the only one recognized by NRMT. We predict that N-terminal methylation is a widespread post-translational modification and that there is interplay between N-terminal acetylation and N-terminal methylation. We also use isothermal calorimetry experiments to demonstrate that NRMT can efficiently recognize and bind to its fully methylated products.

Structure and characterization of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix.

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PS(Ap)) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PS(Ap) would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PS(Ap) in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PS(Ap) is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PS(Ap) follows Michaelis-Menten kinetics at 60 °C, with a K(M) for erythrose 4-phosphate of 280 µM, a K(M) for phosphoenolpyruvate of 891 µM, and a k(cat) of 1.0 s(-1). None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PS(Ap). The structure of DAH7PS(Ap) is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.

Structural analysis of a putative aminoglycoside N-acetyltransferase from Bacillus anthracis.

For the last decade, worldwide efforts for the treatment of anthrax infection have focused on developing effective vaccines. Patients that are already infected are still treated traditionally using different types of standard antimicrobial agents. The most popular are antibiotics such as tetracyclines and fluoroquinolones. While aminoglycosides appear to be less effective antimicrobial agents than other antibiotics, synthetic aminoglycosides have been shown to act as potent inhibitors of anthrax lethal factor and may have potential application as antitoxins. Here, we present a structural analysis of the BA2930 protein, a putative aminoglycoside acetyltransferase, which may be a component of the bacterium's aminoglycoside resistance mechanism. The determined structures revealed details of a fold characteristic only for one other protein structure in the Protein Data Bank, namely, YokD from Bacillus subtilis. Both BA2930 and YokD are members of the Antibiotic_NAT superfamily (PF02522). Sequential and structural analyses showed that residues conserved throughout the Antibiotic_NAT superfamily are responsible for the binding of the cofactor acetyl coenzyme A. The interaction of BA2930 with cofactors was characterized by both crystallographic and binding studies.

The crystal structure of the AF2331 protein from Archaeoglobus fulgidus DSM 4304 forms an unusual interdigitated dimer with a new type of alpha + beta fold.

The structure of AF2331, a 11-kDa orphan protein of unknown function from Archaeoglobus fulgidus, was solved by Se-Met MAD to 2.4 A resolution. The structure consists of an alpha + beta fold formed by an unusual homodimer, where the two core beta-sheets are interdigitated, containing strands alternating from both subunits. The decrease in solvent-accessible surface area upon dimerization is unusually large (3960 A(2)) for a protein of its size. The percentage of the total surface area buried in the interface (41.1%) is one of the largest observed in a nonredundant set of homodimers in the PDB and is above the mean for nearly all other types of homo-oligomers. AF2331 has no sequence homologs, and no structure similar to AF2331 could be found in the PDB using the CE, TM-align, DALI, or SSM packages. The protein has been identified in Pfam 23.0 as the archetype of a new superfamily and is topologically dissimilar to all other proteins with the "3-Layer (BBA) Sandwich" fold in CATH. Therefore, we propose that AF2331 forms a novel alpha + beta fold. AF2331 contains multiple negatively charged surface clusters and is located on the same operon as the basic protein AF2330. We hypothesize that AF2331 and AF2330 may form a charge-stabilized complex in vivo, though the role of the negatively charged surface clusters is not clear.

Biochemical and structural characterization of apolipoprotein A-I binding protein, a novel phosphoprotein with a potential role in sperm capacitation.

The physiological changes that sperm undergo in the female reproductive tract rendering them fertilization-competent constitute the phenomenon of capacitation. Cholesterol efflux from the sperm surface and protein kinase A (PKA)-dependent phosphorylation play major regulatory roles in capacitation, but the link between these two phenomena is unknown. We report that apolipoprotein A-I binding protein (AI-BP) is phosphorylated downstream to PKA activation, localizes to both sperm head and tail domains, and is released from the sperm into the media during in vitro capacitation. AI-BP interacts with apolipoprotein A-I, the component of high-density lipoprotein involved in cholesterol transport. The crystal structure demonstrates that the subunit of the AI-BP homodimer has a Rossmann-like fold. The protein surface has a large two compartment cavity lined with conserved residues. This cavity is likely to constitute an active site, suggesting that AI-BP functions as an enzyme. The presence of AI-BP in sperm, its phosphorylation by PKA, and its release during capacitation suggest that AI-BP plays an important role in capacitation possibly providing a link between protein phosphorylation and cholesterol efflux.

Characterization of the gating brake in the I-II loop of Ca(v)3.2 T-type Ca(2+) channels.

Mutations in the I-II loop of Ca(v)3.2 channels were discovered in patients with childhood absence epilepsy. All of these mutations increased the surface expression of the channel, whereas some mutations, and in particular C456S, altered the biophysical properties of channels. Deletions around C456S were found to produce channels that opened at even more negative potentials than control, suggesting the presence of a gating brake that normally prevents channel opening. The goal of the present study was to identify the minimal sequence of this brake and to provide insights into its structure. A peptide fragment of the I-II loop was purified from bacteria, and its structure was analyzed by circular dichroism. These results indicated that the peptide had a high alpha-helical content, as predicted from secondary structure algorithms. Based on homology modeling, we hypothesized that the proximal region of the I-II loop may form a helix-loop-helix structure. This model was tested by mutagenesis followed by electrophysiological measurement of channel gating. Mutations that disrupted the helices, or the loop region, had profound effects on channel gating, shifting both steady state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure had more modest effects. Taken together, these studies showed that any mutations in the brake, including C456S, disrupted the structural integrity of the brake and its function to maintain these low voltage-activated channels closed at resting membrane potentials.

An extremely SAD case: structure of a putative redox-enzyme maturation protein from Archaeoglobus fulgidus at 3.4 A resolution.

This paper describes the crystal structure of AF0173, a putative redox-enzyme maturation protein (REMP) from Archaeoglobus fulgidus. The REMPs serve as chaperones in the maturation of extracytoplasmic oxidoreductases in archaea and bacteria. The all-helical subunits of AF0173 form a dimer arising from the interaction of residues located in a funnel-shaped cavity on one subunit surface with an uncut expression tag from the other subunit. This cavity is likely to represent a binding site for the twin-arginine motif that interacts with REMPs. The conservation of the overall fold in AF0173 and bacterial REMPs as well as the presence of conserved residues in their putative binding sites indicates that REMPs act in a similar manner in archaea and bacteria despite their limited sequence similarity. A model of the binding of the twin-arginine motif by AF0173 is suggested. The solution of the AF0173 structure by the single anomalous dispersion method represents an extreme case of SAD structure determination: low resolution (3.4 A), the absence of NCS and the presence of only two anomalously scattering atoms in the asymmetric unit. An unusually high solvent content (73%) turned out to be important for the success of the density-modification procedures.

Crystal structure of the reaction complex of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Thermotoga maritima refines the catalytic mechanism and indicates a new mechanism of allosteric regulation.

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) catalyzes the first reaction of the aromatic biosynthetic pathway in bacteria, fungi, and plants, the condensation of phosphoenolpyruvate (PEP) and d-erythrose-4-phosphate (E4P) with the formation of DAHP. Crystals of DAHPS from Thermotoga maritima (DAHPS(Tm)) were grown in the presence of PEP and metal cofactor, Cd(2+), and then soaked with E4P at 4 degrees C where the catalytic activity of the enzyme is negligible. The crystal structure of the "frozen" reaction complex was determined at 2.2A resolution. The subunit of the DAHPS(Tm) homotetramer consists of an N-terminal ferredoxin-like (FL) domain and a (beta/alpha)(8)-barrel domain. The active site located at the C-end of the barrel contains Cd(2+), PEP, and E4P, the latter bound in a non-productive conformation. The productive conformation of E4P is suggested and a catalytic mechanism of DAHPS is proposed. The active site of DAHPS(Tm) is nearly identical to the active sites of the other two known DAHPS structures from Escherichia coli (DAHPS(Ec)) and Saccharomyces cerevisiae (DAHPS(Sc)). However, the secondary, tertiary, and quaternary structures of DAHPS(Tm) are more similar to the functionally related enzyme, 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from E.coli and Aquiflex aeolicus, than to DAHPS(Ec) and DAHPS(Sc). Although DAHPS(Tm) is feedback-regulated by tyrosine and phenylalanine, it lacks the extra barrel segments that are required for feedback inhibition in DAHPS(Ec) and DAHPS(Sc). A sequence similarity search revealed that DAHPSs of phylogenetic family Ibeta possess a FL domain like DAHPS(Tm) while those of family Ialpha have extra barrel segments similar to those of DAHPS(Ec) and DAHPS(Sc). This indicates that the mechanism of feedback regulation in DAHPS(Tm) and other family Ibeta enzymes is different from that of family Ialpha enzymes, most likely being mediated by the FL domain.

The high-resolution structure of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase reveals a twist in the plane of bound phosphoenolpyruvate.

3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS), the first enzyme of the aromatic biosynthetic pathway in microorganisms and plants, catalyzes the aldol-like condensation of phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) with the formation of DAHP. The native and the selenomethionine-substituted forms of the phenylalanine-regulated isozyme [DAHPS(Phe)] from Escherichia coli were crystallized in complex with PEP and a metal cofactor, Mn(2+), but the crystals displayed disorder in their unit cells, preventing satisfactory refinement. However, the crystal structure of the E24Q mutant form of DAHPS(Phe) in complex with PEP and Mn(2+) has been determined at 1.75 A resolution. Unlike the tetrameric wild-type enzyme, the E24Q enzyme is dimeric in solution, as a result of the mutational perturbation of four intersubunit salt bridges that are critical for tetramer formation. The protein chain conformation and subunit arrangement in the crystals of E24Q and wild-type DAHPS are very similar. However, the interaction of Mn(2+) and PEP in the enzymatically active E24Q mutant complex differs from the Pb(2+)-PEP and Mn(2+)-phosphoglycolate interactions in two enzymatically inactive wild-type complexes whose structures have been determined previously. The geometry of PEP bound in the active site of the E24Q enzyme deviates from planarity due to a 30 degrees twist of the carboxylate plane relative to the enol plane. In addition, seven water molecules are within contact distance of PEP, two of which are close enough to its C2 atom to serve as the nucleophile required in the reaction.

Allosteric inhibition of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase alters the coordination of both substrates.

3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS), the first enzyme of the aromatic biosynthetic pathway in microorganisms and plants, catalyzes the aldol-like condensation of phosphoenolpyruvate and D-erythrose-4-phosphate with the formation of 3-deoxy-D-arabino-heptulosonate-7-phosphate. In Escherichia coli, there are three isoforms of DAHPS, each specifically feedback-regulated by one of the three aromatic amino acid end products. The crystal structure of the phenylalanine-regulated DAHPS from E.coli in complex with its inhibitor, L-phenylalanine, phosphoenolpyruvate, and metal cofactor, Mn(2+), has been determined to 2.8A resolution. Phe binds in a cavity formed by residues of two adjacent subunits and is located about 20A from the closest active site. A model for the mechanism of allosteric inhibition has been derived from conformational differences between the Phe-bound and previously determined Phe-free structures. Two interrelated paths of conformational changes transmit the inhibitory signal from the Phe-binding site to the active site of DAHPS. The first path involves transmission within a single subunit due to the movement of adjacent segments of the protein. The second involves alterations in the contacts between subunits. The combination of these two paths changes the conformation of one of the active site loops significantly and shifts the other slightly. This alters the interaction of DAHPS with both of its substrates. Upon binding of Phe, the enzyme loses the ability to bind D-erythrose-4-phosphate and binds phosphoenolpyruvate in a flipped orientation.