Toward Understanding the Mechanism of the Complex Cyclization Reaction Catalyzed by Imidazole Glycerolphosphate Synthase:  Crystal Structures of a Ternary Complex and the Free Enzyme<sup>,</sup>

Chaudhuri, Barnali N.; Lange, Stephanie C.; Myers, Rebecca S.; Davisson, V. Jo; Smith, Janet L.

doi:10.1021/bi034320h

Cited by 71 publications

(121 citation statements)

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“…2) Oxyanion hole formation is a general principle among enzymes with catalytic triads stabilizing the transient negative charge during the hydrolytic reaction. Within the triad GATase family, the oxyanion hole has been conclusively described for ImGPS (43,52). In general, the oxyanion hole is formed by two amide nitrogens, one from the residues following the catalytic nucleophile and the second from an adjacent ␤-strand, referred to as the "oxyanion strand."…”

Section: Discussionmentioning

confidence: 99%

“…In general, the oxyanion hole is formed by two amide nitrogens, one from the residues following the catalytic nucleophile and the second from an adjacent ␤-strand, referred to as the "oxyanion strand." In Pdx2, these amide nitrogens are part of the peptide bonds between Cys 87 -Ala 88 and Gly 51 -Gly 52 . The carbonyl oxygen of the latter peptide bond points toward the putative oxyanion hole and obstructs it (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Vitamin B6 Biosynthesis by the Malaria Parasite Plasmodium falciparum

Gengenbacher

Fitzpatrick

Raschle

et al. 2006

Journal of Biological Chemistry

116

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Vitamin B6 is one of nature's most versatile cofactors. Most organisms synthesize vitamin B6 via a recently discovered pathway employing the proteins Pdx1 and Pdx2. Here we present an in-depth characterization of the respective orthologs from the malaria parasite, Plasmodium falciparum. Expression profiling of Pdx1 and -2 shows that blood-stage parasites indeed possess a functional vitamin B6 de novo biosynthesis. Recombinant Pdx1 and Pdx2 form a complex that functions as a glutamine amidotransferase with Pdx2 as the glutaminase and Pdx1 as pyridoxal-5-phosphate synthase domain. Complex formation is required for catalytic activity of either domain. Pdx1 forms a chimeric bi-enzyme with the bacterial YaaE, a Pdx2 ortholog, both in vivo and in vitro, although this chimera does not attain full catalytic activity, emphasizing that species-specific structural features govern the interaction between the protein partners of the PLP synthase complexes in different organisms. To gain insight into the activation mechanism of the parasite bi-enzyme complex, the three-dimensional structure of Pdx2 was determined at 1.62 Å . The obstruction of the oxyanion hole indicates that Pdx2 is in a resting state and that activation occurs upon Pdx1-Pdx2 complex formation.Plasmodium falciparum is the causative agent of severe malaria. Each year up to two million human deaths and enormous economic losses are attributed to this parasite. Drug resistance in P. falciparum has been aggravating the problem in many parts of the world during the last two decades, which considering the lack of a protective vaccine, is the major obstacle to combat the disease. Hence, new antimalarials are urgently needed. Requirements for nutrients and vitamins have previously been discussed as possible novel targets (1). Indeed the P. falciparum genome contains genes that encode enzymes necessary for the syntheses of the vitamin precursor chorismate (2-4), vitamin B6 (5, 6), and the vitaminlike cofactor lipoic acid (7).Vitamin B6 is renowned in the medical field as being involved in more bodily functions than any other single nutrient. It is required for the maintenance of physical as well as mental health. The term "vitamin B6" collectively refers to the vitamers pyridoxal, pyridoxine, and pyridoxamine, and their respective phosphate esters. The metabolically active form is pyridoxal 5Ј-phosphate (PLP), 6 an essential co-enzyme in numerous pathways such as amino acid metabolism and the biosynthesis of antibiotic compounds. In contrast to mammals, which have to take up vitamin B6 from their diet, bacteria, fungi, plants, and the protozoan P. falciparum have the ability to synthesize the vitamin de novo.Analyses of a number of available genomes has demonstrated that most organisms, including all archaea, fungi, plants, and protozoa and most eubacteria use a class I glutamine amidotransferase (GATase) composed of two domains, a glutaminase and its associated acceptor/ synthase domain to generate vitamin B6 (8 -13). Structural knowledge on class I GATases in genera...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Vitamin B6 Biosynthesis by the Malaria Parasite Plasmodium falciparum

Gengenbacher

Fitzpatrick

Raschle

et al. 2006

Journal of Biological Chemistry

116

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“…In analogy to the reactions catalyzed by imidazole glycerol-phosphate synthase (HisH/HisF) (16) and thiazole synthase (17), it has been hypothesized that a pentulose phosphate imine adduct occurs during the reaction sequence of Pdx1 and Pdx2 (11). The observation of the expected mass for such an adduct with the isolated recombinant protein corroborated this hypothesis.…”

mentioning

confidence: 89%

Reaction Mechanism of Pyridoxal 5′-Phosphate Synthase

Raschle

Arigoni

Brunisholz³

et al. 2007

Journal of Biological Chemistry

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Vitamin B6 is an essential metabolite in all organisms. De novo synthesis of the vitamin can occur through either of two mutually exclusive pathways referred to as deoxyxylulose 5-phosphate-dependent and deoxyxylulose 5-phosphate-independent. The latter pathway has only recently been discovered and is distinguished by the presence of two genes, Pdx1 and Pdx2, encoding the synthase and glutaminase subunit of PLP synthase, respectively. In the presence of ammonia, the synthase alone displays an exceptional polymorphic synthetic ability in carrying out a complex set of reactions, including pentose and triose isomerization, imine formation, ammonia addition, aldol-type condensation, cyclization, and aromatization, that convert C3 and C5 precursors into the cofactor B6 vitamer, pyridoxal 5-phosphate. Here, employing the Bacillus subtilis proteins, we demonstrate key features along the catalytic path. We show that ribose 5-phosphate is the preferred C5 substrate and provide unequivocal evidence that the pent(ul)ose phosphate imine occurs at lysine 81 rather than lysine 149 as previously postulated. While this study was under review, corroborative crystallographic evidence has been provided for imine formation with the corresponding lysine group in the enzyme from Thermotoga maritima (Zein, F., Zhang, Y., Kang, Y.-N., Burns, K., Begley, T. P., and Ealick, S. E. (2006) Biochemistry 45, 14609 -14620). We have detected an unanticipated covalent reaction intermediate that occurs subsequent to imine formation and is dependent on the presence of Pdx2 and glutamine. This step most likely primes the enzyme for acceptance of the triose sugar, ultimately leading to formation of the pyridine ring. Two alternative structures are proposed for the chromophoric intermediate, both of which require substantial modifications of the proposed mechanism.Pyridoxal 5Ј-phosphate (PLP) 2 is an essential cofactor of many enzymes in all living systems. It is involved in amino acid and carbohydrate metabolism and has recently been implicated as an antioxidant with a potent ability to quench singlet oxygen and the superoxide anion (2-4). Two distinct pathways for its de novo biosynthesis have been identified (5-13). One, referred to as the DXP-dependent pathway, is found in a relatively small number of eubacteria and has been extensively studied in Escherichia coli. In this pathway, pyridoxine 5Ј-phosphate is derived from DXP and 4-phosphohydroxy-L-threonine (9, 10). The second pathway, referred to as DXP-independent, has only been identified recently and appears to be far more prevalent, i.e. in archaea, fungi, plants, and the majority of bacteria (2, 13). It is characterized by the presence of two genes, Pdx1 and Pdx2. The corresponding proteins function together as the glutamine amidotransferase, PLP synthase with Pdx2 as the glutaminase domain and Pdx1 as the acceptor domain. As a result of extensive labeling studies in yeast and biochemical analysis with the recombinant enzymes, the substrates of Pdx1 have recently been identified (11,12, 1...

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“…Following the Amadori rearrangement the indole ring is formed [289,290]. This step also is particularly interesting from a molecular evolution perspective.…”

Section: Histidine Biosynthesismentioning

confidence: 99%

Amino Acid Biosynthesis

Parker

Pratt

2010

Amino Acids, Peptides and Proteins in Organic Chemistry

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The ribosomal synthesis of proteins utilizes a family of 20 a-amino acids that are universally coded by the translation machinery; in addition, two further a-amino acids, selenocysteine and pyrrolysine, are now believed to be incorporated into proteins via ribosomal synthesis in some organisms. More than 300 other amino acid residues have been identified in proteins, but most are of restricted distribution and produced via post-translational modification of the ubiquitous protein amino acids [1]. The ribosomally encoded a-amino acids described here ultimately derive from a-keto acids by a process corresponding to reductive amination. The most important biosynthetic distinction relates to whether appropriate carbon skeletons are pre-existing in basic metabolism or whether they have to be synthesized de novo and this division underpins the structure of this chapter.There are a small number of a-keto acids ubiquitously found in core metabolism, notably pyruvate (and a related 3-phosphoglycerate derivative from glycolysis), together with two components of the tricarboxylic acid cycle (TCA), oxaloacetate and a-ketoglutarate (a-KG). These building blocks ultimately provide the carbon skeletons for unbranched a-amino acids of three, four, and five carbons, respectively. a-Amino acids with shorter (glycine) or longer (lysine and pyrrolysine) straight chains are made by alternative pathways depending on the available raw materials. The strategic challenge for the biosynthesis of most straight-chain amino acids centers around two issues: how is the a-amino function introduced into the carbon skeleton and what functional group manipulations are required to generate the diversity of side-chain functionality required for the protein function?The core family of straight-chain amino acids does not provide all the functionality required for proteins. a-Amino acids with branched side-chains are used for two purposes; the primary need is related to protein structural issues. Proteins fold into well-defined three-dimensional shapes by virtue of their amphipathic nature: a significant fraction of the amino acid side-chains are of low polarity and the hydrophobic effect drives the formation of ordered structures in which these side-chains are buried away from water. In contrast to the straight-chain amino

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Toward Understanding the Mechanism of the Complex Cyclization Reaction Catalyzed by Imidazole Glycerolphosphate Synthase: Crystal Structures of a Ternary Complex and the Free Enzyme^,

Cited by 71 publications

References 36 publications

Vitamin B6 Biosynthesis by the Malaria Parasite Plasmodium falciparum

Vitamin B6 Biosynthesis by the Malaria Parasite Plasmodium falciparum

Reaction Mechanism of Pyridoxal 5′-Phosphate Synthase

Amino Acid Biosynthesis

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Toward Understanding the Mechanism of the Complex Cyclization Reaction Catalyzed by Imidazole Glycerolphosphate Synthase: Crystal Structures of a Ternary Complex and the Free Enzyme,

Cited by 71 publications

References 36 publications

Vitamin B6 Biosynthesis by the Malaria Parasite Plasmodium falciparum

Vitamin B6 Biosynthesis by the Malaria Parasite Plasmodium falciparum

Reaction Mechanism of Pyridoxal 5′-Phosphate Synthase

Amino Acid Biosynthesis

Contact Info

Product

Resources

About

Toward Understanding the Mechanism of the Complex Cyclization Reaction Catalyzed by Imidazole Glycerolphosphate Synthase: Crystal Structures of a Ternary Complex and the Free Enzyme^,