Characterization of two 3-deoxy-d-Arabino-Heptulosonate 7-phosphate synthases from Bacillusmethanolicus.

3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with d-erythrose 4-phosphate (E4P) and plays an important role in regulating carbon flux toward aromatic amino acid biosynthesis in bacteria and plants. Sequence analysis of the DAHP synthases AroG1 and AroG2 from Bacillus methanolicus MGA3 suggested this thermophilic, methylotrophic bacterium possesses two type Iß DAHP synthases. This study describes production of AroG1 and AroG2 in Escherichia coli as hexa-histidine fused proteins, which were purified by affinity chromatography. Treatment with TEV protease afforded native proteins for characterization and kinetic analysis. AroG1 and AroG2 are, respectively, 30.1 kDa and 40.0 kDa proteins. Both enzymes have maximal activity over a pH range of 6.3-7.2. The apparent kinetic parameters at 50 °C and pH 7.2 for AroG1 are KmPEP 1100 ± 100 µM, KmE4P 530 ± 100 µM, and kcat 10.3 ± 1.2 s-1. The kinetic parameters for AroG2 are KmPEP 90 ± 20 µM, KmE4P 130 ± 40 µM, and kcat 2.0 ± 0.2 s-1. At 50 °C AroG2 retains 50% of its activity after 96 min whereas AroG1 retains less than 5% of its activity after 10 min. AroG2, which contains an N-terminal regulatory domain, is inhibited by chorismate and prephenate but not l-phenylalanine, l-tyrosine, or l-tryptophan. AroG1 is not inhibited by any of the molecules examined. Understanding DAHP synthase regulation in B. methanolicus is a first step toward generating biocatalysts that exploit the target-rich aromatic amino acid biosynthetic pathway for synthesis of chemicals from methanol.

A stable analog of isochorismate for the study of MenD and other isochorismate-utilizing enzymes.

A novel analog of isochorismate, in which the enolpyruvyl substituent is replaced with a carboxymethoxyl group, has been synthesized in four steps from a known intermediate. This analog is more stable than the natural product, but still acts as a good substrate for the enzyme MenD (SEPHCHC synthase). The enzyme consumes the (+)-enantiomer only, with an apparent turnover similar to that of the natural substrate, and an apparent Michaelis constant conveniently higher than that of isochorismate. This analog will be useful in the study of any isochorismate-utilizing enzyme.

Inhibition studies on salicylate synthase.

Analogues of chorismate and isochorismate were designed and tested as potential inhibitors in the first inhibition study against a salicylate synthase.

Identification of 4-amino-4-deoxychorismate synthase as the molecular target for the antimicrobial action of (6s)-6-fluoroshikimate.

(6S)-6-Fluoroshikimate has antimicrobial activity. The molecular basis of this effect had not been identified, but there was speculation that (6S)-6-fluoroshikimate is first converted in vivo into 2-fluorochorismate, which then could inhibit 4-amino-4-deoxychorismate synthase (ADCS). 2-Fluorochorismate was prepared from E-fluorophosphoenolpyruvate and erythose-4-phosphate by the sequential reactions of DAHP synthase, dehydroquinate synthase, dehydroquinase, shikimate dehydrogenase, EPSP synthase, and chorismate synthase. Inhibition studies on ADCS showed that it was inhibited rapidly and irreversibly by 2-fluorochorismate. Electrospray mass spectrometry of the inactivated enzyme showed an additional mass of 198 +/- 10 Da. A novel peptide of 1087.6 Da was identified in the HPLC trace for the tryptic digest of 2-fluorochorismate-inactivated ADCS. Sequencing of this peptide by MS/MS showed that the peptide corresponded to residues 272-279 with a modification of 206.1 Da on Lys-274. This observation is particularly exciting in the context of a recent proposal for the catalytic mechanism of ADCS.

Microbial origin of plant-type 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate synthases, exemplified by the chorismate- and tryptophan-regulated enzyme from Xanthomonas campestris.

Enzymes performing the initial reaction of aromatic amino acid biosynthesis, 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthases, exist as two distinct homology classes. The three classic Escherichia coli paralogs are AroA(I) proteins, but many members of the Bacteria possess the AroA(II) class of enzyme, sometimes in combination with AroA(I) proteins. AroA(II) DAHP synthases until now have been shown to be specifically dedicated to secondary metabolism (e.g., formation of ansamycin antibiotics or phenazine pigment). In contrast, here we show that the Xanthomonas campestris AroA(II) protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis. X. campestris AroA(II) was cloned in E. coli by functional complementation, and genes corresponding to two possible translation starts were expressed. We developed a 1-day partial purification method (>99%) for the unstable protein. The recombinant AroA(II) protein was found to be subject to an allosteric pattern of sequential feedback inhibition in which chorismate is the prime allosteric effector. L-Tryptophan was found to be a minor feedback inhibitor. An N-terminal region of 111 amino acids may be located in the periplasm since a probable inner membrane-spanning region is predicted. Unlike chloroplast-localized AroA(II) of higher plants, X. campestris AroA(II) was not hysteretically activated by dithiols. Compared to plant AroA(II) proteins, differences in divalent metal activation were also observed. Phylogenetic tree analysis shows that AroA(II) originated within the Bacteria domain, and it seems probable that higher-plant plastids acquired AroA(II) from a gram-negative bacterium via endosymbiosis. The X. campestris AroA(II) protein is suggested to exemplify a case of analog displacement whereby an ancestral aroA(I) species was discarded, with the aroA(II) replacement providing an alternative pattern of allosteric control. Three subgroups of AroA(II) proteins can be recognized: a large, central group containing the plant enzymes and that from X. campestris, one defined by a three-residue deletion near the conserved KPRS motif, and one possessing a larger deletion further downstream.

Escherichia coli chorismate synthase catalyzes the conversion of (6S)-6-fluoro-5-enolpyruvylshikimate-3-phosphate to 6-fluorochorismate. Implications for the enzyme mechanism and the antimicrobial action of (6S)-6-fluoroshikimate.

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. It is the seventh enzyme of the shikimate pathway, which is responsible for the biosynthesis of aromatic metabolites from glucose. The chorismate synthase reaction involves a 1,4-elimination with unusual anti-stereochemistry and requires a reduced flavin cofactor. The substrate analogue (6S)-6-fluoro-5-enolpyruvylshikimate-3-phosphate is a competitive inhibitor of Neurospora crassa chorismate synthase (Balasubramanian, S., Davies, G. M., Coggins, J. R., and Abell, C. (1991) J. Am. Chem. Soc. 113, 8945-8946). We have shown that this analogue is converted to 6-fluorochorismate by Escherichia coli chorismate synthase at a rate 2 orders of magnitude slower than the normal substrate. The decreased rate of reaction is consistent with the destabilization of an allylic cationic intermediate. The formation of chorismate and 6-fluorochorismate involves a common protein-bound flavin intermediate although the fluoro substituent does influence the spectral characteristics of this intermediate. The fluoro substituent also decreased the rate of decay of the flavin intermediate by 280 times. These results are consistent with the antimicrobial activity of (6S)-6-fluoroshikimate not being mediated by the inhibition of chorismate synthase but by the inhibition of 4-aminobenzoic acid synthesis as previously proposed (Davies, G. M., Barrett-Bee, K. J., Jude, D. A., Lehan, M., Nichols, W. W., Pinder, P. E., Thain, J. L., Watkins, W. J., and Wilson, R. G. (1994) Antimicrobial Agents and Chemotherapy 38, 403-406).

p-aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA.

p-Aminobenzoic acid (PABA) is an important precursor in the bacterial biosynthetic pathway for folate enzymes. This biosynthesis requires three separate proteins: PabA, PabB, and PabC. Together PabA and PabB convert glutamine and chorismate to glutamate and 4-amino-4-deoxychorismate. This aminochorismate is subsequently transformed to PABA by PabC. In this study, PabA from Escherichia coli has been purified to homogeneity from an overproducing construct and found to have no detectable glutaminase activity until addition of the E. coli PabB subunit. PabB forms a 1:1 complex with PabA to yield a glutaminase k(cat) of 17 min-1. The addition of chorismate, the substrate of PabB, induces a 2-fold increase of k(cat) as well as a 3-fold increase of Km for glutamine. The PabA/PabB complex has Kd less than 10(-8) M but does not form a stable complex isolable by gel filtration. Studies with the glutamine affinity label diazooxonorleucine (DON) reveal it is an inactivator of the glutaminase activity of the PabA/PabB complex, but DON does not alkylate and inactivate PabA alone. Similarly, while isolated PabA shows no tendency to form a glutamyl-enzyme intermediate, the PabA/PabB complex forms a covalent intermediate with [14C]glutamine on PabA that accumulates to 0.56 mol/mol in hydrolytic turnover. PabA is thus a conditional glutaminase, activated by 1:1 complexation with PabB.

Regulation of the biosynthetic pathway of aromatic amino acids in Nocardia mediterranei.

The regulation of enzymes in the biosynthetic pathway of aromatic amino acids in Norcardia mediterranei was studied. Anthranilate synthase was sensitive to feedback inhibition by very low concentrations of LTrp, and kinetic analysis showed that LTrp was competitive with respect to chorismate; the five enzymes in LTrp biosynthesis pathway, anthranilate synthase (AS), anthranilate-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT), N-5'-phosphoribosylanthranilate isomerase (PRAI), indole-3-glycerol phosphate synthetase (InGPS) and tryptophan synthase (TS), were all repressed by LTrp; LTyr and LPhe inhibited chorismate mutase. Prephenate dehydratase activity was greatly inhibited by LPhe and activated by LTyr, nearly 60% of its activity was inhibited by 5 microM of LPhe, and 20 microM of LTyr increased the activity approx. 3-fold. In addition, the effects of LPhe and LTyr on prephenate dehydratase were highly specific. The regulatory circuit of the biosynthetic pathway of aromatic amino acids in N. mediterranei is presented.

Chorismate aminations: partial purification of Escherichia coli PABA synthase and mechanistic comparison with anthranilate synthase.

Chorismate is converted by regiospecific amination/aromatization sequences to o-aminobenzoate and p-aminobenzoate (PABA) by anthranilate synthase (AS) and PABA synthase (PABS), respectively. We report here the first partial purification of the large subunit of Escherichia coli PABA synthase, previously reported to be quantitatively inactivated in purification attempts. The subunit encoded by the pabB gene was overexpressed from a T7 promoter and purified 9-fold to 25-30% homogeneity. The pabB subunit appears unusually sensitive to inactivation by glycerol so this cosolvent is contraindicated. The Km for chorismate is 42 microM in the ammonia-dependent conversion to PABA, and we estimate a turnover number of 2.6 min-1. A variety of chorismate analogues have been prepared and examined. Of these compounds, cycloheptadienyl analogue 11 has been found to be the most potent inhibitor of Serratia marcescens anthranilate synthase (Ki = 30 microM for an RS mixture) and of the E. coli pabB subunit of PABA synthase (Ki = 226 microM). Modifications in the substituents at C-3 [enolpyruyl ether, (R)- or (S)-lactyl ether, glycolyl ether] or C-4 (O-methyl) of chorismate lead to alternate substrates. The Vmax values for (R)- and (S)-lactyl ethers are down 10-20-fold for each enzyme, and V/K analyses show the (S)-lactyl chorismate analogue to be preferred by 12/1 over (R)-lactyl for anthranilate synthase while a 3/1 preference was observed for (R)-/(S)-lactyl analogues by PABA synthase. The glycolyl ether analogue of chorismate shows 15% Vmax vs. chorismate for anthranilate synthase but is actually a faster substrate (140%) than chorismate with PABA synthase, suggesting the elimination/aromatization step from an aminocyclohexadienyl species may be rate limiting with AS but not with PABS. Indeed, studies with (R)-lactyl analogue 14 and anthranilate synthase led to accumulation of an intermediate, isolable by high-performance liquid chromatography and characterized by NMR and UV-visible spectroscopy as 6-amino-5-[(1-carboxyethyl)oxy]-1,3-cyclohexadiene-1-carboxylic acid (17). This is the anticipated intermediate predicted by our previous work with conversion of synthetic trans-6-amino-5-[(1-carboxyethenyl)oxy]-1,3-cyclohexadiene-1-carbo xylic acid (2) to anthranilate by the enzyme. Compound 17 is quantitatively converted to anthranilate on reincubation with enzyme, but at a 1.3-10-fold lower Vmax than starting lactyl substrate 14 under the conditions investigated; the basis for this kinetic variation is not yet determined.

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: cooperative effects and inhibition by L-tyrosine.

The effects of a variety of structural analogs of L-tyrosine on the mutase and dehydrogenase activities of hydroxyphenylpyruvate synthase have been investigated. From these studies it is concluded that the alpha-NH3+ alpha-COO-, and the 4-OH groups are essential for binding of L-tyrosine as an inhibitor of the dehydrogenase and that the L configuration is also essential. Dixon plots for inhibition of the dehydrogenase activity by some of these analogs were nonlinear and could be described by a velocity equation that is the ratio of quadratic polynomials (a 2/1 function). Dixon plots for inhibition of the mutase by prephenate at low concentrations of chorismate could also be described by a 2/1 function, but at low concentrations of prephenate chorismate acts as an apparent hyperbolic activator of the dehydrogenase activity. Up to concentrations of 300 microM, L-tyrosine activates the mutase but acts as a potent inhibitor of the dehydrogenase. Such data for the dehydrogenase could not be described by a 2/1 function in 1/[prephenate] but could be fitted to the Hill equation with increasing concentrations of L-tyrosine in the presence of 1.0 mM NAD yielding increasing values for the Hill number (n): in the absence of L-tyrosine, n = 1.6 +/- 0.1; at 150 microM L-tyrosine, n = 2.1 +/- 0.1; at 300 microM L-tyrosine, n = 2.3 +/- 0.4. L-Tyrosine bears a close structural resemblance to both prephenate and hydroxyphenylpyruvate, and evidence is presented which is consistent with L-tyrosine acting as a competitive inhibitor with respect to prephenate of the dehydrogenase.

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: positive cooperativity with substrates and inhibitors.

Investigations have been made at pH 6.0 of the effect of chorismate and adamantane derivatives on the mutase and dehydrogenase activities of hydroxyphenylpyruvate synthase from Escherichia coli. When used over a wide range of concentrations, chorismate 5,6-epoxide, chorismate 5,6-diol, adamantane-1,3-diacetate, adamantane-1-acetate, adamantane-1-carboxylate, and adamantane-1-phosphonate give rise to nonlinear plots of the reciprocal of the initial velocity of each reaction as a function of the inhibitor concentration. The inhibitors do not induce the enzyme to undergo polymerization and have only a small effect on the S20,w value of the enzyme as determined by using sucrose density gradient centrifugation. At low substrate concentration, low concentrations of adamantane-1-acetate cause activation of both the mutase and dehydrogenase activities while at higher concentrations this compound functions as an inhibitor. When chorismate and prephenate are varied over a wide range of concentrations, double-reciprocal plots of the data indicate that the reactions exhibit positive cooperativity. The addition of albumin eliminates the cooperative interactions associated with substrates but has little effect on those associated with inhibitors.

Regulatory control of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthetase in Streptomyces antibioticus.

Streptomyces antibioticus possesses a tryptophan-inhibitable 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthetase whose synthesis is also repressed by L-tryptophan. Studies of the DAHP synthetase obtained by ammonium sulfate fractionation of a crude extract derived from S. Antibioticus revealed that the enzymic activity was only partially inhibited by tryptophan. Inhibition of the DAHP synthetase activity was strongly pH dependent at values below 7.0. A number of tryptophan analogues was noted to inhibit the enzyme; by contrast, other aromatic amino acid end products failed to affect DAHP synthetase activity. Chorismic acid, a key intermediate in aromatic amino acid biosynthesis, was ineffective as an inhibitor when used alone; however, if supplied with L-tryptophan, a further reduction of DAHP synthetase activity (15--25%) was routinely observed.

Constitutive and repressivle enzymes of the common pathway of aromatic biosynthesis in Escherichia coli K-12: regulation of enzyme synthesis at different growth rates.

Synthesis of five of the enzymes of the common pathway of aromatic biosynthesis has been shown to be unaffected by either the aromatic amino acids--the product of the first reaction (3-deoxy-D-arabinoheptulosonic acid-7-phosphate) or the product of the last reaction (chorismate)--or by the state of regulator gene loci tyrR. On the other hand, the rate of synthesis of these enzymes, and of several other enzymes for which repression control was inactive because of mutations in relevant regulator genes, was found to change with growth rate. These changes were found to correlate at faster growth rates than those observed in glucose minimal medium with the alterations in the relative frequencies of the corresponding structural genes which occur at these growth rates. It was found that when wild-type cells were grown at these faster growth rates in medium lacking the aromatic amino acids, complete derepression of the tyrosine-inhibitable 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase occurred, in strong contrast to the situation when wild-type cells are grown in glucose minimal medium.

Properties of anthranilate synthetase component II from Pseudomonas putida.

The interaction of Pseudomanas putida anthranilate synthetase Component II (AS II) with glutamine, glutamine analogs, and iodoacetamide has been investigated in order to clarify the initial steps in the mechanism for glutamine utilization. AS II is alkylated and irreversibly inactivated by covalent attachment of approximately 1 eg of L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone) or 1 eq of iodoacetamide. Alkylation of AS II by chloroketone involves initial formation of an enzyme-inhibitor complex having a Ki of 28 muM. Alkylation of AS II by iodoacetamide occurs without initial formation of a reversible complex. In both cases glutamine protects against alkylation and exhibits competitive kinetics. When anthranilate synthetase Component I (AS I) is associated with AS II, the second substrate, chorismate, enhances alkylation of AS II by chloroketone. Alkylation of AS II by iodoacetamide is unaffected by AS I and chorismate. These results suggest a role of chorismate-AS I complex to promote binding of glutamine to AS II or to facilitate conversion of an AS II-glutamine complex to the covalent glutamyl-AS II intermediate. This conclusion is supported by the fact that glutaminase activity of AS II, which requires formation of the glutamyl-AS II intermediate, is stimulated by AS I and chorismate.

Anthranilate synthase from Bacillus subtilis. The role of a reduced subunit X in aggregate formation and amidotransferase activity.

With respect to its sulfhydryl groups, subunit X can exist in at least two forms, oxidized (Xox) and reduced (Xre). The importance of the Xre form for the formation of an EX complex and for amidotransferase activity has been examined. Subunit Xre is rapidly inactivated by p-chloromercuribenzoate and bromopyruvate, whereas subunit Xox, which is not catalytically functional in amidotransferase activity, is not affected. The glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) has no effect on Xre alone but rapidly inactivates the EXre complex. DON-inactivated subunit X cannot be reactivated by 2-mercaptoethanol but can be readily displaced from subunit E by free subunit Xre. The integrity of the EXre complex is maintained following gel filtration on Sephadex G-100 in the presence of glutamine and 2-mercaptoethanol, thus the binding of glutamine to the complex does not require the binding of other substrates. Subunit Xox, however, does not aggregate with subunit E since no EXox complex is found following gel filtration on Sephadex G-100 in the presence of glutamine and in the absence of 2-mercaptoethanol. Thus, a reduced sulfhydryl group(s) is not only essential for amidotransferase activity but also for the formation of the aggregate as well. The following model is proposed to explain these results. Free subunit Xre does not bind DON or glutamine to the catalytically functional sulfhydryl group. Upon aggregation with subunit E, however, the glutamine or DON binds to the glutamine catalytic site on subunit Xre and amidotransfer or alkylation occurs. An EX complex which has been alkylated by DON can be readily dissociated and it is suggested that following catalysis the EX complex may also dissociate.