A thermostable shikimate 5-dehydrogenase from the archaeon Archaeoglobus fulgidus

Shikimate 5-dehydrogenase (SKDH; EC 1.1.1.25) catalyzes the reversible reduction of 3-dehydroshikimate to shikimate and is a key enzyme in the aromatic amino acid biosynthesis pathway. The shikimate 5-dehydrogenase gene, aroE, from Archaeoglobus fulgidus was cloned and overexpressed in Escherichia coli. The recombinant enzyme purified as a homodimer and yielded a maximum specific activity of 732 U/mg at 87 degrees C (with NADP+ as coenzyme). Apparent Km values for shikimate, NADP+, and NAD+ were estimated at 0.17+/-0.03 mM, 0.19+/-0.01 mM, and 11.4+/-0.4 mM, respectively. The half-life of the A. fulgidus SKDH is 2 h at the assay temperature (87 degrees C) and 17 days at 60 degrees C. Addition of 1 M NaCl or KCl stabilized the enzyme's half-life to approximately 70 h at 87 degrees C and approximately 50 days at 60 degrees C. This work presents the first kinetic analysis of an archaeal SKDH.     

Metabolic engineering of Corynebacterium glutamicum for shikimate overproduction by growth-arrested cell reaction

Corynebacterium glutamicum with the ability to simultaneously utilize glucose/pentose mixed sugars was metabolically engineered to overproduce shikimate, a valuable hydroaromatic compound used as a starting material for the synthesis of the anti-influenza drug oseltamivir. To achieve this, the shikimate kinase and other potential metabolic activities for the consumption of shikimate and its precursor dehydroshikimate were inactivated. Carbon flux toward shikimate synthesis was enhanced by overexpression of genes for the shikimate pathway and the non-oxidative pentose phosphate pathway. Subsequently, to improve the availability of the key aromatics precursor phosphoenolpyruvate (PEP) toward shikimate synthesis, the PEP: sugar phosphotransferase system (PTS) was inactivated and an endogenous myo-inositol transporter IolT1 and glucokinases were overexpressed. Unexpectedly, the resultant non-PTS strain accumulated 1,3-dihydroxyacetone (DHA) and glycerol as major byproducts. This observation and metabolome analysis identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-catalyzed reaction as a limiting step in glycolysis. Consistently, overexpression of GAPDH significantly stimulated both glucose consumption and shikimate production. Blockage of the DHA synthesis further improved shikimate yield. We applied an aerobic, growth-arrested and high-density cell reaction to the shikimate production by the resulting strain and notably achieved the highest shikimate titer (141 g/l) and a yield (51% (mol/mol)) from glucose reported to date after 48 h in minimal medium lacking nutrients required for cell growth. Moreover, comparable shikimate productivity could be attained through simultaneous utilization of glucose, xylose, and arabinose, enabling efficient shikimate production from lignocellulosic feedstocks. These findings demonstrate that C. glutamicum has significant potential for the production of shikimate and derived aromatic compounds.     

Expression, purification and properties of shikimate dehydrogenase from Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the main diseases to mankind. It is urgent to discover novel drug targets for appropriate antimicrobial agents against this human pathogen. The shikimate pathway is considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammalian cells. The Mycobacterium tuberculosis aroE-encoded shikimate dehydrogenase was cloned, expressed and purified. Sequence alignment analysis shows that shikimate dehydrogenase of Mycobacterium tuberculosis exhibit the pattern of G-X-(N/S)-V-(T/S)-X-PX-K, which is highly conserved within the shikimate dehydrogenase family. The recombinant shikimate dehydrogenase spectrum determined by CD spectroscopy showed that the percentages for alpha-helix, beta-sheet, beta-turn, and random coil were 29.2 %, 9.3 %, 32.7 %, and 28.8 %, respectively. The enzymatic characterization demonstrates that it appears to be fully active at pH from 9.0 to 12, and temperature 63(o)C. The apparent Michaelis constant for shikimic acid and NADP(+) were calculated to be about 29.5 microM and 63 microM. The recombinant shikimate dehydrogenase catalyzes the substrate in the presence of NADP(+) with an enzyme turnover number of 399 s(-1). Zymological studies suggest that the cloned shikimate dehydrogenase from M. tuberculosis has a pretty activity, and the work should help in the discovery of enzyme inhibitors and further of possible antimicrobial agents against Mycobacterium tuberculosis.     

Synthesis,biological activity and molecular modelling studies of shikimic acid derivatives as inhibitors of the shikimate dehydrogenase enzyme of Escherichia coli

Shikimic acid (SA) pathway is the common route used by bacteria, plants, fungi, algae, and certain Apicomplexa parasites for the biosynthesis of aromatic amino acids and other secondary metabolites. As this essential pathway is absent in mammals designing inhibitors against implied enzymes may lead to the development of antimicrobial and herbicidal agents harmless to humans. Shikimate dehydrogenase (SDH) is the fourth enzyme of the SA pathway. In this contribution, a series of SA amide derivatives were synthesised and evaluated for in vitro SDH inhibition and antibacterial activity against Escherichia coli. All tested compounds showed to be mixed type inhibitors; diamide derivatives displayed more inhibitory activity than synthesised monoamides. Among the evaluated compounds, molecules called 4a and 4b were the most active derivatives with IC₅₀ 588 and 589?µM, respectively. Molecular modelling studies suggested two different binding modes of monoamide and diamide derivatives to the SDH enzyme of E. coli.     

Archaeal shikimate kinase, a new member of the GHMP-kinase family

Shikimate kinase (EC 2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi1500322 in Methanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC 2.7.1.39) of the GHMP-kinase superfamily. The latter functionality in M. jannaschii is assigned to another gene (gi591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed in Escherichia coli and purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases: K(m,shikimate) = 414 +/- 33 microM, K(m,ATP) = 48 +/- 4 microM, and k(cat) = 57 +/- 2 s(-1) for the predicted shikimate kinase and K(m,homoserine) = 188 +/- 37 microM, K(m,ATP) = 101 +/- 7 microM, and k(cat) = 28 +/- 1 s(-1) for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes.     

In vivo activity, purification, and characterization of shikimate kinase from sorghum

Sorghum bicolor stems and shoots were observed to phosphorylate exogenously supplied shikimate, forming a product which could not be distinguished from shikimate 3-phosphate by anion exchange and thin layer chromatography. Upon treatment with phosphatase, this product gave rise to a compound which co-chromatographed with shikimate.     

Broad-specificity quinate (shikimate) dehydrogenasefrom Pinus taeda needles

Two proteins having quinate dehydrogenase (QDH, quinate:NAD(P)⁺-oxidoreductase, EC 1.1.1.24) and shikimate dehydrogenase (SDH, shikimate:NADP⁺-oxidoreductase, EC 1.1.1.25) activities were purified about 3 000-fold from young loblolly pine (Pinus taeda L.) needles. A combination of ammonium sulfate solubilization, and chromatographies on DEAE-cellulose, 2′, 5′ ADP-Sepharose and Mono-Q was used. Throughout all purification steps, the QDH activity consistently co-purified with the activity of the first of three forms of SDH, and the ratio of QDH/SDH was constant (variation from 1.63 to 1.89). These data indicate that both QDH and SDH activities are catalyzed by a single broad-specificity quinate (shikimate) dehydrogenase. Gel chromatography on Superdex 75 was used to estimate the native molecular mass of two forms of the enzyme as 35 and 53 kDa.     

Enzymatic preparation of metabolic intermediates, 3-dehydroquinate and 3-dehydroshikimate, in the shikimate pathway

A method for enzymatic preparation of 3-dehydroquinate and 3-dehydroshikimate in the shikimate pathway was established by controlling the enzyme activity of 3-dehydroquinate dehydratase. When quinate was incubated with the membrane fraction of acetic acid bacteria at pH 5.0, 3-dehydroquinate was formed as the predominant product. 3-Dehydroshikimate was the sole product when incubated at pH 8.0. Mutual separation of the metabolic intermediates was also exemplified.     

Synthesis and separation of tritium-labeled intermediates of the shikimate pathway

[5-³H]Shikimate (sp radioact 2000 Ci/mol) has been synthesized by reduction of the methyl ester of 5-dehydroshikimate with NaB³H₄ and subsequent hydrolysis of the ester group (M. M. Leduc, P. M. Dansette, and R. G. Azerad (1970) Eur. J. Biochem.15, 428–435). The [5-³H]shikimate has been converted enzymatically to [5-³H]chorismate and [5-³H]prephenate of similar high specific radioactivity by using a cell-free extract of Aerobacter aerogenes 62-1. In addition, a chromatograhic procedure, which utilizes polyethyleneimine-cellulose thin-layer chromatograms, has been developed for the separation of intermediates along the shikimate pathway between shikimate and hydroxyphenylpyruvate or phenylpyruvate. Since the method allows quantitative measurement of tritium-labeled intermediates, it provides the basis for sensitive radioassays of the individual enzymes and allows study of the reaction flux along the overall pathway. The same intermediates can be separated on a large scale by use of a column of DEAE-Sephacel.     

Purification and properties of NADP-dependent shikimate dehydrogenase from Gluconobacter oxydans IFO 3244 and its application to enzymatic shikimate production

NADP-Dependent shikimate dehydrogenae (SKDH, EC 1.1.1.25) was purified from Gluconobacter oxydans IFO 3244. SKDH showed a single protein band on native-PAGE accompanying enzyme activity. It required NADP exclusively and catalyzed only the shuttle reaction between shikimate and 3-dehydroshikimate. The optimum pH for shikimate oxidation and 3-dehydroshikimate reduction was found at pH 10 and 7 respectively. SKDH proved to be a useful catalyst for shikimate production from 3-dehydroshikimate.     

Annotating the Plasmodium genome and the enigma of the shikimate pathway

The completion of the Plasmodium falciparum genome sequence heralds a new era in the effort to identify all the parasite's genes along with their cellular functions. A combination of bioinformatics and experimental proof will facilitate this process. Many enzymes in metabolic processes have been identified, but several examples exist of incomplete pathways, such as the shikimate pathway. This review uses the example of the shikimate pathway to examine the application of bioinformatics to lead experimental design in post-genomic biology.     

The metabolism of shikimate in the rat.

In the rat, shikimate was metabolized and excreted as hippurate, hexahydrohippurate, 3,4,5,6-tetrahydrohippurate, t-3,t-4-dihydroxycyclohexane-r-1-carboxylate and c-3,t-4-dihydroxycyclophexane-r-1-carboxylate, conjugates of catechol and CO2. The metabolism was entirely dependent on various initial microbial transformations in the gut, metabolite formation being suppressed in animals pretreated with antibiotics. Shikimate was not metabolized by mammalian tissues, and products of microbial metabolism were excreted either unchanged or after further biotransformation in the animal tissues.     

Localization, purification, and characterization of shikimate oxidoreductase-dehydroquinate hydrolyase from stroma of spinach chloroplasts

The stroma of chloroplasts is probably the sole site of the shikimate pathway enzymes shikimate oxidoreductase/dehydroquinate hydrolyase (SORase/DHQase) in spinach leaves. (a) The chromatographic behavior of the bifunctional protein SORase/DHQase on several separation materials with extracts from stroma compared with leaf extracts showed only one peak of enzymic activity originating from the stroma. (b) Polyacrylamide gel electrophoresis (PAGE) of these extracts followed by specific staining resulted in the same pattern without a band of extraplastidic enzyme. (c) In protoplast fractionation experiments it was shown that SORase/DHQase was present only in the soluble chloroplast protein fraction.

An improved purification procedure for SORase/DHQase from stroma of chloroplasts, yield 40%, 1600 times as pure, gave essentially one protein band on sodium dodecyl sulfate-PAGE. Our results demonstrate that both enzyme functions are carried out by a single polypeptide. Nondenaturing PAGE exhibited a pattern of four bands with SORase/DHQase showing that they differ in charge but not in their molecular weight. Molecular weight was determined to be 67 kilodaltons (gel filtration) and 59 kilodaltons (PAGE) for all four forms. It was proven they were not due to artifacts. The four forms show similar kinetic properties, their K_m and pH optima differing only very slightly. Response to some metabolites is reported.

     

Genetic analysis of Triticeae shikimate dehydrogenase

Starch gel electrophoresis and polyacrylamide gel isoelectric focusing (IEF) were used to investigate the genetic control of Triticeae shikimate dehydrogenase-1 (SKDH-1). Studies of wheat-alien species chromosome addition lines established thatSkdh-1 ofHordeum vulgare cv. Betzes is located in chromosome 5H,Skdh-V1 ofDasypyrum villosum in 5V,Skdh-R1 ofSecale cereale cvs. Dakold and King II in 5R, andSkdh-S ¹1 ofTriticum longissimum in 5S¹S. Also, the chromosomal locations of the genes that encode SKDH-1 inT. aestivum cv. Chinese Spring,T. umbellulatum, andS. cereale cv. Imperial, determined earlier using zone electrophoresis, were reconfirmed using IEF. Zone electrophoresis and IEF do not differ markedly in their ability to detect the expression of alienSkdh-1 genes in wheat-alien species chromosome addition lines. However, IEF may be superior to zone electrophoresis as a technique for detecting and analyzing SKDH-1 genetic variants within Triticeae species; among the species studied, IEF generally resolved two or more isozymes perSkdh-1 allele present, while zone electrophoresis resolved only one.Technical article No. 22791 of the Texas Agricultural Experiment Station. This paper is based upon research supported in part by the U.S. Department of Agriculture under Agreement No. 83-CRCR-1-1322.     

The role of quinate and shikimate in the metabolism of lactobacilli

The metabolism of (–)-quinate and shikimate by one heterofermentative strain,actobacillus pastorianus, and by one homofermentative strain,Lactobacillus plantarum, has been studied using growing and washed cells. Both organisms reduced quinate and shikimate under anaerobic conditions in the presence of suitable hydrogen donors including fructose, glucose andd(–) andl(+)-lactates. The end-product ofL.pastorianus metabolism was dihydroshikimate butL.plantarum carried the reduction a stage further tocis-3,4-dihydroxycyclohexanecarboxylate and formed, simultaneously, catechol. The enzymes involved in these reductions are induced; their importance in the metabolism of lactobacilli is discussed.     

Aggregation and separability of the shikimate pathway enzymes in yeasts

Gel filtration was employed to estimate the molecular weights and to determine possible physical aggregation of enzymes [5-dehydroquinate synthase (DHQ synthase), 5-dehydroquinase (DHQase, EC 4.2.1.10), shikimate: NADP oxidoreductase (EC 1.1.1.25), shikimate kinase (EC 2.7.1.71), 3-enolpyruvylshikimate 5-phosphate synthase (EPSP synthase)] in the shikimate pathway in eleven species of yeasts. The five enzymes were not aggregated in extracts of Hansenula henricii, H. fabianii, H. anomala, Candida utilis, Pichia guilliermondii, and Lodderomyces elongisporus. Two enzymes (DHQase and shikimate:NADP oxidoreductase) were not separable by this method and by ion exchange chromatography, and we conclude that they exist as an aggregate in these yeasts. Evidence is presented for an enzyme aggregate containing five activities, with a molecular weight of approximately 280,000 in Rhodosporidium spaerocarpum, Rh. toruloides, Rhodotorula rubra, Saccharomycopsis lipolytica, and Saccharomyces cerevisiae. Similarities between the enzymes in the shikimate pathway of plants, bacteria, and other fungi and those of investigated yeasts are discussed.     

Insights into the function of RifI2: Structural and biochemical investigation of a new shikimate dehydrogenase family protein

The shikimate dehydrogenase (SDH) family consists of enzymes with diverse roles in secondary metabolism. The two most widespread members of the family, AroE and YdiB, function in amino acid biosynthesis and quinate catabolism, respectively. Here, we have determined the crystal structure of an SDH homolog belonging to the RifI class, a group of enzymes with proposed roles in antibiotic biosynthesis. The structure of RifI2 from Pseudomonas putida exhibits a number of distinctive features, including a substantial C-terminal truncation and an atypical mode of oligomerization. The active site of the enzyme contains substrate- and cofactor-binding motifs that are significantly different from those of any previously characterized member of the SDH family. These features are reflected in the novel kinetic properties of the enzyme. RifI2 exhibits much lower activity using shikimate as a substrate than AroE, and a strong preference for NAD⁺ instead of NADP⁺ as a cofactor. Moreover, the enzyme has only trace activity using quinate, unlike YdiB. Cocrystallization of RifI2 with NAD⁺ provided the opportunity to determine the mode of cofactor selectivity employed by the enzyme. We complemented this analysis by probing the role of a strictly conserved residue in the cofactor-binding domain, Asn193, by site directed mutagenesis. This study presents the first crystal structure and formal kinetic characterization of a new NAD⁺-dependent member of the SDH family.     

Evolutionary origins of the eukaryotic shikimate pathway: gene fusions, horizontal gene transfer, and endosymbiotic replacements

Currently the shikimate pathway is reported as a metabolic feature of prokaryotes, ascomycete fungi, apicomplexans, and plants. The plant shikimate pathway enzymes have similarities to prokaryote homologues and are largely active in chloroplasts, suggesting ancestry from the plastid progenitor genome. Toxoplasma gondii, which also possesses an alga-derived plastid organelle, encodes a shikimate pathway with similarities to ascomycete genes, including a five-enzyme pentafunctional arom. These data suggests that the shikimate pathway and the pentafunctional arom either had an ancient origin in the eukaryotes or was conveyed by eukaryote-to-eukaryote horizontal gene transfer (HGT). We expand sampling and analyses of the shikimate pathway genes to include the oomycetes, ciliates, diatoms, basidiomycetes, zygomycetes, and the green and red algae. Sequencing of cDNA from Tetrahymena thermophila confirmed the presence of a pentafused arom, as in fungi and T. gondii. Phylogenies and taxon distribution suggest that the arom gene fusion event may be an ancient eukaryotic innovation. Conversely, the Plantae lineage (represented here by both Viridaeplantae and the red algae) acquired different prokaryotic genes for all seven steps of the shikimate pathway. Two of the phylogenies suggest a derivation of the Plantae genes from the cyanobacterial plastid progenitor genome, but if the full Plantae pathway was originally of cyanobacterial origin, then the five other shikimate pathway genes were obtained from a minimum of two other eubacterial genomes. Thus, the phylogenies demonstrate both separate HGTs and shared derived HGTs within the Plantae clade either by primary HGT transfer or secondarily via the plastid progenitor genome. The shared derived characters support the holophyly of the Plantae lineage and a single ancestral primary plastid endosymbiosis. Our analyses also pinpoints a minimum of 50 gene/domain loss events, demonstrating that loss and replacement events have been an important process in eukaryote genome evolution.