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1.
Evolution of a secondary metabolic pathway from primary metabolism: shikimate and quinate biosynthesis in plants
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Yuriko Carrington Jia Guo Cuong H. Le Alexander Fillo Junsu Kwon Lan T. Tran Jürgen Ehlting 《The Plant journal : for cell and molecular biology》2018,95(5):823-833
The shikimate pathway synthesizes aromatic amino acids essential for protein biosynthesis. Shikimate dehydrogenase (SDH) is a central enzyme of this primary metabolic pathway, producing shikimate. The structurally similar quinate is a secondary metabolite synthesized by quinate dehydrogenase (QDH). SDH and QDH belong to the same gene family, which diverged into two phylogenetic clades after a defining gene duplication just prior to the angiosperm/gymnosperm split. Non‐seed plants that diverged before this duplication harbour only a single gene of this family. Extant representatives from the chlorophytes (Chlamydomonas reinhardtii), bryophytes (Physcomitrella patens) and lycophytes (Selaginella moellendorfii) encoded almost exclusively SDH activity in vitro. A reconstructed ancestral sequence representing the node just prior to the gene duplication also encoded SDH activity. Quinate dehydrogenase activity was gained only in seed plants following gene duplication. Quinate dehydrogenases of gymnosperms, represented here by Pinus taeda, may be reminiscent of an evolutionary intermediate since they encode equal SDH and QDH activities. The second copy in P. taeda maintained specificity for shikimate similar to the activity found in the angiosperm SDH sister clade. The codon for a tyrosine residue within the active site displayed a signature of positive selection at the node defining the QDH clade, where it changed to a glycine. Replacing the tyrosine with a glycine in a highly shikimate‐specific angiosperm SDH was sufficient to gain some QDH function. Thus, very few mutations were necessary to facilitate the evolution of QDH genes. 相似文献
2.
Jia Guo ) Yuriko Carrington Annette Alber Jürgen Ehlting 《The Journal of biological chemistry》2014,289(34):23846-23858
The shikimate pathway leads to the biosynthesis of aromatic amino acids essential for protein biosynthesis and the production of a wide array of plant secondary metabolites. Among them, quinate is an astringent feeding deterrent that can be formed in a single step reaction from 3-dehydroquinate catalyzed by quinate dehydrogenase (QDH). 3-Dehydroquinate is also the substrate for shikimate biosynthesis through the sequential actions of dehydroquinate dehydratase (DQD) and shikimate dehydrogenase (SDH) contained in a single protein in plants. The reaction mechanism of QDH resembles that of SDH. The poplar genome encodes five DQD/SDH-like genes (Poptr1 to Poptr5), which have diverged into two distinct groups based on sequence analysis and protein structure prediction. In vitro biochemical assays proved that Poptr1 and -5 are true DQD/SDHs, whereas Poptr2 and -3 instead have QDH activity with only residual DQD/SDH activity. Poplar DQD/SDHs have distinct expression profiles suggesting separate roles in protein and lignin biosynthesis. Also, the QDH genes are differentially expressed. In summary, quinate (secondary metabolism) and shikimate (primary metabolism) metabolic activities are encoded by distinct members of the same gene family, each having different physiological functions. 相似文献
3.
Pinus taeda 《Plant Physiology and Biochemistry》2000,38(12)
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. 相似文献
4.
《Bioscience, biotechnology, and biochemistry》2013,77(12):2438-2444
The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases. 相似文献
5.
Structural and biochemical approaches uncover multiple evolutionary trajectories of plant quinate dehydrogenases
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Artyom Gritsunov James Peek Julio Diaz Caballero David Guttman Dinesh Christendat 《The Plant journal : for cell and molecular biology》2018,95(5):812-822
Quinate is produced and used by many plants in the biosynthesis of chlorogenic acids (CGAs). Chlorogenic acids are astringent and serve to deter herbivory. They also function as antifungal agents and have potent antioxidant properties. Quinate is produced at a branch point of shikimate biosynthesis by the enzyme quinate dehydrogenase (QDH). However, little information exists on the identity and biochemical properties of plant QDHs. In this study, we utilized structural and bioinformatics approaches to establish a QDH‐specific primary sequence motif. Using this motif, we identified QDHs from diverse plants and confirmed their activity by recombinant protein production and kinetic assays. Through a detailed phylogenetic analysis, we show that plant QDHs arose directly from bifunctional dehydroquinate dehydratase–shikimate dehydrogenases (DHQD‐SDHs) through different convergent evolutionary events, illustrated by our findings that eudicot and conifer QDHs arose early in vascular plant evolution whereas Brassicaceae QDHs emerged later. This process of recurrent evolution of QDH is further demonstrated by the fact that this family of proteins independently evolved NAD+ and NADP+ specificity in eudicots. The acquisition of QDH activity by these proteins was accompanied by the inactivation or functional evolution of the DHQD domain, as verified by enzyme activity assays and as reflected in the loss of key DHQD active site residues. The implications of QDH activity and evolution are discussed in terms of plant growth and development. 相似文献
6.
Shihomi Nishikura-Imamura Minenosuke Matsutani Chayatip Insomphun Alisa S. Vangnai Hirohide Toyama Toshiharu Yakushi Tetsuya Abe Osao Adachi Kazunobu Matsushita 《Applied microbiology and biotechnology》2014,98(7):2955-2963
Shikimate and 3-dehydroshikimate are useful chemical intermediates for the synthesis of various compounds, including the antiviral drug oseltamivir. Here, we show an almost stoichiometric biotransformation of quinate to 3-dehydroshikimate by an engineered Gluconobacter oxydans strain. Even under pH control, 3-dehydroshikimate was barely detected during the growth of the wild-type G. oxydans strain NBRC3244 on the medium containing quinate, suggesting that the activity of 3-dehydroquinate dehydratase (DHQase) is the rate-limiting step. To identify the gene encoding G. oxydans DHQase, we overexpressed the gox0437 gene from the G. oxydans strain ATCC621H, which is homologous to the aroQ gene for type II DHQase, in Escherichia coli and detected high DHQase activity in cell-free extracts. We identified the aroQ gene in a draft genome sequence of G. oxydans NBRC3244 and constructed G. oxydans NBRC3244 strains harboring plasmids containing aroQ and different types of promoters. All recombinant G. oxydans strains produced a significant amount of 3-dehydroshikimate from quinate, and differences between promoters affected 3-dehydroshikimate production levels with little statistical significance. By using the recombinant NBRC3244 strain harboring aroQ driven by the lac promoter, a sequential pH adjustment for each step of the biotransformation was determined to be crucial because 3-dehydroshikimate production was enhanced. Under optimal conditions with a shift in pH, the strain could efficiently produce a nearly equimolar amount of 3-dehydroshikimate from quinate. In the present study, one of the important steps to convert quinate to shikimate by fermenting G. oxydans cells was investigated. 相似文献
7.
Adachi O Ano Y Toyama H Matsushita K 《Bioscience, biotechnology, and biochemistry》2006,70(10):2579-2582
3-Dehydroshikimate was formed with a yield of 57-77% from quinate via 3-dehydroquinate by two successive enzyme reactions, quinoprotein quinate dehydrogenase (QDH) and 3-dehydroquinate dehydratase, in the cytoplasmic membranes of acetic acid bacteria. 3-Dehydroshikimate was then reduced to shikimate (SKA) with NADP-dependent SKA dehydrogenase (SKDH) from the same organism. When SKDH was coupled with NADP-dependent D-glucose dehydrogenase (GDH) in the presence of excess D-glucose as an NADPH re-generating system, SKDH continued to produce SKA until 3-dehydroshikimate added initially in the reaction mixture was completely converted to SKA. Based on the data presented, a strategy for high SKA production was proposed. 相似文献
8.
Acinetobacter calcoaceticus LMD 79.41 produced significant amounts of pyrrolo-quinoline quinone (PQQ) in its culture medium when grown on quinic acid or shikimic acid. Studies with LMD 79.41 and PQQ--mutants of this strain demonstrated that this organism contains an NAD(P)-independent quinate dehydrogenase (QDH) (EC 1.1.99.-), catalyzing the first degradation step of these compounds, and that the enzyme contains PQQ as a cofactor, i.e. is a quinoprotein. Synthesis of QDH was induced by protocatechuate and the enzyme appeared to be particle-bound. Acinetobacter lwoffi RAG-1 produced a quinoprotein QDH apoenzyme since growth on quinic acid only occurred in the presence of PQQ. The results obtained with the PQQ--mutants of strain LMD 79.41 also provided some insight into the regulation of PQQ biosynthesis and assemblage of quinoprotein enzymes in the periplasmic space. Since two species of Pseudomonas also contained a quinoprotein QDH, it is assumed that bacterial NAD(P)-independent quinate dehydrogenase is a quinoprotein.Abbreviations DCPIP
2,6-dichlorophenolindophenol 相似文献
9.
Takeshi Kubota Yuya Tanaka Norihiko Takemoto Akira Watanabe Kazumi Hiraga Masayuki Inui Hideaki Yukawa 《Molecular microbiology》2014,92(2):356-368
The qsu operon of Corynebacterium glutamicum comprises four genes (qsuABCD) that underpin the microorganism's quinate/shikimate utilization pathways. The genes encode enzymes that catalyse reactions at the metabolic branch point between the biosynthesis route for synthesis of aromatic compounds and the catabolic route for degradation of quinate and shikimate for energy production. A qsuR gene located immediately upstream of qsuA encodes a protein (QsuR) which activates the operon in the presence of quinate or shikimate. Three observations support chorismate, an intermediate of the biosynthesis route, as a direct effector of QsuR: First, induction of qsuA mRNA in the presence of either quinate or shikimate disappears upon deletion of the gene encoding chorismate synthase. Second, chorismate accumulates when the operon is induced. Third, a DNase I‐protected segment by QsuR is shortened in the presence of chorismate. The QsuR tetramer senses the accumulation of chorismate and activates qsu genes that promote the quinate/shikimate catabolic instead of the aromatic compounds biosynthetic route. Such chorismate‐dependent control of carbon flow has not been previously described. 相似文献
10.
Purification and characterization of membrane-bound quinoprotein quinate dehydrogenase 总被引:2,自引:0,他引:2
Adachi O Yoshihara N Tanasupawat S Toyama H Matsushita K 《Bioscience, biotechnology, and biochemistry》2003,67(10):2115-2123
Several bacterial strains carrying quinoprotein quinate dehydrogenase (QDH) were screened through acetic acid bacteria and other bacteria. Strong enzyme activity was found in the membrane fraction of Gluconobacter melanogenus IFO 3294, G. oxydans IFO 3292, G. oxydans IFO 3244, and some strains of Acinetobacter calcoaceticus. Interestingly, in the membrane fraction of A. calcoaceticus AC3, which is unable to produce pyrroloquinoline quinone (PQQ), fairly large amounts of apo-QDH were formed, and were converted to holo-QDH only by the addition of PQQ. It was difficult to detach PQQ from the holo-QDH by EDTA treatment, and EDTA treatment with apo-QDH prior to PQQ addition gave no significant holo-QDH. For QDH purification, Gluconobacter strains were not suitable due to the presence of huge amounts of quinohemoprotein alcohol dehydrogenase (ADH) in the same membrane, which was co-solubilized with QDH and disturbed purification of QDH. Purification of holo-QDH was done with Acinetobacter sp. SA1 instead, which contained no ADH. Apo-QDH was purified from A. aclcoaceticus AC3. This is the first report dealing with QDH purification, and two different criteria of QDH purification were given. A combination of two steps using butyl-Toyopearl and hydroxyapatite columns gave a highly purified holo-QDH which was monodispersed and showed enough purity, though the specific activity did not increase as much as expected. When QDH purification was done with A. calcoaceticus AC3 in the absence of PQQ, purified apo-QDH appeared to be a dimer, which was converted to the monomer on addition of PQQ. Since QDH was highly hydrophobic, one-step chromatography on a DEAE-Sepharose column was tried. Purified holo-QDH of higher specific activity was obtained with a higher yield. The molecular mass of QDH was estimated to be 88 kDa. There was no characteristic absorption spectrum with the purified QDH except for a small bump around 420 nm. QDH oxidized only quinate and shikimate so far examined. The optimal QDH activity was found at pH 6-7 when assayed with artificial electron acceptors. QDH was formed in the presence or absence of quinate in the culture medium, although stronger induction was usually observed in the presence of quinate. 相似文献
11.
Vangnai AS Toyama H De-Eknamkul W Yoshihara N Adachi O Matsushita K 《FEMS microbiology letters》2004,241(2):157-162
Quinoprotein quinate dehydrogenase (QDH) is a membrane-bound enzyme containing pyrroloquinoline quinone (PQQ) as the prosthetic group. QDH in Gluconobacter oxydans IFO3244 was found to be inducible by quinate and it is not constitutively expressed in the absence of quinate. The purification of holo-form of QDH to nearly homogeneity was achieved. The purified QDH appears to have two subunits of approximately 65 and 21 kDa, which could be the result of proteolysis of single polypeptide. Kinetic analysis indicated that the purified enzyme is much more specific to quinate than QDH from Acinetobacter calcoaceticus. The efficiency of the artificial electron acceptor was also determined. 相似文献
12.
Structurally diverse dehydroshikimate dehydratase variants participate in microbial quinate catabolism
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James Peek Joseph Roman Graham R. Moran Dinesh Christendat 《Molecular microbiology》2017,103(1):39-54
Quinate and shikimate can be degraded by a number of microbes. Dehydroshikimate dehydratases (DSDs) play a central role in this process, catalyzing the conversion of 3‐dehydroshikimate to protocatechuate, a common intermediate of aromatic degradation pathways. DSDs have applications in metabolic engineering for the production of valuable protocatechuate‐derived molecules. Although a number of Gram‐negative bacteria are known to catabolize quinate and shikimate, only limited information exists on the quinate/shikimate catabolic enzymes found in these organisms. Here, we have functionally and structurally characterized a putative DSD designated QuiC1, which is present in some pseudomonads. The QuiC1 protein is not related by sequence with previously identified DSDs from the Gram‐negative genus, Acinetobacter, but instead shows limited sequence identity in its N‐terminal half with fungal DSDs. Analysis of a Pseudomonas aeruginosa quiC1 gene knock‐out demonstrates that it is important for growth on either quinate or shikimate. The structure of a QuiC1 enzyme from P. putida reveals that the protein is a fusion of two distinct modules: an N‐terminal sugar phosphate isomerase‐like domain associated with DSD activity and a novel C‐terminal hydroxyphenylpyruvate dioxygenase‐like domain. The results of this study highlight the considerable diversity of enzymes that participate in quinate/shikimate catabolism in different microbes. 相似文献
13.
14.
Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase. 总被引:4,自引:0,他引:4
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The Escherichia coli aroE gene encoding shikimate dehydrogenase was sequenced. The deduced amino acid sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced protein. The complete polypeptide chain has 272 amino acid residues and has a calculated Mr of 29,380. E. coli shikimate dehydrogenase is homologous to the shikimate dehydrogenase domain of the fungal arom multifunctional enzymes and to the catabolic quinate dehydrogenase of Neurospora crassa. 相似文献
15.
Quinate grown cells of Rhodococcus rhodochrous N75 metabolized both quinate and shikimate via protocatechuate to succinate and acetyl CoA. The initial enzyme of the hydroaromatic pathway, quinate dehydrogenase was purified 188-fold to electrophoretic homogeneity. The enzyme is a monomer with a native relative molecular mass of 44,000 and is NAD-dependent. The enzyme is highly stereospecific with regard to hydroaromatic substrates, oxidising only the axial hydroxyl group at C-3 of (-)-isomers of quinate, shikimate, dihydroshikimate and t-3,t-4-dihydroxycyclohexane-c-1-carboxylate, but shows activity with several NAD analogues. 相似文献
16.
Molecular organisation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans 总被引:9,自引:0,他引:9
Alastair R. Hawkins Heather K. Lamb Melanie Smith John W. Keyte Clive F. Roberts 《Molecular & general genetics : MGG》1988,214(2):224-231
Summary The functional integrity of the QUTB gene (encoding quinate dehydrogenase) has been confirmed by transformation of a qutB mutant strain. The DNA sequence of the contiguous genes QUTD (quinate permease), QUTB and QUTG (function unknown) has been determined and analysed, together with that of QUTE (catabolic 3-dehydroquinase). The QUTB sequence shows significant homology with the shikimate dehydrogenase function of the complex AROM locus of Aspergillus nidulans, and with the QA-3 quinate dehydrogenase and QA-1S (repressor) genes of Neurospora crassa. The QUTD gene shows strong homology with the N. crassa QA-Y gene and QUTG with the QA-X gene. QUTD, QUTB, and QUTG, QUTE form two pairs of divergently transcribed genes, and conserved sequence motifs identified in the two common 5 non-coding regions show significant homology with UAS
GAL
and UAS
QA
sequences of the Saccharomyces cerevisiae and N. crassa Gal and QA systems. In addition, conserved 5 sequences homologous to the mammalian CAAT box are noted and a previously unreported conserved 22 nucleotide motif is presented. 相似文献
17.
Adachi O Tanasupawat S Yoshihara N Toyama H Matsushita K 《Bioscience, biotechnology, and biochemistry》2003,67(10):2124-2131
3-Dehydroquinate production from quinate by oxidative fermentation with Gluconobacter strains of acetic acid bacteria was analyzed for the first time. In the bacterial membrane, quinate dehydrogenase, a typical quinoprotein containing pyrroloquinoline quinone (PQQ) as the coenzyme, functions as the primary enzyme in quinate oxidation. Quinate was oxidized to 3-dehydroquinate with the final yield of almost 100% in earlier growth phase. Resting cells, dried cells, and immobilized cells or an immobilized membrane fraction of Gluconobacter strains were found to be useful biocatalysts for quinate oxidation. 3-Dehydroquinate was further converted to 3-dehydroshikimate with a reasonable yield by growing cells and also immobilized cells. Strong enzyme activities of 3-dehydroquinate dehydratase and NADP-dependent shikimate dehydrogenase were detected in the soluble fraction of the same organism and partially fractionated from each other. Since the shikimate pathway is remote from glucose in the metabolic pathway, the entrance into the shikimate pathway from quinate to 3-dehydroquinate looks advantageous to produce metabolic intermediates in the shikimate pathway. 相似文献
18.
Takeshi Kubota Yuya Tanaka Kazumi Hiraga Masayuki Inui Hideaki Yukawa 《Applied microbiology and biotechnology》2013,97(18):8139-8149
The function of three Corynebacterium glutamicum shikimate dehydrogenase homologues, designated as qsuD (cgR_0495), cgR_1216, and aroE (cgR_1677), was investigated. A disruptant of aroE required shikimate for growth, whereas a qsuD-deficient strain did not grow in medium supplemented with either quinate or shikimate as sole carbon sources. There was no discernible difference in growth rate between wild-type and a cgR_1216-deficient strain. Enzymatic assays showed that AroE both reduced 3-dehydroshikimate, using NADPH as cofactor, and oxidized shikimate, the reverse reaction, using NADP+ as cofactor. The reduction reaction was ten times faster than the oxidation. QsuD reduced 3-dehydroquinate using NADH and oxidized quinate using NAD+ as cofactor. Different from the other two homologues, the product of cgR_1216 displayed considerably lower enzyme activity for both the reduction and the oxidation. The catalytic reaction of QsuD and AroE was highly susceptible to pH. Furthermore, reduction of 3-dehydroshikimate by AroE was inhibited by high concentrations of shikimate, but neither quinate nor aromatic amino acids had any effect on the reaction. Expression of qsuD mRNA was strongly enhanced in the presence of shikimate, whereas that of cgR_1216 and aroE decreased. We conclude that while AroE is the main catalyst for shikimate production in the shikimate pathway, QsuD is essential for quinate/shikimate utilization. 相似文献
19.
A novel protein from Gluconobacter oxydans DSM2003 which shows 60–70% similarity with members of aldo–keto reductase (AKR) superfamily was overexpressed in Escherichia coli BL21 (DE3) and purified by one step affinity chromatography with a Ni-NTA agarose column. The recombinant protein (named
GOX0644) consists of 279 amino acids with an apparent molecular mass of 32 kDa in the soluble fraction, and the gene sequence
encoding the protein GOX0644 is 100% identical to the ORF of gox0644 in G. oxydans 621H (DSM2343). For a detailed analysis of its enzymatic activity, the substrate specificity of the recombinant protein GOX0644
was determined. With NADPH as a cofactor, GOX0644 exhibited better activity to aromatic aldehydes, especially o-chlorobenzaldehyde, compared to aliphatic aldehydes. It showed almost no activity toward glyceraldehyde, xylose, glucose,
and ketones. The protein was unable to oxidize primary- or secondary alcohols. Based on these results, GOX0644 was defined
as a novel NADPH-dependent aldehyde reductase. Kinetic parameters of the protein and the dependence of its activity on temperature
and pH were also determined. 相似文献
20.
cDNAs and genes encoding a hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase (CbRAS; rosmarinic acid synthase)
and a hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (CbHST) were isolated from Coleus blumei Benth. (syn. Solenostemon scutellarioides (L.) Codd; Lamiaceae). The proteins were expressed in E. coli and the substrate specificity of both enzymes was tested. CbRAS accepted several CoA-activated phenylpropenoic acids as donor
substrates and d-(hydroxy)phenyllactates as acceptors resulting in ester formation while shikimate and quinate were not accepted. Unexpectedly,
amino acids (d-phenylalanine, d-tyrosine, d-DOPA) also yielded products, showing that RAS can putatively catalyze amide formation. CbHST was able to transfer cinnamic,
4-coumaric, caffeic, ferulic as well as sinapic acid from CoA to shikimate but not to quinate or acceptor substrates utilized
by CbRAS. In addition, 3-hydroxyanthranilate, 3-hydroxybenzoate and 2,3-dihydroxybenzoate were used as acceptor substrates.
The reaction product with 3-aminobenzoate putatively is an amide. For both enzymes, structural requirements for donor and
acceptor substrates were deduced. The acceptance of unusual acceptor substrates by CbRAS and CbHST resulted in the formation
of novel compounds. The rather relaxed substrate as well as reaction specificity of both hydroxycinnamoyltransferases opens
up possibilities for the evolution of novel enzymes forming novel secondary metabolites in plants and for the in vitro formation
of new compounds with putatively interesting biological activities. 相似文献