Enzyme Alterations in Tyrosine and Phenylalanine Auxotrophs of Salmonella typhimurium

The enzyme activities specified by the tyrA and pheA genes were studied in wildtype strain Salmonella typhimurium and in phenylalanine and tyrosine auxotrophs. As in Aerobacter aerogenes and Escherichia coli, the wild-type enzymes of Salmonella catalyze two consecutive reactions: chorismate --> prephenate --> 4-hydroxy-phenylpyruvate (tyrA), and chorismate --> prephenate --> phenylpyruvate (pheA). A group of tyrA mutants capable of interallelic complementation had altered enzymes which retained chorismate mutase T activity but lacked prephenate dehydrogenase. Similarly, pheA mutants (in which interallelic complementation does not occur) had one group with altered enzymes which retained chorismate mutase P but lacked prephenate dehydratase. Tyrosine and phenylalanine auxotrophs outside of these categories showed loss of both activities of their respective bifunctional enzyme. TyrA mutants which had mutase T were considerably derepressed in this activity by tyrosine starvation and consequently excreted prephenate. A new and specific procedure was developed for assaying prephenate dehydrogenase activity.     

组合代谢调控提高大肠杆菌对氨基苯甲酸产量

对氨基苯甲酸是一种重要的有机合成中间体,广泛应用于医药、染料等行业。近年来对氨基苯甲酸作为一种潜在的高强度共聚物单体越来越受到重视。对氨基苯甲酸作为叶酸合成的前体之一,其合成在大肠杆菌体内由叶酸合成途径的pabA、pabB和pabC三个基因负责,催化分支酸合成对氨基苯甲酸。本研究以实验室构建的酪氨酸高产工程菌TYR002作为出发菌株,首先弱化双功能分支酸突变酶/预苯酸脱氢酶TyrA的表达,以减少酪氨酸积累,然后利用3种不同强度的组成型启动子分别调控pabA、pabB和pabC的表达。摇瓶发酵表明不同的组合调控模式下大肠杆菌发酵培养基中的对氨基苯甲酸积累量存在显著差异,最高可获得0.67 g/L的摇瓶发酵产量。进一步通过发酵条件优化和分批补料发酵,在5L发酵罐中获得了6.4g/L的对氨基苯甲酸产量。本研究为改善对氨基苯甲酸生物合成效率提供了重要理论参考。     

Absolute dependence of phenylalanine and tyrosine biosynthetic enzyme on tryptophan in Candida maltosa

Candida maltosa synthesizes phenylalanine and tyrosine only via phenylpyruvate and p-hydroxyphenylpyruvate. Tryptophan is absolutely necessary for the enzymatic reaction of chorismate mutase and prephenate dehydrogenase; activity of prephenate dehydratase can be increased 2.5-fold in the presence of tryptophan. Activation of the chorismate mutase, prephenate dehydratase and prephenate dehydrogenase by tryptophan is competitive with respect to chorismate and prephenate with Ka 0.06mM, 0.56mM and 1.7mM. In addition tyrosine is a competitive inhibitor of chorismate mutase (Ki = 0.55mM) and prephenate dehydrogenase (Ki = 5.5mM).     

基于结构域活性分析的大肠杆菌T蛋白结构研究

大肠杆菌T蛋白含有三个结构域:分支酸变位酶、预苯酸脱氢酶和调节结构域。文章作者分段克隆了T蛋白的分支酸变位酶、预苯酸脱氢酶和调节结构域等片段,并对其进行了活性研究。研究发现,定位于N末端的分支酸变位酶结构域的比活性虽然不高,而稳定性较好;同时拥有调节结构域和预苯酸脱氢酶结构域的C末端片段,其预苯酸脱氢酶比活性的剩余百分率虽然高于分支酸变位酶结构域,但稳定性较差。作者进而表达了C末端切除38个氨基酸的T/1-336片段,发现预苯酸脱氢酶活性彻底丧失,而其分支酸变位酶和调节结构域的活性却基本保留。这说明T蛋白中分支酸变位酶结构域拥有一个相对独立、完整的结构,而预苯酸脱氢酶结构域和调节结构域交织共存,结构松散。     

Kinetic studies on the mechanism of chorismate mutase/prephenate dehydratase from Escherichia coli K12

The effect of pH on chorismate mutase/prephenate dehydratase (chorismate pyruvate mutase/prephenate hydro-lyase (decarboxylating) EC 5.4.99.5/EC 4.2.1.51) from Escherichia coli K12 has been studied. While the maximum velocity of both activities is independent of pH, Km for chorismate or prephenate shows a complex pH dependence. Differences in mutase activity in acetate/phosphate/borate and citrate/phosphate/borate buffers were traced to inhibition by citrate. When a variety of analogues of citrate were tested as possible inhibitors of the enzyme, several were found to inhibit mutase and dehydratase activities to different extents, and by different mechanisms. Thus citrate competitively inhibits mutase activity, but inhibits dehydratase activity by either a non-competitive or an uncompetitive mechanism. Conversely, cis- and trans-aconitate competitively inhibit dehydratase activity, but are partially competitive inhibitors of mutase activity. The differential effects of these inhibitors on the two activities are consistent with the existence of two distinct active sites, but additionally suggest some degree of interconnection between them. The implications of these results for possible mechanisms of catalysis by chorismate mutase/prephenate dehydratase are discussed.     

A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K12 defective in the PheA gene

Three classes of mutant strains of Escherichia coli K12 defective in pheA, the gene coding for chorismate mutase/prephenate dehydratase, have been isolated: (1) those lacking prephenate dehydratase activity, (2) those lacking chorismate mutase activity, and (3) those lacking both activities. Chorismate mutase/prephenate dehydratase from the second class of mutants was less sensitive to inhibition by phenylalanine than wild-type enzyme and, along with the defective enzyme from the third class of mutants, could not be purified by affinity chromatography on Sepharosyl-phenylalanine. Pure chorismate mutase/prephenate dehydratase protein was prepared from two strains belonging to the first class. The chorismate mutase activity of these enzymes is kinetically similar to that of the wild-type enzyme except for a two- to threefold increase in both the K_a for chorismate and the K_is for inhibition by prephenate. In both cases only one change in the tryptic fingerprint was detected, resulting from a substitution of the threonine residue in the peptide Gln·Asn·Phe·Thr·Arg. This suggests that this residue is catalytically or structurally essential for the dehydratase activity.     

Production of tyrosine from sucrose or glucose achieved by rapid genetic changes to phenylalanine-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> strains

Escherichia coli K12 strains producing l-phenylalanine were converted to l-tyrosine-producing strains using a novel genetic method for gene replacement. We deleted a region of the E. coli K12 chromosome including the pheA gene encoding chorismate mutase/prephenate dehydratase, its leader peptide (pheL), and its promoter using a new polymerase chain reaction-based method that does not leave a chromosomal scar. For high level expression of tyrA, encoding chorismate mutase/prephenate dehydrogenase, its native promoter was replaced with the strong trc promoter. The linked ΔpheLA and Ptrc-tyrA::Kan^R genetic modifications were moved into l-phenylalanine producing strains by generalized transduction to convert l-phenylalanine-producing strains to l-tyrosine-producing strains. Moreover, introduction of a plasmid carrying genes responsible for sucrose degradation into these strains enabled l-tyrosine-production from sucrose.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: spatial relationship of the mutase and dehydrogenase sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase (4-hydroxyphenylpyruvate synthase) by substrate analogues has been investigated at pH 6.0 with the aim of elucidating the spatial relationship that exists between the sites at which each reaction occurs. Several chorismate and adamantane derivatives, as well as 2-hydroxyphenyl acetate and diethyl malonate, act as linear competitive inhibitors with respect to chorismate in the mutase reaction and with respect to chorismate in the mutase reaction and with respect to prephenate in the dehydrogenase reaction. The similarity of the dissociation constants for the interaction of these compounds with the free enzyme, as determined from the mutase and dehydrogenase reactions, indicates that the reaction of these inhibitors at a single site prevents the binding of both chorismate and prephenate. However, not all the groups on the enzyme, which are responsible for the binding of these two substrates, can be identical. At lower concentrations, citrate or malonate prevents reaction of the enzyme with prephenate, but not with chorismate. Nevertheless, the combining sites for chorismate and prephenate are in such close proximity that the diethyl derivative of malonate prevents the binding of both substrates. The results lead to the proposal that the sites at which chorismate and prephenate react on hydroxyphenylpyruvate synthase share common features and can be considered to overlap.     

Modular Optimization of Heterologous Pathways for De Novo Synthesis of (2S)-Naringenin in Escherichia coli

Due to increasing concerns about food safety and environmental issues, bio-based production of flavonoids from safe, inexpensive, and renewable substrates is increasingly attracting attention. Here, the complete biosynthetic pathway, consisting of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydrogenase (CM/PDH), tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was constructed using pre-made modules to overproduce (2S)-naringenin from D-glucose. Modular pathway engineering strategies were applied to the production of the flavonoid precursor (2S)-naringenin from L-tyrosine to investigate the metabolic space for efficient conversion. Modular expression was combinatorially tuned by modifying plasmid gene copy numbers and promoter strengths to identify an optimally balanced pathway. Furthermore, a new modular pathway from D-glucose to L-tyrosine was assembled and re-optimized with the identified optimal modules to enable de novo synthesis of (2S)-naringenin. Once this metabolic balance was achieved, the optimum strain was capable of producing 100.64 mg/L (2S)-naringenin directly from D-glucose, which is the highest production titer from D-glucose in Escherichia coli. The fermentation system described here paves the way for the development of an economical process for microbial production of flavonoids.     

Use of the valine biosynthetic pathway to convert glucose into isobutanol

Microbiological synthesis of higher alcohols (1-butanol, isobutanol, 2-methyl-1-butanol, etc.) from plant biomass is critically important due to their advantages over ethanol as a motor fuel. In recent years, the use of branched-chain amino acid (BCAA) biosynthesis pathways together with heterologous Ehrlich pathway enzyme system (Hazelwood et al. in Appl Environ Microbiol 74:2259–2266, 2008) has been proposed by the Liao group as an alternative approach to aerobic production of higher alcohols as new-generation biofuels (Atsumi et al. in Nature 451:86–90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89–98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769–5775, 2008; Shen and Liao in Metab Eng 10:312–320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471–479, 2009). On the basis of these remarkable investigations, we re-engineered Escherichia coli valine-producing strain H-81, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar into isobutanol. To redirect valine biosynthesis to the production of alcohol, we also—as has been demonstrated previously (Atsumi et al. in Nature 451:86–90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89–98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769–5775, 2008; Shen and Liao in Metab Eng 10:312–320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471–479, 2009)—used enzymes of Ehrlich pathway. In particular, in our study, the following heterologous proteins were exploited: branched-chain 2-keto acid decarboxylase (BCKAD) encoded by the kdcA gene from Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by the ADH2 gene from Saccharomyces cerevisiae. We show that expression of both of these genes in the valine-producing strain H-81 results in accumulation of isobutanol instead of valine. Expression of BCKAD alone also resulted in isobutanol accumulation in the culture broth, supporting earlier obtained data (Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010) that native ADHs of E. coli are also capable of isobutanol production. Thus, in this work, isobutanol synthesis by E. coli was achieved using enzymes similar to but somewhat different from those previously used.     

Chorismate mutase-prephenate dehydratase from Escherichia coli: active sites of a bifunctional enzyme.

The relationship between the active sites of the bifunctional enzyme chorismate mutase-prephenate dehydratase has been examined. Steady-state kinetic investigations of the reactions with chorismate or prephenate as substrate and studies of the overall conversion of chorismate to phenylpyruvate indicate that there are two distinct active sites. One site is responsible for the mutase activity and the other for the dehydratase activity. Studies of the overall reaction using radioactive chorismate show that prephenate, which is formed from chorismate, dissociates from the mutase site and equilibrates with the bulk medium before combining at the dehydratase site. No evidence was obtained for direct channeling of prephenate from one site to the other, or for any strong interaction between the sites.     

Relationships Among Mycobacteria and Nocardiae Based upon Deoxyribonucleic Acid Reassociation

Chorismate mutase from Streptomyces aureofaciens was purified 12-fold. This enzyme preparation did not show any activity when tested for anthranilate synthetase, prephenate dehydrogenase, or prephenate dehydratase. The catalytic activity of chorismate mutase has a broad optimum between pH 7 and 8. The initial velocity data followed regular Michaelis-Menten kinetics with a K(m) of 5.3 x 10(-4) M, and the molecular weight of the enzyme was determined by sucrose gradient centrifugation to be 50,000. Heat inactivation of chorismate mutase, which occurs above temperatures of 60 C, is reversible. The enzyme activity can be restored even when chorismate mutase is treated at the temperature of a boiling-water bath for 15 min. Heat-denatured and renatured enzymes showed the same Michaelis constant and the same molecular weight as the native enzyme. l-Phenylalanine, l-tyrosine, l-tryptophan, and metabolites of the aromatic amino acid pathway were tested as potential modifiers of chorismate mutase activity. The activity of the enzyme was inhibited by none of these substances. Chorismate mutase of S. aureofaciens was not repressed in cells grown in minimal medium supplemented with l-phenylalanine, l-tyrosine, or l-tryptophan.     

Chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes: evidence that the two reactions occur at one active site.

The relationship between the sites for catalysis of two reactions by the bifunctional enzyme chorismate mutase--prephenate dehydrogenase has been investigated. The results are consistent with the occurrence of both reactions at one active site. Comparisons have been made between experimental data for the time course of the overall reaction and computer simulations, according to various models for the relationship between the mutase and dehydrogenase sites. A model based on a single active site is consistent with the time course data if a minor proportion of the chorismate that reacts can be converted through to (hydroxyphenyl)pyruvate without the intermediate release of prephenate. Consistent with this requirement, some channeling of radioactivity from chorismate to (hydroxyphenyl)pyruvate has been detected. A model based on two separate sites has also been considered; the simulations show that if this model applies there is no need to postulate any channeling of the intermediate, prephenate, between the sites and there must be marked inhibition of the dehydrogenase reaction by chorismate. Since channeling has been observed and chorismate increases the dehydrogenase rate under all conditions, the two-site model appears unlikely. Consistent with the one-site model are the observations that a variety of inactivating conditions cause parallel loss of mutase and dehydrogenase activity and that identical protection against inactivation of both mutase and dehydrogenase by iodoacetamide is afforded by prephenate.     

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

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.     

Synthesis of chiral arogenic acid via immobilized microbial proteins

Although l-(8S)-arogenate has been recognized as a potential precursor of l-phenylalanine or l-tyrosine biosynthesis for only a few years, it is widely distributed in nature. The biochemical formation of arogenate has involved its isolation from the culture supernatant of a mutant strain of Neurospora crassa, a lengthy procedure of 20-day duration. We now report an improved approach using immobilized crude enzyme extracts from a cyanobacterium. The starting materials, chorismic acid or prephenic acid, are readily available, and overall yields ranging from 40 to 60% are obtained. The whole procedure takes only 1 day. Crude, unfractionated enzyme extracts from Synechocystis sp. ATCC 29108 are immobilized on a phenoxyacetyl cellulose solid support. The hydrophobic binding of the extract proteins did not denature chorismate mutase or prephenate aminotransferase, the enzymes catalyzing the conversion of chorismate to prephenate and prephenate to arogenate, respectively. This microbial system was ideally suited for preparation of arogenate, since other enzyme activities which might compete for prephenate or chorismate as substrates, or which might further metabolize arogenate, were absent or inactive under the conditions used. In addition to the substrates prephenate or chorismate, pyridoxal-5′-phosphate (the coenzyme required for transamination), as well as leucine (amino donor for transamination of prephenate), was added. The reaction product, arogenate, was separated from the starting materials by preparative thin-layer chromatography.     

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16. II. The isolation and characterization of mutants auxotrophic for phenylalanine and tyrosine.

1. Mutants derived from the hydrogen bacterium Alcaligenes eutrophus strain H 16 auxotrophic for phenylalanine and tyrosine were isolated employing mutagenic agents (EMS, nitrite), the colistine counterselection technique and the "pin-point" isolation method. Three different types of mutants were found: (1) Mutants, requiring phenylalanine or phenylpyruvate for growth, were affected in chorismate mutase as well as prephenate dehydratase. Both activities were regained by reversion to prototrophy. The auxotrophic strains accumulated chorismic acid. (2) Strains with a growth response similar to that of the first group lacked only prephenate dehydratase activity which was partially regained by reversion. Chorismate mutase and prephenate dehydrogenase were derepressed up to two-fold. Mutants grown in minimal medium excreted prephenic acid. (3) The third type of mutants required phenylalanine or phenylpyruvate and grew slowly when supplemented with chorismate or prephenate. The enzymes involved in the specific pathway of phenylalanine and tyrosine were found to be present. Some of them were even more active than in the wild-type. 2. Mutants accumulating chorismic acid or prepheric acid were able to grow on minimal medium when incubated long enough. The chemical instability of the excretion products resulted in their nonenzymatic conversion to subsequent intermediates which were taken up by the cells, allowing growth. 3. A method is described for preparing barium prephenate using the auxotrophic mutant 6B-1 derived from A.eutrophus H 16. Prephenic acid, excreted by this strain, was obtained from the culture filtrate with a purity of at least 70% and a yield of approximately 180 mg per 21 of medium.     

Chorismate mutase:prephenate dehydratase from Acinetobacter calcoaceticus. Purification, properties and immunological cross-reactivity

The bifunctional P protein (chorismate mutase: prephenate dehydratase) from Acinetobacter calcoaceticus has been purified. It was homogeneous in polyacrylamide gels and was more than 95% pure on the basis of the immunostaining of purified P protein with the antibodies raised against the P protein. The native enzyme is a homodimer (Mr = 91,000) composed of 45-kDa subunits. A twofold increase in the native molecular mass of the P protein occurred in the presence of L-phenylalanine (inhibitor of both activities) or L-tyrosine (activator of the dehydratase activity) during gel filtration. Chorismate mutase activity followed Michaelis-Menten kinetics with a Km of 0.55 mM for chorismate. L-Phenylalanine was a relatively poor non-competitive inhibitor of the mutase activity. The chorismate mutase activity was also competitively inhibited by prephenate (reaction product). Substrate-saturation curves for the dehydratase activity were sigmoidal showing positive cooperativity among the prephenate-binding sites. L-Tyrosine activated prephenate dehydratase strongly but did not abolish positive cooperativity with respect to prephenate. L-Phenylalanine inhibited the dehydratase activity, and the substrate-saturation curves became increasingly sigmoidal as phenylalanine concentrations were increased with happ values changing from 2.0 (no phenylalanine) to 4.0 (0.08 mM L-phenylalanine). A sigmoidal inhibition curve of the dehydratase activity by L-phenylalanine gave Hill plots having a slope of -2.9. Higher ionic strength increased the dehydratase activity by reducing the positive cooperative binding of prephenate, and the sigmoidal substrate-saturation curves were changed to near-hyperbolic form. The happ values decreased with increase in ionic strength. Antibodies raised against the purified P protein showed cross-reactivity with the P proteins from near phylogenetic relatives of A. calcoaceticus. At a greater phylogenetic distance, cross-reaction was superior with P protein from Neisseria gonorrhoeae than with that from the more closely related Escherichia coli.     

Design, synthesis, and evaluation of aza inhibitors of chorismate mutase

A series of aza inhibitors (4-9) of chorismate mutase (E.C. 5.4.99.5) was designed, prepared, and evaluated against the enzyme by monitoring the direct inhibition of the chorismate, 1, to prephenate, 2, conversion. None of these aza inhibitors displayed tighter binding to the enzyme than the native substrate chorismate or greater inhibitory action than the previously reported ether analogue, 3. Furthermore, no time-dependent loss of enzyme activity was observed in the presence of the two potentially reactive aza inhibitors (7 and 9). These results in conjunction with inhibition data from a broader series of chorismate mutase inhibitors allowed a novel proposal for the mechanistic role of chorismate mutase to be developed. This proposed mechanism was computationally verified and correlated with crystallographic studies of various chorismate mutases.