Structural, Biochemical, and In Vivo Investigations of the Threonine Synthase from Mycobacterium tuberculosis

Covarrubias, Adrian Suarez; Högbom, Martin; Bergfors, Terese; Carroll, Paul; Mannerstedt, Karin; Oscarson, Stefan; Parish, Tanya; Jones, T. Alwyn; Mowbray, Sherry L.

doi:10.1016/j.jmb.2008.05.086

Cited by 19 publications

(27 citation statements)

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“…Structurally, these enzymes form fold type II PLP enzymes (4). Because TS is found only in bacteria (5), yeasts (6), and plants (4,7), it can be a target for developing antibacterial drugs (5). For this purpose, elucidation of the mechanism of action of TS is crucial.…”

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confidence: 99%

Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase

Murakawa

Machida

Hayashi

2011

Journal of Biological Chemistry

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Threonine synthase (TS), which is a pyridoxal 5-phosphate (PLP)-dependent enzyme, catalyzes the elimination of the ␥-phosphate group from O-phospho-L-homoserine (OPHS) and the subsequent addition of water at C␤ to form L-threonine. The catalytic course of TS is the most complex among the PLP enzymes, and it is an intriguing problem how the elementary steps are controlled in TS to carry out selective reactions. When L-vinylglycine was added to Thermus thermophilus HB8 TS in the presence of phosphate, L-threonine was formed with k cat and reaction specificity comparable with those when OPHS was used as the substrate. However, in the absence of phosphate or when sulfate was used in place of phosphate, only the side reaction product, ␣-ketobutyrate, was formed. Global analysis of the spectral changes in the reaction of TS with L-threonine showed that compared with the more acidic sulfate ion, the phosphate ion decreased the energy levels of the transition states of the addition of water at the C␤ of the PLP-␣-aminocrotonate aldimine (AC) and the transaldimination to form L-threonine. The x-ray crystallographic analysis of TS complexed with an analog for AC gave a distinct electron density assigned to the phosphate ion derived from the solvent near the C␤ of the analog. These results indicated that the phosphate ion released from OPHS by ␥-elimination acts as the base catalyst for the addition of water at C␤ of AC, thereby providing the basis of the reaction specificity. The phosphate ion is also considered to accelerate the protonation/ deprotonation at C␥. Threonine synthase (TS)2 catalyzes the last step of the Lthreonine biosynthesis, conversion of O-phospho-L-homoserine (OPHS) into L-threonine and inorganic phosphate (1). TS is a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme and, together with the L-and D-serine dehydratases, threonine dehydratase, tryptophan synthase, and cysteine synthase, constitutes the ␤-family of PLP enzymes (2, 3). Structurally, these enzymes form fold type II PLP enzymes (4). Because TS is found only in bacteria (5), yeasts (6), and plants (4, 7), it can be a target for developing antibacterial drugs (5). For this purpose, elucidation of the mechanism of action of TS is crucial.The reaction mechanism of TS is considered to be the most complicated one catalyzed by the PLP enzymes, proceeding through all the types of intermediates formed during the catalysis of the PLP enzymes (summarized in Scheme 1 (1, 3, 8, 9)). In this mechanism, OPHS reacts with the PLP-Lys aldimine (internal aldimine 1) to form the external aldimine (2) and liberates the side chain of the Lys residue (transaldimination). The intermediate 2 is then converted to the ketimine (3) via a 1,3-prototropic shift. The electron-withdrawing imino group promotes deprotonation at C␤, and after the formation of the enamine (4), the ␥-phosphate group is eliminated, yielding ␤,␥-unsaturated ketimine (5) and a phosphate ion. In 5, deprotonation at C4Ј of the cofactor and protonation at C␥ of the substrate moiety occur to form the PLP-...

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confidence: 99%

Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase

Murakawa

Machida

Hayashi

2011

Journal of Biological Chemistry

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“…Again, the structure of the complex suggests that the only residue capable of performing the role of protonating the phosphate ester oxygen is K281. This is reminiscent of the role proposed for lysine K69 in the PLP-dependent threonine synthase, [16] which also eliminates phosphate from its substrate (l-homoserine phosphate), although its structural fold (Type II) is very different.…”

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confidence: 85%

Structural Basis for Phospholyase Activity of a Class III Transaminase Homologue

et al. 2016

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Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a remarkable diversity of chemical reactions in nature. A1RDF1 from Arthrobacter aurescens TC1 is a fold type I, PLP-dependent enzyme in the class III transaminase (TA) subgroup. Despite sharing 28 % sequence identity with its closest structural homologues, including b-alanine:pyruvate and g-aminobutyrate:a-ketoglutarate TAs, A1RDF1 displayed no TA activity. Activity screening revealed that the enzyme possesses phospholyase (E.C. 4.2.3.2) activity towards O-phosphoethanolamine (PEtN), an activity described previously for vertebrate enzymes such as human AGXT2L1, enzymes for which no structure has yet been reported. In order to shed light on the distinctive features of PLP-dependent phospholyases, structures of A1RDF1 in complex with PLP (internal aldimine) and PLP·PEtN (external aldimine) were determined, revealing the basis of substrate binding and the structural factors that distinguish the enzyme from class III homologues that display TA activity.Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a wide range of chemical reactions, including the racemisation and decarboxylation of amino acids and transamination between amino acid donors and keto acid acceptors, [1,2] and new reactions, including oxidations, [3] continue to be discovered. Some of these enzymes, notably those of the "PLP fold type I" and belonging to the class III transaminase (TA) subgroup, have become extremely useful in biotechnology, because some members possess the ability to form chiral amines from ketone precursors, [4] whereas others catalyse the useful racemisation of amino acid amides. [5,6] In a review in 2015, Steffen-Munsberg and co-workers also drew attention to more obscure and uncharacterised reactions of the class III transaminase group, [1] including roles as phospholyases (E.C. 4.2.3.2). Phospholyases had been identified and partially characterised in early work by Jones [7] and Faulkner [8] in strains of Erwinia and Pseudomonas.The Erwinia enzyme was reported to catalyse the transformation of phosphoethanolamine (PEtN, 1) to yield acetaldehyde (2), ammonia and phosphate (Scheme 1).The involvement of PLP in the elimination of phosphate from 1 has since been established for vertebrate enzymes such as human AGXT2L1 and AGXT2L2 by the groups of Schaftingen [9] and Peracchi.[10] These enzymes have a role in phospholipid metabolism, and are of interest as playing a role in neuropsychiatric disorders, [9] although no structure of such a PLPdependent phospholyase has yet been reported. As part of an ongoing study of the structural and catalytic diversity displayed by PLP-dependent enzymes, we cloned the complement of genes encoding predicted transaminase enzymes from the bacterium Arthrobacter aurescens TC1 [11] into Escherichia coli, and many of the genes were expressed in the soluble fraction. From detailed bioinformatics analysis of this enzyme complement by previously described methods, [1] and from direct comparison with the AGXT2L1 sequence, [9,10] it was predicted that th...

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“…Owing to its potential significance for the development of antibiotics to treat tuberculosis [114], the class I TS from M. tuberculosis has been studied in detail [115]. In the resting state, the PLP cofactor forms a Schiff base with Lys69.…”

Section: Threonine Synthasementioning

confidence: 99%

“…While Lys69 is well placed to undertake the initial proton transfers, the molecular gymnastics required to also mediate the latter proton transfer is beyond its reach and there has been debate in the literature about the catalytic group required for this chemistry. Recent detailed structural studies show that the most likely acid-base catalyst is the 5 0 -phosphate of the PLP cofactor (Figure 1.35) [115]. This phosphate moiety is less than 5 Å away from both the benzylic position of the PLP and the c-carbon of the substrate.…”

Section: Threonine Synthasementioning

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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Structural, Biochemical, and In Vivo Investigations of the Threonine Synthase from Mycobacterium tuberculosis

Cited by 19 publications

References 55 publications

Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase

Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase

Structural Basis for Phospholyase Activity of a Class III Transaminase Homologue

Amino Acid Biosynthesis

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