Thiamine Biosynthesis in <i>Escherichia coli</i>: Identification of the Intermediate and By‐Product Derived from Tyrosine

Kriek, Marco; Martins, Filipa; Challand, Martin R.; Croft, Anna K.; Roach, Peter L.

doi:10.1002/ange.200702554

Cited by 22 publications

(39 citation statements)

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“…[6,10] HydG has recently been shown to catalyze radical-mediated tyrosine cleavage and generate p-cresol, [11] which is a known fermentation product of several anaerobes. [14][15][16] It was hypothesized by Pilet et al [11] that two molecules of dehydroglycine condense at an FeS cluster on HydG and result in generation of the dithiolate ligand. [14][15][16] It was hypothesized by Pilet et al [11] that two molecules of dehydroglycine condense at an FeS cluster on HydG and result in generation of the dithiolate ligand.…”

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“…[11] Cyanide was detected and quantified after derivatization by a modification of the method of Tracqui et al [17] in which the cyanide anion reacts with naphthalene-2,3-dicarbaldehyde (NDA) 5 and a primary amine (either taurine (6) or N 1 ,N 1 -dimethylethane-1,2-diamine (7)), thus generating the fluorescent 1-cyanobenz[f]isoindole (CBI) derivatives 8 and 9 (Figure 1 A). To confirm that tyrosine is the source of cyanide, l-U-[ 13 C, 15 N]tyrosine was incubated with HydG, AdoMet, and dithionite. The components required for cyanide-forming activity were tyrosine, AdoMet, a reducing agent, and HydG.…”

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“…The components required for cyanide-forming activity were tyrosine, AdoMet, a reducing agent, and HydG. [15] Purified HydG protein was denatured, and approximately 10 mol % per mole glyoxylate was detected in the solution. In these experiments, 5'-methylthioadenosine/ Scheme 1.…”

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“…0 = (20 AE 2) 10 À4 s À1 ) is comparable to the observed activity of the closely related ThiH (for p-cresol formation, k cat. [15,16] ThiH, HydG, and NosL share the common feature of cleaving the C a ÀC b bond of aromatic amino acids (Scheme 2), and it is likely that these transformations proceed by related mechanisms, potentially resulting in a benzylic radical intermediate. [16] The ratio of DOA to p-cresol and cyanide was observed to be 1.3:1, indicating some uncoupled turnover.…”

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“…D) Mass spectrum of CBI derivative 9 from an assay mixture incubated with l-U-[ 13 C,15 N]tyrosine. A) Formation of cyanide from tyrosine by HydG and subsequent precolumn derivatization with amine 6 for fluorescent HPLC or amine 7 for LC-MS analysis.…”

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[FeFe]‐Hydrogenase Cyanide Ligands Derived From S‐Adenosylmethionine‐Dependent Cleavage of Tyrosine

et al. 2010

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Hydrogenases catalyze the reversible reduction of protons to yield molecular hydrogen (H 2 ) and occur in three evolutionarily unrelated forms termed the [Fe]-, [FeFe]-, and [NiFe]hydrogenases. [1,2] [FeFe]-hydrogenases all contain a complex active-site cofactor termed the H cluster (1, Scheme 1) that consists of a regular [4Fe4S] cluster bridged by a shared cysteine thiolate sulfur atom to a 2Fe subcluster with biologically unique carbon monoxide, cyanide, and dithiolate ligands. [3,4] The H cluster is biosynthesized in a stepwise process in which generalized host cell machinery [5] is directed towards the synthesis of a [4Fe4S] subcluster with subsequent synthesis and insertion of the 2Fe subcluster by specialized hyd encoded proteins, HydE, HydF, and HydG. [1,6] HydE and HydG are radical S-adenosylmethionine (AdoMet) enzymes thought to be responsible for the synthesis and proper incorporation of the nonprotein ligands. [1,[7][8][9] HydF has been proposed to function as a scaffold for assembly of the H cluster 2Fe subcluster and also to mediate its subsequent insertion into HydA. [6,10] HydG has recently been shown to catalyze radical-mediated tyrosine cleavage and generate p-cresol, [11] which is a known fermentation product of several anaerobes. [12,13] The HydG-catalyzed reaction is proposed to be similar to that catalyzed by the thiamine biosynthetic enzyme ThiH and to yield dehydroglycine as an intermediate. [14][15][16] It was hypothesized by Pilet et al. [11] that two molecules of dehydroglycine condense at an FeS cluster on HydG and result in generation of the dithiolate ligand. Herein, we demonstrate that cyanide is a product of HydGcatalyzed tyrosine cleavage, a result which clarifies the role of HydG and indicates that tyrosine is the source of the cyanide ligands in the H cluster.To investigate the reaction products formed by HydG, enzyme activity assays containing chemically reconstituted HydG (on average 5.1 AE 0.5 Fe per HydG), tyrosine, AdoMet, and sodium dithionite were prepared. At selected time points, assays were stopped by acidification, and the precipitated protein was removed by centrifugation. HPLC-based analysis methods were then used to measure the concentration of reaction products in the supernatant, thus confirming the turnover of AdoMet to yield deoxyadenosine (DOA), whilst tyrosine was cleaved to yield p-cresol. [11] Cyanide was detected and quantified after derivatization by a modification of the method of Tracqui et al. [17] in which the cyanide anion reacts with naphthalene-2,3-dicarbaldehyde (NDA) 5 and a primary amine (either taurine (6) or N 1 ,N 1 -dimethylethane-1,2-diamine (7)), thus generating the fluorescent 1-cyanobenz[f]isoindole (CBI) derivatives 8 and 9 (Figure 1 A). After derivatization, HPLC analysis showed a fluorescent peak which coeluted with a standard of the CBI derivative (Figure 1 B). The components required for cyanide-forming activity were tyrosine, AdoMet, a reducing agent, and HydG. Omission of any of these components resulted in loss of activity, consi...

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confidence: 99%

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confidence: 99%

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[FeFe]‐Hydrogenase Cyanide Ligands Derived From S‐Adenosylmethionine‐Dependent Cleavage of Tyrosine

et al. 2010

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Die Entschlüsselung der Biosynthese der schnellsten natürlichen Hydrogenase

Rauchfuss

2010

Angewandte Chemie

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[2] drei CO-Liganden und zwei Cyanid-Liganden (Schema 1). Die Biosynthesen dieser Liganden, abgesehen von dem 4Fe4S-Cluster, sind unbekannt. Diese Situation ändert sich jetzt rasch durch die Aufklärung des Reaktionswegs zu den Cyanidliganden. [3,4] Cyanid wird von Anorganikern als der "starke Ligand" schlechthin angesehen; es ist ein Bestandteil der ersten synthetischen Koordinationsverbindung, Berliner Blau. Der Ligand Cyanid steht an der Spitze vieler Listen, sei es bei den Stabilitätskonstanten seiner Komplexe oder in der spektrochemischen Reihe. Als Konsequenz hieraus handelt es sich bei Cyanid um ein starkes Gift, weil es wichtige Metalloenzyme der Atmungskette außer Kraft setzt.Industrielle Cyanidsynthesen beginnen mit der Platinkatalysierten Reaktion von Gemischen aus Ammoniak, Methan und Sauerstoff. Bei der gängigsten Variante dieses Verfahrens, einer Aza-analogen Dampfreformierung, wird Sauerstoff ausgeschlossen, und außerdem wird H 2 gebildet. Durch diesen Prozess werden jährlich mehr als eine Milliarde Kilogramm an HCN hergestellt. [5] Es sind viele Wege bekannt, die zu Metallcyaniden füh-ren, [6,7] was nicht überraschend ist, da es sich bei solchen Komplexen um äußerst stabile Verbindungen handelt. Typischerweise werden Metallcyanide ausgehend von Cyanidsalzen erhalten. Außerdem können sie aus vielen CN-haltigen Vorstufen wie Cyanwasserstoff, Dicyan, Halogencyanen und Thiocyanaten und sogar Nitrilen [8] und Isonitrilen [9] hervorgehen. Interessantere und weniger offensichtliche Wege zu Metallcyaniden gehen nicht von einer Vorstufe mit einer CN-Bindung aus. [Fe(CO) 5 ] reagiert zum Beispiel mit Ammoniak (und seinen ¾quivalenten [10] ) unter Bildung von Cyanidkomplexen. Es scheint, dass solche Reaktionen über Carbamoyl-Intermediate ablaufen. [7,11] Metallkomplexe von Methylaminen neigen zur oxidativen Dehydrierung zu den entsprechenden Cyanidkomplexen (Schema 2). [12] In Pflanzen und Gliederfüßern wird Cyanid ausgehend von mehreren Aminosäuren, aber nicht aus Glycin, produziert. Die Oxidation des Amins wird durch zwei verschiedene Enzyme der Cytochrom-P450-Familie katalysiert und führt zu a-Hydroxynitrilen. Diese Verbindungen oder ihre Derivate werden von ihren Wirten als HCN-Vorstufen für die Abwehr von Fressfeinden verwendet.[13] Bakterien hingegen erzeugen Cyanid im Allgemeinen aus Glycin [Gl. (1)

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Crystal Structure of Tryptophan Lyase (NosL): Evidence for Radical Formation at the Amino Group of Tryptophan

et al. 2014

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Streptomyces actuosus tryptophan lyase (NosL) is a radical SAM enzyme which catalyzes the synthesis of 3-methyl-2-indolic acid, a precursor in the synthesis of the promising antibiotic nosiheptide. The reaction involves cleavage of the tryptophan CaÀCb bond and recombination of the amino-acid-derived -COOH fragment at the indole ring. Reported herein is the 1.8 resolution crystal structure of NosL complexed with its substrate. Unexpectedly, only one of the tryptophan amino hydrogen atoms is optimally placed for H abstraction by the SAM-derived 5'-deoxyadenosyl radical. This orientation, in turn, rules out the previously proposed delocalized indole radical as the species which undergoes Ca À Cb bond cleavage. Instead, stereochemical considerations indicate that the reactive intermediate is a CNH tryptophanyl radical. A structure-based amino acid sequence comparison of NosL with the tyrosine lyases ThiH and HydG strongly suggests that an equivalent CNH radical operates in the latter enzymes.Scheme 1. Reaction catalyzed by NosL. The carboxylate fragment in MIA (depicted in bold) originates directly from tryptophan. [4]

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Thiamine Biosynthesis in Escherichia coli: Identification of the Intermediate and By‐Product Derived from Tyrosine

Cited by 22 publications

References 24 publications

[FeFe]‐Hydrogenase Cyanide Ligands Derived From S‐Adenosylmethionine‐Dependent Cleavage of Tyrosine

[FeFe]‐Hydrogenase Cyanide Ligands Derived From S‐Adenosylmethionine‐Dependent Cleavage of Tyrosine

Die Entschlüsselung der Biosynthese der schnellsten natürlichen Hydrogenase

Crystal Structure of Tryptophan Lyase (NosL): Evidence for Radical Formation at the Amino Group of Tryptophan

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