A “Radical Dance” in Thiamin Biosynthesis: Mechanistic Analysis of the Bacterial Hydroxymethylpyrimidine Phosphate Synthase

Chatterjee, Abhishek; Hazra, Amrita B.; Abdelwahed, Sameh H.; Hilmey, David G.; Begley, Tadhg P.

doi:10.1002/anie.201003419

Cited by 48 publications

(52 citation statements)

References 20 publications

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“…The reduction of [4Fe–4S] 2+ to [4Fe–4S] + state in the process of the reaction was monitored by UV - vis spectroscopy. The decreasing shoulder at 420 nm and a remarkable shift at 360 nm indicate the presence of the [4Fe–4S] + cluster (Figure 3) as reported for other radical SAM enzymes, such as BtrN [24] in butirosin biosynthesis, ThiC [25] in thiamin pyrimidine biosynthesis and NosL [21] in nosiheptide biosynthesis.…”

Section: Resultssupporting

confidence: 72%

Discovery and Characterization of BlsE, a Radical S-Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway

Feng

Dai

et al. 2013

PLoS ONE

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BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe–4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe–4S]2+ cluster. Mutagenesis studies demonstrated that Cys31, Cys35, Cys38 in the C×××C×MC motif and Gly73, Gly74, Glu75, Pro76 in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met37 had little effect on its function. Our results indicate that BlsE represents a typical [4Fe–4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.

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Section: Resultssupporting

confidence: 72%

Discovery and Characterization of BlsE, a Radical S-Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway

Feng

Dai

et al. 2013

PLoS ONE

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“…In bacteria the thiazole is formed from deoxy-D-xylulose 5-phosphate, glycine (or tyrosine) and cysteine and the pyrimidine is formed from aminoimidazole ribotide 3 . In contrast, in S. cerevisiae the thiazole 7 is formed from NAD, glycine and an active site cysteine of the THI4 protein 4 and the the 4-amino-2-methyl-5-hydroxymethyl pyrimidine phosphate (HMP-P)( 10 ) is formed from histidine and pyridoxal phosphate (PLP) 5–9 .…”

mentioning

confidence: 99%

Thiamin Pyrimidine Biosynthesis in Candida albicans: A Remarkable Reaction between Histidine and Pyridoxal Phosphate

Lai

Huang²,

Fenwick³

et al. 2012

J. Am. Chem. Soc.

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In S. cerevisiae the thiamin pyrimidine is formed from histidine and pyridoxal phosphate. The origin of all of the pyrimidine atoms has been determined using labeling studies and suggests that the pyrimidine is formed using remarkable chemistry that is without chemical or biochemical precedent. Here we report the overexpression of the closely related Candida albicans pyrimidine synthase (THI5p) and the reconstitution and preliminary characterization of the enzymatic activity. A structure of the C. albicans THI5p shows PLP bound at the active site via an imine with Lys62 and His66 in close proximity to the PLP. Our data suggest that His66 of the THI5 protein is the histidine source for pyrimidine formation and that the pyrimidine synthase is a single turnover enzyme.

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“…The ThiC reaction uses SAM as a cosubstrate and is sensitive to fluctuations in CoA levels in vivo (14,23,24,50). Nutritional studies led to the hypothesis that one metabolic consequence of AICAR accumulation was decreased CoA levels.…”

Section: Discussionmentioning

confidence: 99%

Amino-4-Imidazolecarboxamide Ribotide Directly Inhibits Coenzyme A Biosynthesis in Salmonella enterica

Bazurto

Downs

2014

J Bacteriol

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Historically, cellular metabolism has been described in the context of discrete metabolic units (e.g., biosynthetic or degradative pathways), a perspective that facilitated efforts to understand metabolic components such as enzymes and metabolites. In reality, these units are integrated to form a complex metabolic network whose connectivity is mediated extensively by metabolites. Under appropriate circumstances (i.e., growth conditions or strain background), dynamic changes of intracellular metabolite levels can have significant impacts on cellular fitness, by providing unexpected mechanisms for survival (1, 2) or compromising growth. Dissecting metabolic integration in bacteria and understanding the diverse roles of metabolites can reveal paradigms that are conserved across phylogenetic kingdoms. Defining these paradigms provides insights into the consequences of perturbing the metabolic network that can be exploited for desired outcomes.

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A “Radical Dance” in Thiamin Biosynthesis: Mechanistic Analysis of the Bacterial Hydroxymethylpyrimidine Phosphate Synthase

Cited by 48 publications

References 20 publications

Discovery and Characterization of BlsE, a Radical S-Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway

Discovery and Characterization of BlsE, a Radical S-Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway

Thiamin Pyrimidine Biosynthesis in Candida albicans: A Remarkable Reaction between Histidine and Pyridoxal Phosphate

Amino-4-Imidazolecarboxamide Ribotide Directly Inhibits Coenzyme A Biosynthesis in Salmonella enterica

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