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Isolation and biochemical characterization of a metagenome-derived 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase gene from subtropical marine mangrove wetland sediments.

ABSTRACT: 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) is a key rate-limiting enzyme in aromatic amino acid anabolism. A new Iβ-type DAHPS gene (aro1A) was identified in a metagenomic library from subtropical marine mangrove sediment. The gene encoded a polypeptide composed of 272 amino acids and had a maximum similarity of 52.4% to a known DAHPS at the amino acid level. Multiple sequence alignment, homologous modeling, and molecular docking showed that Aro1A had the typical (β/α)8 barrel-shaped catalytic structural domain of DAHPS. The motifs and amino acid residues involved in the combination of substrates and metal ligand were highly conservative with the known DAHPS. The putative DAHPS gene was subcloned into a pET-30a(+) vector and was overexpressed in Escherichia coli Rosetta (DE3) cells. The recombinant protein was purified to homogeneity. The maximum activity for the recombinant Aro1A protein occurred at pH 8.0 and 40 °C. Ba2+ and Ca2+ stimulated the activity of Aro1A protein. The enzyme showed high affinity and catalytic efficiency (K m PEP = 19.58 μM, V max PEP = 29.02 μM min-1, and k cat PEP /K m PEP = 0.88 s-1 μM-1) under optimal reaction conditions. The enzymatic property of Aro1A indicates its potential in aromatic amino acid industrial production.

SUBMITTER: Zhao H

PROVIDER: S-EPMC6362186 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Corynebacterium glutamicum contains 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that display novel biochemical features.

Project description:3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 2.5.1.54) catalyzes the first step of the shikimate pathway that finally leads to the biosynthesis of aromatic amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). In Corynebacterium glutamicum ATCC 13032, two chromosomal genes, NCgl0950 (aroF) and NCgl2098 (aroG), were located that encode two putative DAHP synthases. The deletion of NCgl2098 resulted in the loss of the ability of C. glutamicum RES167 (a restriction-deficient strain derived from C. glutamicum ATCC 13032) to grow in mineral medium; however, the deletion of NCgl0950 did not result in any observable phenotypic alteration. Analysis of DAHP synthase activities in the wild type and mutants of C. glutamicum RES167 indicated that NCgl2098, rather than NCgl0950, was involved in the biosynthesis of aromatic amino acids. Cloning and expression in Escherichia coli showed that both NCgl0950 and NCgl2098 encoded active DAHP synthases. Both the NCgl0950 and NCgl2098 DAHP synthases were purified from recombinant E. coli cells and characterized. The NCgl0950 DAHP synthase was sensitive to feedback inhibition by Tyr and, to a much lesser extent, by Phe and Trp. The NCgl2098 DAHP synthase was slightly sensitive to feedback inhibition by Trp, but not sensitive to Tyr and Phe, findings that were in contrast to the properties of previously known DAHP synthases from C. glutamicum subsp. flavum. Both Co2+ and Mn2+ significantly stimulated the NCgl0950 DAHP synthase's activity, whereas Mn2+ was much more stimulatory than Co2+ to the NCgl2098 DAHP synthase's activity.

| S-EPMC2546626 | biostudies-literature

Protein engineering for feedback resistance in 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

Project description:The shikimate pathway delivers aromatic amino acids (AAAs) in prokaryotes, fungi, and plants and is highly utilized in the industrial synthesis of bioactive compounds. Carbon flow into this pathway is controlled by the initial enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS). AAAs produced further downstream, phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), regulate DAHPS by feedback inhibition. Corynebacterium glutamicum, the industrial workhorse for amino acid production, has two isoenzymes of DAHPS, AroF (Tyr sensitive) and AroG (Phe and Tyr sensitive). Here, we introduce feedback resistance against Tyr in the class I DAHPS AroF (AroFcg). We pursued a consensus approach by drawing on structural modeling, sequence and structural comparisons, knowledge of feedback-resistant variants in E. coli homologs, and computed folding free energy changes. Two types of variants were predicted: Those where substitutions putatively either destabilize the inhibitor binding site or directly interfere with inhibitor binding. The recombinant variants were purified and assessed in enzyme activity assays in the presence or absence of Tyr. Of eight AroFcg variants, two yielded > 80% (E154N) and > 50% (P155L) residual activity at 5 mM Tyr and showed > 50% specific activity of the wt AroFcg in the absence of Tyr. Evaluation of two and four further variants at positions 154 and 155 yielded E154S, completely resistant to 5 mM Tyr, and P155I, which behaves similarly to P155L. Hence, feedback-resistant variants were found that are unlikely to evolve by point mutations from the parental gene and, thus, would be missed by classical strain engineering. KEY POINTS: • We introduce feedback resistance against Tyr in the class I DAHPS AroF • Variants at position 154 (155) yield > 80% (> 50%) residual activity at 5 mM Tyr • The variants found are unlikely to evolve by point mutations from the parental gene.

| S-EPMC9529685 | biostudies-literature

The Functional Unit of Neisseria meningitidis 3-Deoxy-?-Arabino-Heptulosonate 7-Phosphate Synthase Is Dimeric.

Project description:Neisseria meningitidis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (NmeDAH7PS) adopts a homotetrameric structure consisting of an extensive and a less extensive interface. Perturbation of the less extensive interface through a single mutation of a salt bridge (Arg126-Glu27) formed at the tetramer interface of all chains resulted in a dimeric DAH7PS in solution, as determined by small angle X-ray scattering, analytical ultracentrifugation and analytical size-exclusion chromatography. The dimeric NmeDAH7PSR126S variant was shown to be catalytically active in the aldol-like condensation reaction between D-erythrose 4-phosphate and phosphoenolpyruvate, and allosterically inhibited by L-phenylalanine to the same extent as the wild-type enzyme. The dimeric NmeDAH7PSR126S variant exhibited a slight reduction in thermal stability by differential scanning calorimetry experiments and a slow loss of activity over time compared to the wild-type enzyme. Although NmeDAH7PSR126S crystallised as a tetramer, like the wild-type enzyme, structural asymmetry at the less extensive interface was observed consistent with its destabilisation. The tetrameric association enabled by this Arg126-Glu27 salt-bridge appears to contribute solely to the stability of the protein, ultimately revealing that the functional unit of NmeDAH7PS is dimeric.

| S-EPMC4735112 | biostudies-literature

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

Project description: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.

| S-EPMC3260417 | biostudies-literature

Bacillus subtilis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase revisited: resolution of two long-standing enigmas.

Project description:The mono/bifunctional and metallo/non-metallo properties of Bacillus subtilis DAHPS (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) have been controversial for several decades. The present study investigated the DAHPSs from both the B. subtilis parent Marburg strain and the derivative strain 168 in detail and clarified the above two long-standing questions. The DAHPSs from the parent and the derivative 168 strains have identical sequence and are both bifunctional enzymes with a CM (chorismate mutase) activity and a DAHPS activity. The parent strain expresses a second independent monofunctional CM, encoded by aroH, that is highly active, while the 168 strain expresses an aroH containing a single residue mutation (A112V) that is significantly less active thus leading to previous confusion regarding the mono/bifunctionality of DAHPS. Metal analysis showed that B. subtilis DAHPS as isolated contained iron and zinc and is inactivated by dipicolinic acid; the inactive apoenzyme can be reactivated by bivalent metal ions, indicating that the enzyme is a metalloenzyme. The enzyme-bound metal is insensitive to EDTA treatment, leading to the previous conclusion that this DAHPS does not require a metal. The enzyme displays a homotetrameric structure in solution and appears to follow Michaelis-Menten kinetics with K(m)(PEP)=139+/-11.4 microM for phosphoenolpyruvate, K(m)(E4P)=1760+/-110 microM for D-erythrose 4-phosphate, kcat=4.6+/-0.1 s(-1) for DAHPS activity and K(m)(chorismate)=850+/-97 microM, kcat=0.41+/-0.01 s(-1) for CM activity. B. subtilis DAHPS is inhibited by the Shikimate pathway intermediates prephenate and chorismate.

| S-EPMC1198938 | biostudies-literature

Characterization of a recombinant type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Helicobacter pylori.

Project description:DAH7P (3-Deoxy-D-arabino-heptulosonate 7-phosphate) synthase catalyses the condensation reaction between phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) as the first committed step in the biosynthesis of aromatic compounds in plants and micro-organisms. Previous work has identified two families of DAH7P synthases based on sequence similarity and molecular mass, with the majority of the mechanistic and structural studies being carried out on the type I paralogues from Escherichia coli. Whereas a number of organisms possess genes encoding both type I and type II DAH7P synthases, the pathogen Helicobacter pylori has only a single, type II, enzyme. Recombinant DAH7P synthase from H. pylori was partially solubilized by co-expression with chaperonins GroEL/GroES in E. coli, and purified to homogeneity. The enzyme reaction follows an ordered sequential mechanism with the following kinetic parameters: K(m) (PEP), 3 microM; K(m) (E4P), 6 microM; and kcat, 3.3 s(-1). The enzyme reaction involves interaction of the si face of PEP with the re face of E4P. H. pylori DAH7P synthase is not inhibited by phenylalanine, tyrosine, tryptophan or chorismate. EDTA inactivates the enzyme, and activity is restored by a range of bivalent metal ions, including (in order of decreasing effectiveness) Co2+, Mn2+, Ca2+, Mg2+, Cu2+ and Zn2+. Analysis of type II DAH7P synthase sequences reveals several highly conserved motifs, and comparison with the type I enzymes suggests that catalysis by these two enzyme types occurs on a similar active-site scaffold and that the two DAH7P synthase families may indeed be distantly related.

| S-EPMC1184578 | biostudies-literature

Evolution of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase-encoding genes in the yeast Saccharomyces cerevisiae.

Project description:The shikimate pathway resulting in three aromatic amino acids is initiated in different organisms by two and three 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases, respectively. Aro3p and Aro4p are the yeast enzymes feedback-inhibited by phenylalanine and tyrosine, respectively. A yeast strain deficient in the general control transcriptional regulatory system of amino acid biosynthesis is unable to live in the presence of high amounts of phenylalanine and tyrosine. Here, we show that this yeast strain can be rescued by the expression of aroH from Escherichia coli encoding the tryptophan-regulated AroH as third isoenzyme. Yeast carrying Ec AroH as the only enzyme for the initial step of the shikimate pathway can grow in the absence of tryptophan. Without aromatic amino acids, this yeast strain survives only when the yeast ARO3 promoter instead of the ARO4 promoter drives E. coli aroH. The detailed analysis of Aro3p and Aro4p revealed a triple feedback control by tyrosine/phenylalanine and tryptophan. Dissecting this control allowed engineering of Aro4p S195A as an enzyme, which is inhibited like AroH only by tryptophan. In addition, Aro4p variants were constructed that show an equally strong inhibition by tyrosine and tryptophan (Aro4p P165G Q302R) and in which the regulation by tyrosine and tryptophan was reversed (Aro4p P165G). Our data suggest that yeast possesses only two instead of three isogenes encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases because both isoenzymes can be fine tuned by tryptophan as additional effector and because transcriptional regulation by the general control system can be induced as backup when aromatic amino acids in the environment are imbalanced.

| S-EPMC1175010 | biostudies-literature

Evaluation of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS) as a Vulnerable Target in Mycobacterium tuberculosis.

Project description:Tuberculosis (TB) remains one of the leading causes of death due to a single pathogen. The emergence and proliferation of multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) represent compelling reasons to invest in the pursuit of new anti-TB agents. The shikimate pathway, responsible for chorismate biosynthesis, which is a precursor of important aromatic compounds, is required for Mycobacterium tuberculosis growth. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (MtbDAHPS) catalyzes the first step in the shikimate pathway and it is an attractive target for anti-tubercular agents. Here, we used a CRISPRi system to evaluate the DAHPS as a vulnerable target in M. tuberculosis. The silencing of aroG significantly reduces the M. tuberculosis growth in both rich medium and, especially, in infected murine macrophages. The supplementation with amino acids was only able to partially rescue the growth of bacilli, whereas the Aro supplement (aromix) was enough to sustain the bacterial growth at lower rates. This study shows that MtbDAHPS protein is vulnerable and, therefore, an attractive target to develop new anti-TB agents. In addition, the study contributes to a better understanding of the biosynthesis of aromatic compounds and the bacillus physiology. IMPORTANCE Determining the vulnerability of a potential target allows us to assess whether its partial inhibition will impact bacterial growth. Here, we evaluated the vulnerability of the enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) from M. tuberculosis by silencing the DAHPS-coding aroG gene in different contexts. These results could lead to the development of novel and potent anti-tubercular agents in the near future.

| S-EPMC9430761 | biostudies-literature

Neisseria meningitidis expresses a single 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase that is inhibited primarily by phenylalanine.

Project description:Neisseria meningitidis is the causative agent of meningitis and meningococcal septicemia is a major cause of disease worldwide, resulting in brain damage and hearing loss, and can be fatal in a large proportion of cases. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first reaction in the shikimate pathway leading to the biosynthesis of aromatic metabolites including the aromatic acids l-Trp, l-Phe, and l-Tyr. This pathway is absent in humans, meaning that enzymes of the pathway are considered as potential candidates for therapeutic intervention. As the entry point, feedback inhibition of DAH7PS by pathway end products is a key mechanism for the control of pathway flux. The structure of the single DAH7PS expressed by N. meningitidis was determined at 2.0 Å resolution. In contrast to the other DAH7PS enzymes, which are inhibited only by a single aromatic amino acid, the N. meningitidis DAH7PS was inhibited by all three aromatic amino acids, showing greatest sensitivity to l-Phe. An N. meningitidis enzyme variant, in which a single Ser residue at the bottom of the inhibitor-binding cavity was substituted to Gly, altered inhibitor specificity from l-Phe to l-Tyr. Comparison of the crystal structures of both unbound and Tyr-bound forms and the small angle X-ray scattering profiles reveal that N. meningtidis DAH7PS undergoes no significant conformational change on inhibitor binding. These observations are consistent with an allosteric response arising from changes in protein motion rather than conformation, and suggest ligands that modulate protein dynamics may be effective inhibitors of this enzyme.

| S-EPMC3832045 | biostudies-literature

Structural analysis of a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase with an N-terminal chorismate mutase-like regulatory domain.

Project description:3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase-an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis-and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn(2+) and Mn(2+) + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.

| S-EPMC3403423 | biostudies-literature

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