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Papers by Adelbert Bacher
Biochemical Journal, 1994
GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthes... more GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthesis. Its expression is regulated by interferon-gamma or kit ligand in a tissue-specific manner. Three different cDNA forms have been reported for human GTP cyclohydrolase I [Togari, Ichinose, Matsumoto, Fujita and Nagatsu (1992) Biochem. Biophys. Res. Commun. 187, 359-365]. We have isolated, from a human liver cDNA library, two clones which contained inserts identical with two of the cDNAs reported by Togari et al. (1992). The three open reading frames corresponding to all reported cDNA sequences were expressed in Escherichia coli. Only the recombinant protein corresponding to the longest reading frame catalysed the conversion of GTP into dihydroneopterin triphosphate. The proteins corresponding to the shorter reading frames failed to catalyse not only the generation of dihydroneopterin triphosphate but also the release of formate from GTP, an intermediate step of the reaction. Recombinan...
![Research paper thumbnail of Biosynthesis of Isoprenoids: Crystal Structure of the [4Fe–4S] Cluster Protein IspG](https://attachments.academia-assets.com/117307942/thumbnails/1.jpg)
](Journal of Molecular Biology, 2010
4-Diphosphocytidyl-2C-methyl-D-erythritol kinase, an essential enzyme in the nonmevalonate pathwa... more 4-Diphosphocytidyl-2C-methyl-D-erythritol kinase, an essential enzyme in the nonmevalonate pathway of isopentenyl diphosphate and dimethylallyl diphosphate biosynthesis, catalyzes the single ATP-dependent phosphorylation stage affording 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. The 2-Å resolution crystal structure of the Escherichia coli enzyme in a ternary complex with substrate and a nonhydrolyzable ATP analogue reveals the molecular determinants of specificity and catalysis. The enzyme subunit displays the ␣͞ fold characteristic of the galactose kinase͞ homoserine kinase͞mevalonate kinase͞phosphomevalonate kinase superfamily, arranged into cofactor and substrate-binding domains with the catalytic center positioned in a deep cleft between domains. Comparisons with related members of this superfamily indicate that the core regions of each domain are conserved, whereas there are significant differences in the substrate-binding pockets. The nonmevalonate pathway is essential in many microbial pathogens and distinct from the mevalonate pathway used by mammals. The high degree of sequence conservation of the enzyme across bacterial species suggests similarities in structure, specificity, and mechanism. Our model therefore provides an accurate template to facilitate the structure-based design of broad-spectrum antimicrobial agents. galactose kinase͞homoserine kinase͞mevalonate kinase͞ phosphomevalonate kinase ͉ enzyme mechanism ͉ nonmevalonate ͉ phosphorylation
Scientific Reports, 2021
Flavocoenzymes are nearly ubiquitous cofactors that are involved in the catalysis and regulation ... more Flavocoenzymes are nearly ubiquitous cofactors that are involved in the catalysis and regulation of a wide range of biological processes including some light-induced ones, such as the photolyase-mediated DNA repair, magnetoreception of migratory birds, and the blue-light driven phototropism in plants. One of the factors that enable versatile flavin-coenzyme biochemistry and biophysics is the fine-tuning of the cofactor’s frontier orbital by interactions with the protein environment. Probing the singly-occupied molecular orbital (SOMO) of the intermediate radical state of flavins is therefore a prerequisite for a thorough understanding of the diverse functions of the flavoprotein family. This may be ultimately achieved by unravelling the hyperfine structure of a flavin by electron paramagnetic resonance. In this contribution we present a rigorous approach to obtaining a hyperfine map of the flavin’s chromophoric 7,8-dimethyl isoalloxazine unit at an as yet unprecedented level of reso...
Biosynthesis of 5-hydroxybenzimidazolylcobamid (factor III) in Methanobacterium thermoautotrophicum
Journal of Biological Chemistry, 1991
Metabolites, 2016
13 CO 2 pulse-chase experiments monitored by high-resolution NMR spectroscopy and mass spectromet... more 13 CO 2 pulse-chase experiments monitored by high-resolution NMR spectroscopy and mass spectrometry can provide 13 C-isotopologue compositions in biosynthetic products. Experiments with a variety of plant species have documented that the isotopologue profiles generated with 13 CO 2 pulse-chase labeling are directly comparable to those that can be generated by the application of [U-13 C 6 ]glucose to aseptically growing plants. However, the application of the 13 CO 2 labeling technology is not subject to the experimental limitations that one has to take into account for experiments with [U-13 C 6 ]glucose and can be applied to plants growing under physiological conditions, even in the field. In practical terms, the results of biosynthetic studies with 13 CO 2 consist of the detection of pairs, triples and occasionally quadruples of 13 C atoms that have been jointly contributed to the target metabolite, at an abundance that is well above the stochastic occurrence of such multiples. Notably, the connectivities of jointly transferred 13 C multiples can have undergone modification by skeletal rearrangements that can be diagnosed from the isotopologue data. As shown by the examples presented in this review article, the approach turns out to be powerful in decoding the carbon topology of even complex biosynthetic pathways.
The EMBO Journal, 1997
Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the es... more Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the essential cofactor of aromatic amino acid hydroxylases and nitric oxide synthases. We have determined the crystal structure of mouse sepiapterin reductase by multiple isomorphous replacement at a resolution of 1.25 Å in its ternary complex with oxaloacetate and NADP. The homodimeric structure reveals a single-domain α/βfold with a central four-helix bundle connecting two seven-stranded parallel β-sheets, each sandwiched between two arrays of three helices. Ternary complexes with the substrate sepiapterin or the product tetrahydrobiopterin were studied. Each subunit contains a specific aspartate anchor (Asp258) for pterin-substrates, which positions the substrate side chain C1Јcarbonyl group near Tyr171 OH and NADP C4ЈN. The catalytic mechanism of SR appears to consist of a NADPH-dependent proton transfer from Tyr171 to the substrate C1Ј and C2Ј carbonyl functions accompanied by stereospecific side chain isomerization. Complex structures with the inhibitor N-acetyl serotonin show the indoleamine bound such that both reductase and isomerase activity for pterins is inhibited, but reaction with a variety of carbonyl compounds is possible. The complex structure with N-acetyl serotonin suggests the possibility for a highly specific feedback regulatory mechanism between the formation of indoleamines and pteridines in vivo.
Structure, 1995
Background: Tetrahydrobiopterin serves as the cofactor for enzymes involved in neurotransmitter b... more Background: Tetrahydrobiopterin serves as the cofactor for enzymes involved in neurotransmitter biosynthesis and as a regulatory factor in immune cell proliferation and the biosynthesis of melanin. The biosynthetic pathway to tetrahydrobiopterin consists of three steps starting from GTP. The initial reaction is catalyzed by GTP cyclohydrolase I (GTP-CH-I) and involves the chemically complex transformation of the purine into the pterin ring system. Results: The crystal structure of the Escherichia coli GTP-CH-I was solved by single isomorphous replacement and molecular averaging at 3.0 A resolution. The functional enzyme is a homodecameric complex with D. symmetry, forming a torus with dimensions 65 A x 100 A. The pentameric subunits are constructed via an unprecedented cyclic arrangement of the four-stranded antiparallel [3-sheets of the five monomers to form a 20-stranded antiparallel [3-barrel of 35 A diameter. Two pentamers are tightly associated by intercalation of two antiparallel helix pairs positioned close to the subunit N termini. The C-terminal domain of the GTP-CH-I monomer is topologically identical to a subunit of the homohexameric 6-pyruvoyl tetrahydropterin synthase, the enzyme catalyzing the second step in tetrahydrobiopterin biosynthesis. Conclusions: The active site of GTP-CH-I is located at the interface of three subunits. It represents a novel GTP-binding site, distinct from the one found in G proteins, with a catalytic apparatus that suggests involvement of histidines and, possibly, a cystine in the unusual reaction mechanism. Despite the lack of significant sequence homology between GTP-CH-I and 6-pyruvoyl tetrahydropterin synthase, the two proteins, which catalyze consecutive steps in tetrahydrobiopterin biosynthesis, share a common subunit fold and oligomerization mode. In addition, the active centres have an identical acceptor site for the 2-amino-4-oxo pyrimidine moiety of their substrates which suggests an evolutionarily conserved protein fold designed for pterin biosynthesis.
Structure, 1999
Background: Dihydroneopterin triphosphate (H 2 NTP) is the central substrate in the biosynthesis ... more Background: Dihydroneopterin triphosphate (H 2 NTP) is the central substrate in the biosynthesis of folate and tetrahydrobiopterin. Folate serves as a cofactor in amino acid and purine biosynthesis and tetrahydrobiopterin is used as a cofactor in amino acid hydroxylation and nitric oxide synthesis. In bacteria, H 2 NTP enters the folate biosynthetic pathway after nonenzymatic dephosphorylation; in vertebrates, H 2 NTP is used to synthesize tetrahydrobiopterin. The dihydroneopterin triphosphate epimerase of Escherichia coli catalyzes the inversion of carbon 2′ of H 2 NTP. Results: The crystal structure of the homo-octameric protein has been solved by a combination of multiple isomorphous replacement, Patterson search techniques and cyclic averaging and has been refined to a crystallographic R factor of 18.8% at 2.9 Å resolution. The enzyme is a torus-shaped, D 4 symmetric homo-octamer with approximate dimensions of 65 × 65 Å. Four epimerase monomers form an unusual 16-stranded antiparallel β barrel by tight association between the N-and C-terminal β strands of two adjacent subunits. Two tetramers associate in a head-to-head fashion to form the active enzyme complex. Conclusions: The folding topology, quaternary structure and amino acid sequence of epimerase is similar to that of the dihydroneopterin aldolase involved in the biosynthesis of the vitamin folic acid. The monomer fold of epimerase is also topologically similar to that of GTP cyclohydrolase I (GTP CH-1), 6-pyrovoyl tetrahydropterin synthase (PTPS) and uroate oxidase (UO). Despite a lack of significant sequence homology these proteins share a common subunit fold and oligomerize to form central β barrel structures employing different cyclic symmetry elements, D 4 , D 5 , D 3 and D 2 , respectively. Moreover, these enzymes have a topologically equivalent acceptor site for the 2-amino-4-oxo pyrimidine (2-oxo-4-oxo pyrimidine in uroate oxidase) moiety of their respective substrates.
Proceedings of the National Academy of Sciences, 2002
Isopentenyl diphosphate and dimethylallyl diphosphate serve as the universal precursors for the b... more Isopentenyl diphosphate and dimethylallyl diphosphate serve as the universal precursors for the biosynthesis of terpenes. Although their biosynthesis by means of mevalonate has been studied in detail, a second biosynthetic pathway for their formation by means of 1-deoxy- d -xylulose 5-phosphate has been discovered only recently in plants and certain eubacteria. Earlier in vivo experiments with recombinant Escherichia coli strains showed that exogenous 1-deoxy- d -xylulose can be converted into 1-hydroxy-2-methyl-2-( E )-butenyl 4-diphosphate by the consecutive action of enzymes specified by the xylB and ispCDEFG genes. This article describes the transformation of exogenous [U- 13 C 5 ]1-deoxy- d -xylulose into a 5:1 mixture of [U- 13 C 5 ]isopentenyl diphosphate and [U- 13 C 5 ]dimethylallyl diphosphate by an E. coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes.
Proceedings of the National Academy of Sciences, 2000
In many microorganisms, the putative orthologs of the Escherichia coli ygbB gene are tightly link... more In many microorganisms, the putative orthologs of the Escherichia coli ygbB gene are tightly linked or fused to putative orthologs of ygbP , which has been shown earlier to be involved in terpenoid biosynthesis. The ygbB gene of E. coli was expressed in a recombinant E. coli strain and was shown to direct the synthesis of a soluble, 17-kDa polypeptide. The recombinant protein was found to convert 4-diphosphocytidyl-2C-methyl- d -erythritol 2-phosphate into 2C-methyl- d -erythritol 2,4-cyclodiphosphate and CMP. The structure of the reaction product was established by NMR spectroscopy using 13 C-labeled substrate samples. The enzyme-catalyzed reaction requires Mn 2+ or Mg 2+ but no other cofactors. Radioactivity from [2- 14 C]2C-methyl- d -erythritol 2,4-cyclodiphosphate was diverted efficiently to carotenoids by isolated chromoplasts from Capsicum annuum and, thus, was established as an intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis. YgbB protein also ...
Proceedings of the National Academy of Sciences, 2000
The crystal structure of recombinant human GTP cyclohydrolase I was solved by Patterson search me... more The crystal structure of recombinant human GTP cyclohydrolase I was solved by Patterson search methods by using the coordinates of the Escherichia coli enzyme as a model. The human as well as bacterial enzyme were shown to contain an essential zinc ion coordinated to a His side chain and two thiol groups in each active site of the homodecameric enzymes that had escaped detection during earlier studies of the E. coli enzyme. The zinc ion is proposed to generate a hydroxyl nucleophile for attack of imidazole ring carbon atom eight of the substrate, GTP. It may also be involved in the hydrolytic release of formate from the intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3 H )-pyrimidinone 5′-triphosphate, and in the consecutive Amadori rearrangement of the ribosyl moiety.
Proceedings of the National Academy of Sciences, 1996
A cell culture of Taxus chinensis was established to produce the diterpene 2alpha,5alpha,10beta,1... more A cell culture of Taxus chinensis was established to produce the diterpene 2alpha,5alpha,10beta,14beta-tetra-acetoxy4 ++ +(20),11-taxadiene (taxuyunnanine C) in 2.6% (dry weight) yield. The incorporation of [U-13C6]glucose, [1-13C]glucose, and [1,2-13C2]acetate into this diterpene was analyzed by NMR spectroscopy. Label from [1,2-13C2]acetate was diverted to the four acetyl groups of taxuyunnanine C, but not to the taxane ring system. Label from [1-13C]glucose and [U-13C6]glucose was efficiently incorporated into both the taxane ring system and the acetyl groups. The four isoprenoid moieties of the diterpene showed identical labeling patterns. The analysis of long-range 13C13C couplings in taxuyunnanine C obtained from an experiment with [U-13C6]glucose documents the involvement of an intramolecular rearrangement in the biosynthesis of the isoprenoid precursor. The labeling patterns are inconsistent with the mevalonate pathway. The taxoid data share important features with the alter...
Plant Physiology, 2002
The seeds of cereals represent an important sink for metabolites during the accumulation of stora... more The seeds of cereals represent an important sink for metabolites during the accumulation of storage products, and seeds are an essential component of human and animal nutrition. Understanding the metabolic interconversions (networks) underpinning storage product formation could provide the foundation for effective metabolic engineering of these primary nutritional sources. In this paper, we describe the use of retrobiosynthetic nuclear magnetic resonance analysis to establish the metabolic history of the glucose (Glc) units of starch in maize (Zea mays) kernels. Maize kernel cultures were grown with [U-13C6]Glc, [U-13C12]sucrose, or [1,2-13C2]acetate as supplements. After 19 d, starch was hydrolyzed, and the isotopomer composition of the resulting Glc was determined by quantitative nuclear magnetic resonance analysis. [1,2-13C2]Acetate was not incorporated into starch. [U-13C6]Glc or [U-13C12]sucrose gave similar labeling patterns of polysaccharide Glc units, which were dominated by...
Plant Physiology, 2004
Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to... more Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to be cloned and characterized from plants. This enzyme catalyzes conversion of 7,8-dihydroneopterin (DHN) to 6-hydroxymethyl-7,8-dihydropterin, and is encoded by the folB gene in Escherichia coli. The E. coli FolB protein also mediates epimerization of DHN to 7,8-dihydromonapterin. Searches of the Arabidopsis genome detected three genes encoding substantially diverged FolB homologs (AtFolB1–3, sharing 57%–73% identity), for which cDNAs were isolated. A fourth cDNA specifying a FolB-like protein (LeFolB1) was obtained from tomato (Lycopersicon esculentum) by reverse transcription-PCR. When overproduced in E. coli, recombinant AtFolB1, AtFolB2, and LeFolB1 proteins all had both dihydroneopterin aldolase and epimerase activities, and carried out the aldol cleavage reaction on the epimerization product, 7,8-dihydromonapterin, as well as on DHN. AtFolB3, however, could not be expressed in acti...
Organic & Biomolecular Chemistry, 2008
Take-down policy If you believe that this document breaches copyright please contact us providing... more Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Nature Communications, 2012
The final step of the methylerythritol phosphate isoprenoid biosynthesis pathway is catalysed by ... more The final step of the methylerythritol phosphate isoprenoid biosynthesis pathway is catalysed by the iron-sulphur enzyme IspH, producing the universal precursors of terpenes: isopentenyl diphosphate and dimethylallyl diphosphate. Here we report an unforeseen reaction discovered during the investigation of the interaction of IspH with acetylene inhibitors by X-ray crystallography, mößbauer, and nuclear magnetic resonance spectroscopy. In addition to its role as a 2H + /2e − reductase, IspH can hydrate acetylenes to aldehydes and ketones via anti-markovnikov/markovnikov addition. The reactions only occur with the oxidised protein and proceed via η 1-o-enolate intermediates. one of these is characterized crystallographically and contains a C4 ligand oxygen bound to the unique, fourth iron in the 4Fe-4s cluster: this intermediate subsequently hydrolyzes to produce an aldehyde product. This unexpected side to IspH reactivity is of interest in the context of the mechanism of action of other acetylene hydratases, as well as in the design of antiinfectives targeting IspH.
The Journal of Organic Chemistry, 2006
The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development.... more The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2Cmethyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of g0.86. 13 C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13 C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.
Journal of Molecular Biology, 2003
This is a Full wwPDB X-ray Structure Validation Report for a publicly released PDB entry.
Journal of Molecular Biology, 2002
Elsevier Science Ltd.GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopter... more Elsevier Science Ltd.GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopterin 3 H-triphosphate, which is the ®rst committed precursor of tetrahydrofolate and tetrahydrobiopterin. The kinetically competent reaction intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone, was used as substrate for single turnover experiments monitored by multiwavelength photometry. The early reaction phase is characterized by the rapid appearance of an optical transient with an absorption maximum centred at 320. This species is likely to represent a Schiff base intermediate at the initial stage of the Amadori rearrangement of the carbohydrate side-chain. Deconvolution of the optical spectra suggested four linearly independent processes. A ®fth reaction step was attributed to photodecomposition of the enzyme product. Presteady state experiments were also performed with the H179A mutant which can catalyse a reversible conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone but is unable to form the ®nal product, dihydroneopterin triphosphate. Optical spectroscopy failed to detect any intermediate in the reversible reaction sequence catalysed by the mutant protein. The data obtained with the wild-type and mutant protein in conjunction with earlier quenched¯ow studies show that the enzyme-catalysed opening of the imidazole ring of GTP and the hydrolytic release of formate from the resulting formamide type intermediate are both rapid reactions by comparison with the subsequent rearrangement of the carbohydrate side-chain which precedes the formation of the dihydropyrazine ring of dihydroneopterin triphosphate.
Journal of Molecular Biology, 2001
Tetrahydrobiopterin, the cofactor required for hydroxylation of aromatic amino acids regulates it... more Tetrahydrobiopterin, the cofactor required for hydroxylation of aromatic amino acids regulates its own synthesis in mammals through feedback inhibition of GTP cyclohydrolase I. This mechanism is mediated by a regulatory subunit called GTP cyclohydrolase I feedback regulatory protein (GFRP). The 2.6 A Ê resolution crystal structure of rat GFRP shows that the protein forms a pentamer. This indicates a model for the interaction of mammalian GTP cyclohydrolase I with its regulator, GFRP. Kinetic investigations of human GTP cyclohydrolase I in complex with rat and human GFRP showed similar regulatory effects of both GFRP proteins.
Biochemical Journal, 1994
GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthes... more GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthesis. Its expression is regulated by interferon-gamma or kit ligand in a tissue-specific manner. Three different cDNA forms have been reported for human GTP cyclohydrolase I [Togari, Ichinose, Matsumoto, Fujita and Nagatsu (1992) Biochem. Biophys. Res. Commun. 187, 359-365]. We have isolated, from a human liver cDNA library, two clones which contained inserts identical with two of the cDNAs reported by Togari et al. (1992). The three open reading frames corresponding to all reported cDNA sequences were expressed in Escherichia coli. Only the recombinant protein corresponding to the longest reading frame catalysed the conversion of GTP into dihydroneopterin triphosphate. The proteins corresponding to the shorter reading frames failed to catalyse not only the generation of dihydroneopterin triphosphate but also the release of formate from GTP, an intermediate step of the reaction. Recombinan...
Journal of Molecular Biology, 2010
4-Diphosphocytidyl-2C-methyl-D-erythritol kinase, an essential enzyme in the nonmevalonate pathwa... more 4-Diphosphocytidyl-2C-methyl-D-erythritol kinase, an essential enzyme in the nonmevalonate pathway of isopentenyl diphosphate and dimethylallyl diphosphate biosynthesis, catalyzes the single ATP-dependent phosphorylation stage affording 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. The 2-Å resolution crystal structure of the Escherichia coli enzyme in a ternary complex with substrate and a nonhydrolyzable ATP analogue reveals the molecular determinants of specificity and catalysis. The enzyme subunit displays the ␣͞ fold characteristic of the galactose kinase͞ homoserine kinase͞mevalonate kinase͞phosphomevalonate kinase superfamily, arranged into cofactor and substrate-binding domains with the catalytic center positioned in a deep cleft between domains. Comparisons with related members of this superfamily indicate that the core regions of each domain are conserved, whereas there are significant differences in the substrate-binding pockets. The nonmevalonate pathway is essential in many microbial pathogens and distinct from the mevalonate pathway used by mammals. The high degree of sequence conservation of the enzyme across bacterial species suggests similarities in structure, specificity, and mechanism. Our model therefore provides an accurate template to facilitate the structure-based design of broad-spectrum antimicrobial agents. galactose kinase͞homoserine kinase͞mevalonate kinase͞ phosphomevalonate kinase ͉ enzyme mechanism ͉ nonmevalonate ͉ phosphorylation
Scientific Reports, 2021
Flavocoenzymes are nearly ubiquitous cofactors that are involved in the catalysis and regulation ... more Flavocoenzymes are nearly ubiquitous cofactors that are involved in the catalysis and regulation of a wide range of biological processes including some light-induced ones, such as the photolyase-mediated DNA repair, magnetoreception of migratory birds, and the blue-light driven phototropism in plants. One of the factors that enable versatile flavin-coenzyme biochemistry and biophysics is the fine-tuning of the cofactor’s frontier orbital by interactions with the protein environment. Probing the singly-occupied molecular orbital (SOMO) of the intermediate radical state of flavins is therefore a prerequisite for a thorough understanding of the diverse functions of the flavoprotein family. This may be ultimately achieved by unravelling the hyperfine structure of a flavin by electron paramagnetic resonance. In this contribution we present a rigorous approach to obtaining a hyperfine map of the flavin’s chromophoric 7,8-dimethyl isoalloxazine unit at an as yet unprecedented level of reso...
Biosynthesis of 5-hydroxybenzimidazolylcobamid (factor III) in Methanobacterium thermoautotrophicum
Journal of Biological Chemistry, 1991
Metabolites, 2016
13 CO 2 pulse-chase experiments monitored by high-resolution NMR spectroscopy and mass spectromet... more 13 CO 2 pulse-chase experiments monitored by high-resolution NMR spectroscopy and mass spectrometry can provide 13 C-isotopologue compositions in biosynthetic products. Experiments with a variety of plant species have documented that the isotopologue profiles generated with 13 CO 2 pulse-chase labeling are directly comparable to those that can be generated by the application of [U-13 C 6 ]glucose to aseptically growing plants. However, the application of the 13 CO 2 labeling technology is not subject to the experimental limitations that one has to take into account for experiments with [U-13 C 6 ]glucose and can be applied to plants growing under physiological conditions, even in the field. In practical terms, the results of biosynthetic studies with 13 CO 2 consist of the detection of pairs, triples and occasionally quadruples of 13 C atoms that have been jointly contributed to the target metabolite, at an abundance that is well above the stochastic occurrence of such multiples. Notably, the connectivities of jointly transferred 13 C multiples can have undergone modification by skeletal rearrangements that can be diagnosed from the isotopologue data. As shown by the examples presented in this review article, the approach turns out to be powerful in decoding the carbon topology of even complex biosynthetic pathways.
The EMBO Journal, 1997
Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the es... more Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the essential cofactor of aromatic amino acid hydroxylases and nitric oxide synthases. We have determined the crystal structure of mouse sepiapterin reductase by multiple isomorphous replacement at a resolution of 1.25 Å in its ternary complex with oxaloacetate and NADP. The homodimeric structure reveals a single-domain α/βfold with a central four-helix bundle connecting two seven-stranded parallel β-sheets, each sandwiched between two arrays of three helices. Ternary complexes with the substrate sepiapterin or the product tetrahydrobiopterin were studied. Each subunit contains a specific aspartate anchor (Asp258) for pterin-substrates, which positions the substrate side chain C1Јcarbonyl group near Tyr171 OH and NADP C4ЈN. The catalytic mechanism of SR appears to consist of a NADPH-dependent proton transfer from Tyr171 to the substrate C1Ј and C2Ј carbonyl functions accompanied by stereospecific side chain isomerization. Complex structures with the inhibitor N-acetyl serotonin show the indoleamine bound such that both reductase and isomerase activity for pterins is inhibited, but reaction with a variety of carbonyl compounds is possible. The complex structure with N-acetyl serotonin suggests the possibility for a highly specific feedback regulatory mechanism between the formation of indoleamines and pteridines in vivo.
Structure, 1995
Background: Tetrahydrobiopterin serves as the cofactor for enzymes involved in neurotransmitter b... more Background: Tetrahydrobiopterin serves as the cofactor for enzymes involved in neurotransmitter biosynthesis and as a regulatory factor in immune cell proliferation and the biosynthesis of melanin. The biosynthetic pathway to tetrahydrobiopterin consists of three steps starting from GTP. The initial reaction is catalyzed by GTP cyclohydrolase I (GTP-CH-I) and involves the chemically complex transformation of the purine into the pterin ring system. Results: The crystal structure of the Escherichia coli GTP-CH-I was solved by single isomorphous replacement and molecular averaging at 3.0 A resolution. The functional enzyme is a homodecameric complex with D. symmetry, forming a torus with dimensions 65 A x 100 A. The pentameric subunits are constructed via an unprecedented cyclic arrangement of the four-stranded antiparallel [3-sheets of the five monomers to form a 20-stranded antiparallel [3-barrel of 35 A diameter. Two pentamers are tightly associated by intercalation of two antiparallel helix pairs positioned close to the subunit N termini. The C-terminal domain of the GTP-CH-I monomer is topologically identical to a subunit of the homohexameric 6-pyruvoyl tetrahydropterin synthase, the enzyme catalyzing the second step in tetrahydrobiopterin biosynthesis. Conclusions: The active site of GTP-CH-I is located at the interface of three subunits. It represents a novel GTP-binding site, distinct from the one found in G proteins, with a catalytic apparatus that suggests involvement of histidines and, possibly, a cystine in the unusual reaction mechanism. Despite the lack of significant sequence homology between GTP-CH-I and 6-pyruvoyl tetrahydropterin synthase, the two proteins, which catalyze consecutive steps in tetrahydrobiopterin biosynthesis, share a common subunit fold and oligomerization mode. In addition, the active centres have an identical acceptor site for the 2-amino-4-oxo pyrimidine moiety of their substrates which suggests an evolutionarily conserved protein fold designed for pterin biosynthesis.
Structure, 1999
Background: Dihydroneopterin triphosphate (H 2 NTP) is the central substrate in the biosynthesis ... more Background: Dihydroneopterin triphosphate (H 2 NTP) is the central substrate in the biosynthesis of folate and tetrahydrobiopterin. Folate serves as a cofactor in amino acid and purine biosynthesis and tetrahydrobiopterin is used as a cofactor in amino acid hydroxylation and nitric oxide synthesis. In bacteria, H 2 NTP enters the folate biosynthetic pathway after nonenzymatic dephosphorylation; in vertebrates, H 2 NTP is used to synthesize tetrahydrobiopterin. The dihydroneopterin triphosphate epimerase of Escherichia coli catalyzes the inversion of carbon 2′ of H 2 NTP. Results: The crystal structure of the homo-octameric protein has been solved by a combination of multiple isomorphous replacement, Patterson search techniques and cyclic averaging and has been refined to a crystallographic R factor of 18.8% at 2.9 Å resolution. The enzyme is a torus-shaped, D 4 symmetric homo-octamer with approximate dimensions of 65 × 65 Å. Four epimerase monomers form an unusual 16-stranded antiparallel β barrel by tight association between the N-and C-terminal β strands of two adjacent subunits. Two tetramers associate in a head-to-head fashion to form the active enzyme complex. Conclusions: The folding topology, quaternary structure and amino acid sequence of epimerase is similar to that of the dihydroneopterin aldolase involved in the biosynthesis of the vitamin folic acid. The monomer fold of epimerase is also topologically similar to that of GTP cyclohydrolase I (GTP CH-1), 6-pyrovoyl tetrahydropterin synthase (PTPS) and uroate oxidase (UO). Despite a lack of significant sequence homology these proteins share a common subunit fold and oligomerize to form central β barrel structures employing different cyclic symmetry elements, D 4 , D 5 , D 3 and D 2 , respectively. Moreover, these enzymes have a topologically equivalent acceptor site for the 2-amino-4-oxo pyrimidine (2-oxo-4-oxo pyrimidine in uroate oxidase) moiety of their respective substrates.
Proceedings of the National Academy of Sciences, 2002
Isopentenyl diphosphate and dimethylallyl diphosphate serve as the universal precursors for the b... more Isopentenyl diphosphate and dimethylallyl diphosphate serve as the universal precursors for the biosynthesis of terpenes. Although their biosynthesis by means of mevalonate has been studied in detail, a second biosynthetic pathway for their formation by means of 1-deoxy- d -xylulose 5-phosphate has been discovered only recently in plants and certain eubacteria. Earlier in vivo experiments with recombinant Escherichia coli strains showed that exogenous 1-deoxy- d -xylulose can be converted into 1-hydroxy-2-methyl-2-( E )-butenyl 4-diphosphate by the consecutive action of enzymes specified by the xylB and ispCDEFG genes. This article describes the transformation of exogenous [U- 13 C 5 ]1-deoxy- d -xylulose into a 5:1 mixture of [U- 13 C 5 ]isopentenyl diphosphate and [U- 13 C 5 ]dimethylallyl diphosphate by an E. coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes.
Proceedings of the National Academy of Sciences, 2000
In many microorganisms, the putative orthologs of the Escherichia coli ygbB gene are tightly link... more In many microorganisms, the putative orthologs of the Escherichia coli ygbB gene are tightly linked or fused to putative orthologs of ygbP , which has been shown earlier to be involved in terpenoid biosynthesis. The ygbB gene of E. coli was expressed in a recombinant E. coli strain and was shown to direct the synthesis of a soluble, 17-kDa polypeptide. The recombinant protein was found to convert 4-diphosphocytidyl-2C-methyl- d -erythritol 2-phosphate into 2C-methyl- d -erythritol 2,4-cyclodiphosphate and CMP. The structure of the reaction product was established by NMR spectroscopy using 13 C-labeled substrate samples. The enzyme-catalyzed reaction requires Mn 2+ or Mg 2+ but no other cofactors. Radioactivity from [2- 14 C]2C-methyl- d -erythritol 2,4-cyclodiphosphate was diverted efficiently to carotenoids by isolated chromoplasts from Capsicum annuum and, thus, was established as an intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis. YgbB protein also ...
Proceedings of the National Academy of Sciences, 2000
The crystal structure of recombinant human GTP cyclohydrolase I was solved by Patterson search me... more The crystal structure of recombinant human GTP cyclohydrolase I was solved by Patterson search methods by using the coordinates of the Escherichia coli enzyme as a model. The human as well as bacterial enzyme were shown to contain an essential zinc ion coordinated to a His side chain and two thiol groups in each active site of the homodecameric enzymes that had escaped detection during earlier studies of the E. coli enzyme. The zinc ion is proposed to generate a hydroxyl nucleophile for attack of imidazole ring carbon atom eight of the substrate, GTP. It may also be involved in the hydrolytic release of formate from the intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3 H )-pyrimidinone 5′-triphosphate, and in the consecutive Amadori rearrangement of the ribosyl moiety.
Proceedings of the National Academy of Sciences, 1996
A cell culture of Taxus chinensis was established to produce the diterpene 2alpha,5alpha,10beta,1... more A cell culture of Taxus chinensis was established to produce the diterpene 2alpha,5alpha,10beta,14beta-tetra-acetoxy4 ++ +(20),11-taxadiene (taxuyunnanine C) in 2.6% (dry weight) yield. The incorporation of [U-13C6]glucose, [1-13C]glucose, and [1,2-13C2]acetate into this diterpene was analyzed by NMR spectroscopy. Label from [1,2-13C2]acetate was diverted to the four acetyl groups of taxuyunnanine C, but not to the taxane ring system. Label from [1-13C]glucose and [U-13C6]glucose was efficiently incorporated into both the taxane ring system and the acetyl groups. The four isoprenoid moieties of the diterpene showed identical labeling patterns. The analysis of long-range 13C13C couplings in taxuyunnanine C obtained from an experiment with [U-13C6]glucose documents the involvement of an intramolecular rearrangement in the biosynthesis of the isoprenoid precursor. The labeling patterns are inconsistent with the mevalonate pathway. The taxoid data share important features with the alter...
Plant Physiology, 2002
The seeds of cereals represent an important sink for metabolites during the accumulation of stora... more The seeds of cereals represent an important sink for metabolites during the accumulation of storage products, and seeds are an essential component of human and animal nutrition. Understanding the metabolic interconversions (networks) underpinning storage product formation could provide the foundation for effective metabolic engineering of these primary nutritional sources. In this paper, we describe the use of retrobiosynthetic nuclear magnetic resonance analysis to establish the metabolic history of the glucose (Glc) units of starch in maize (Zea mays) kernels. Maize kernel cultures were grown with [U-13C6]Glc, [U-13C12]sucrose, or [1,2-13C2]acetate as supplements. After 19 d, starch was hydrolyzed, and the isotopomer composition of the resulting Glc was determined by quantitative nuclear magnetic resonance analysis. [1,2-13C2]Acetate was not incorporated into starch. [U-13C6]Glc or [U-13C12]sucrose gave similar labeling patterns of polysaccharide Glc units, which were dominated by...
Plant Physiology, 2004
Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to... more Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to be cloned and characterized from plants. This enzyme catalyzes conversion of 7,8-dihydroneopterin (DHN) to 6-hydroxymethyl-7,8-dihydropterin, and is encoded by the folB gene in Escherichia coli. The E. coli FolB protein also mediates epimerization of DHN to 7,8-dihydromonapterin. Searches of the Arabidopsis genome detected three genes encoding substantially diverged FolB homologs (AtFolB1–3, sharing 57%–73% identity), for which cDNAs were isolated. A fourth cDNA specifying a FolB-like protein (LeFolB1) was obtained from tomato (Lycopersicon esculentum) by reverse transcription-PCR. When overproduced in E. coli, recombinant AtFolB1, AtFolB2, and LeFolB1 proteins all had both dihydroneopterin aldolase and epimerase activities, and carried out the aldol cleavage reaction on the epimerization product, 7,8-dihydromonapterin, as well as on DHN. AtFolB3, however, could not be expressed in acti...
Organic & Biomolecular Chemistry, 2008
Take-down policy If you believe that this document breaches copyright please contact us providing... more Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Nature Communications, 2012
The final step of the methylerythritol phosphate isoprenoid biosynthesis pathway is catalysed by ... more The final step of the methylerythritol phosphate isoprenoid biosynthesis pathway is catalysed by the iron-sulphur enzyme IspH, producing the universal precursors of terpenes: isopentenyl diphosphate and dimethylallyl diphosphate. Here we report an unforeseen reaction discovered during the investigation of the interaction of IspH with acetylene inhibitors by X-ray crystallography, mößbauer, and nuclear magnetic resonance spectroscopy. In addition to its role as a 2H + /2e − reductase, IspH can hydrate acetylenes to aldehydes and ketones via anti-markovnikov/markovnikov addition. The reactions only occur with the oxidised protein and proceed via η 1-o-enolate intermediates. one of these is characterized crystallographically and contains a C4 ligand oxygen bound to the unique, fourth iron in the 4Fe-4s cluster: this intermediate subsequently hydrolyzes to produce an aldehyde product. This unexpected side to IspH reactivity is of interest in the context of the mechanism of action of other acetylene hydratases, as well as in the design of antiinfectives targeting IspH.
The Journal of Organic Chemistry, 2006
The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development.... more The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2Cmethyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of g0.86. 13 C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13 C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.
Journal of Molecular Biology, 2003
This is a Full wwPDB X-ray Structure Validation Report for a publicly released PDB entry.
Journal of Molecular Biology, 2002
Elsevier Science Ltd.GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopter... more Elsevier Science Ltd.GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopterin 3 H-triphosphate, which is the ®rst committed precursor of tetrahydrofolate and tetrahydrobiopterin. The kinetically competent reaction intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone, was used as substrate for single turnover experiments monitored by multiwavelength photometry. The early reaction phase is characterized by the rapid appearance of an optical transient with an absorption maximum centred at 320. This species is likely to represent a Schiff base intermediate at the initial stage of the Amadori rearrangement of the carbohydrate side-chain. Deconvolution of the optical spectra suggested four linearly independent processes. A ®fth reaction step was attributed to photodecomposition of the enzyme product. Presteady state experiments were also performed with the H179A mutant which can catalyse a reversible conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone but is unable to form the ®nal product, dihydroneopterin triphosphate. Optical spectroscopy failed to detect any intermediate in the reversible reaction sequence catalysed by the mutant protein. The data obtained with the wild-type and mutant protein in conjunction with earlier quenched¯ow studies show that the enzyme-catalysed opening of the imidazole ring of GTP and the hydrolytic release of formate from the resulting formamide type intermediate are both rapid reactions by comparison with the subsequent rearrangement of the carbohydrate side-chain which precedes the formation of the dihydropyrazine ring of dihydroneopterin triphosphate.
Journal of Molecular Biology, 2001
Tetrahydrobiopterin, the cofactor required for hydroxylation of aromatic amino acids regulates it... more Tetrahydrobiopterin, the cofactor required for hydroxylation of aromatic amino acids regulates its own synthesis in mammals through feedback inhibition of GTP cyclohydrolase I. This mechanism is mediated by a regulatory subunit called GTP cyclohydrolase I feedback regulatory protein (GFRP). The 2.6 A Ê resolution crystal structure of rat GFRP shows that the protein forms a pentamer. This indicates a model for the interaction of mammalian GTP cyclohydrolase I with its regulator, GFRP. Kinetic investigations of human GTP cyclohydrolase I in complex with rat and human GFRP showed similar regulatory effects of both GFRP proteins.