Thiamine biosynthesis in Escherichia coli: Identification of the intermediate and by-product derived from tyrosine

DOI: 10.1002/anie.200702554

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Angewandte Chemie - International Edition p. 9223 - 9226 (2007)

Update date:2022-08-02

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Authors:

Kriek, Marco

Martins, Filipa

Challand, Martin R.

Croft, Anna

Roach, Peter L.

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Article abstract of DOI:10.1002/anie.200702554

(Chemical Equation Presented) In anaerobic organisms such as E. coli the tyrosine lyase ThiH is essential for the biosynthesis of the thiazole moiety of the vitamin thiamine. ThiH is a member of the radical AdoMet family. The products formed by cleavage of tyrosine in vitro have been identified and suggest a radical-mediated cleavage resulting in p-cresol and dehydroglycine which is hydrolyzed to glyoxylate.

Full text of DOI:10.1002/anie.200702554

Angewandte

Chemie

DOI: 10.1002/anie.200702554

Thiamine Biosynthesis

Thiamine Biosynthesis in Escherichia coli: Identification of the

Intermediate and By-Product Derived from Tyrosine**

Marco Kriek, Filipa Martins, Martin R. Challand, Anna Croft, and Peter L. Roach*

Thiamine is an essential cofactor in many metabolic pathways

and is required by all living organisms. The biosynthesis of

thiamine phosphate is convergent, linking two independently

biosynthesized heterocyclic precursors (Scheme 1, 6 and 7) in

(Scheme 1). This hydrolytically unstable intermediate is

incorporated into the thiazole 6 in a multistep reaction

requiring the thiazole synthase (ThiG),^[7]the sulfur donor

ThiFS thiocarboxylate,^[8,9]and 1-deoxyxylulose 5-phosphate

(Dxp, 5). Dehydroglycine 2 has been

proposed as the first common inter-

mediate for bacterial thiazole biosyn-

thesis,^[10]but microorganisms growing

under anaerobic conditions probably

cannot oxidize glycine. Instead anae-

robes, such as Escherichia coli, utilize

an alternative pathway forming dehy-

droglycine from tyrosine 1 in a ThiH-

dependent reaction.^[11,12]This biosyn-

thetic step therefore requires the

ꢀ

cleavage of the C_aC_bbond and

release of the aromatic side chain.

ThiH shows sequence similarity to the

“radical

S-adenosylmethionine

(AdoMet)” family of proteins,^[13]

including conserved ligands to an

essential [4Fe-4S] cluster^[14]and has

been shown to form a complex with

ThiG.^[15]Studies on the reconstitution

of the thiazole-forming reaction^[10,16]

with purified E. coli proteins have

Scheme 1. The biosynthesis of thiamine phosphate 8.

shown that ThiGH requires AdoMet

the final step.^[1–4]The biosynthesis of the thiazole moiety of

thiamine has been well characterized in the aerobe Bacillus

subtilis,^[4,5]which obtains the C2–N3 fragment of the thiazole

by oxidation of glycine 9 to dehydroglycine 2 using ThiO^[6]

and a reductant for activity, but (like other proteins in the

“radical Adomet” family^[17,18]) it is not catalytically active in

vitro. In this paper, we elucidate the reaction products formed

in vitro, demonstrating that the aromatic by-product derived

from the side chain of tyrosine is p-cresol and the remaining

fragment of tyrosine yields glyoxylate, the expected product

of in situ hydrolysis of dehydroglycine. Finally, these experi-

ments are shown to be relevant to in vivo metabolism by the

detection of p-cresol in E. coli that are expressing ThiH.

To follow the fate of tyrosine in the reaction, ¹⁴C-labeled

tyrosine was incubated with the heterodimeric ThiGH com-

plex, AdoMet and the natural electron-donor system consist-

ing of flavodoxin (FldA), flavodoxin reductase (Fpr), and

NADPH. After 2 h, the reaction was analyzed by thin-layer

chromatography (TLC) and monitored by autoradiography

(Figure 1). By using two TLC systems, two radiolabeled

products with very different polarities were observed. The

highly polar component co-eluted with glyoxylate (4 in

Scheme 1 and Figure 1A) and comparison of the less polar

product with several aromatic standards showed that it co-

eluted with p-cresol (3 in Scheme 1 and Figure 1B). This

experiment also demonstrated the absolute requirement for

AdoMet as the negative control showed no activity.

[*] Dr. M. Kriek, F. Martins, M. R. Challand, Dr. P. L. Roach

School of Chemistry

University of Southampton, Highfield

Southampton SO17 1BJ (UK)

Fax: (+44)2380-596-805

E-mail: plr2@soton.ac.uk

Homepage: http://www.soton.ac.uk /~roachweb/home/index.htm

Dr. A. Croft

School of Chemistry

University of Wales, Bangor

Gwynedd LL57 2UW (UK)

[**] This work was supported by grants from the Biotechnology and

Biological Sciences Research Council (M.K. and M.R.C.), the

Engineering and Physical Sciences Research Council (F.T.M.), and

the Royal Society (University Research Fellowship to P.L.R.). We

thank J. Street, N. Wells, G. J. Langley, and J. Herniman for help with

spectroscopy and spectrometry.

Supporting information for this article is available on the WWW

under http://www.angewandte.org or from the author.

Angew. Chem. Int. Ed. 2007, 46, 9223 –9226

ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

9223

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Communications

Figure 1. Autoradiograms of a ThiH assay. Two TLC systems (plates A

and B) were used to resolve the products. The complete assay (lane 1

on each plate) contained l-[U-¹⁴C]tyrosine, reconstituted ThiGH,

AdoMet, and a reductant. The negative control (lane 2) lacks AdoMet.

Spots corresponding to p-cresol, tyrosine, and glyoxylate standards are

indicated by C, Tyr, and G, respectively.

Figure 2. ¹³C NMR spectra of ThiGH reactions. The reaction mixtures

contained reconstituted ThiGH, l-[U-¹³C]tyrosine, AdoMet, and a

reductant, and were incubated at 378C for 2 h. Panel A shows the ¹³C

NMR spectrum of the supernatant obtained after the proteins were

removed by acid precipitation and centrifugation. Glyoxylate gives

signals at 173 and 86 ppm under these conditions, observed as

doublets in here because of the ¹³C labeling. Panel B shows the ¹³C

NMR spectrum of an organic extract prepared from the supernatant

and is consistent with ¹³C-labeled p-cresol [¹³C NMR (100 MHz; CDCl₃)

d=153.2 (t, J=66 Hz, COH), 130.1 (m, 2CH, CCH₃), 115.0 (m, 2CH),

20.4 ppm (m, CH₃)].

The formation of these two products was confirmed by

¹³C NMR spectra of reactions containing [U-¹³C]tyrosine.

After precipitation of the protein components, the spectrum

of the aqueous supernatant showed signals typical of glyox-

ylate (Figure 2A). The spectrum of an organic extract

prepared from this supernatant confirmed the formation of

p-cresol (Figure 2B). This sample was also analyzed by

GCMS, which showed the retention time (13.38 min) and

expected mass of p-cresol derived from [¹³C]tyrosine. Taken

together, the results demonstrate unequivocally that p-cresol

and glyoxylate were produced from tyrosine during in vitro

reactions.

The role of ThiH in the reaction can be explained by the

mechanism proposed in Scheme 2. The reductive cleavage of

AdoMet yields the highly reactive 5’-deoxyadenosyl radical,

which can abstract the phenolic hydrogen atom from tyrosine.

The resultant radical 10 may react by at least two pathways.

ꢀ

Homolytic cleavage of the C_aC_bbond yields the quinone

methide 11 and the radical 12. The alternative heterolytic

pathway leads directly to 2 and the stabilized radical anion

13a$13b. To form p-cresol and dehydroglycine by either of

these pathways requires further electron-transfer steps, which

may be facilitated by the proximity of the intermediates to the

[4Fe-4S] cluster in the active site of ThiH. DFT and ab initio

calculations suggest that the homolytic mechanism is ther-

Scheme 2. Proposed mechanism for ThiH. The 5’-deoxyadenosyl radical (DOAC) is derived from the reductive cleavage of AdoMet.

ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2007, 46, 9223 –9226

9224 www.angewandte.org

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Angewandte

Chemie

modynamically more favorable, with increased polarity

improving the relative stability of the heterolytic pathway,

but not enough for this to be identified as the preferred

pathway. Judicious placement of specific residues may there-

fore result in enough stabilization to tip this balance, and

further experimental data will be required to differentiate

between these pathways. The subsequent fate of the dehy-

droglycine 2 depends on the reaction conditions: if the

components required for thiazole formation are present

(including ThiG, the sulfur donor ThiFS thiocarboxylate,

and Dxp), then it will be incorporated into the thiazole

phosphate 6.^[10]If any of these components are absent, the

dehydroglycine is likely released from the ThiGH complex

into the aqueous medium, where it is rapidly hydrolyzed to

glyoxylate and ammonia.

the amount of 5’-deoxyadenosine formed after 2 h was

measured as 1.3 mol equivalents relative to p-cresol, suggest-

ing some uncoupled turnover of AdoMet had occurred.

To demonstrate the formation of p-cresol in vivo, we used

a mutant E. coli K strain that lacks a genomic copy of thiH.^[22]

Two derivative strains of this mutant were prepared, one

transformed with a ThiH expression plasmid^[15]and the other

with a control plasmid containing no thiamine biosynthetic

genes. These strains were cultured in defined medium for

24 h, and an organic extract was prepared from the cleared

medium. Comparison of the extracts by GCMS analysis

(Figure 4) revealed a new peak at 13.38 min, corresponding to

p-cresol, in the sample derived from the ThiH-expressing

cells. In contrast p-cresol was not detectable in the negative

control. Using cultures of an E. coli B strain and [3,3-

²H₂]tyrosine, White^[23]reported the isolation of 4-hydroxy-

benzyl alcohol as a by-product from thiamine biosynthesis, a

result that seemingly contradicts our observations. However,

ꢀ

ThiH is not unique in mediating this type of C C bond

cleavage of a phenolic compound. For example, Clostridium

difficile produces p-cresol by the decarboxylation of p-

hydroxyphenylacetate using a glycyl radical enzyme p-

hydroxyphenylacetate decarboxylase (HPAD).^[19–21]It seems

probable that ThiH and HPAD use different carbon-centered

radicals to initiate mechanistically related cleavage reactions.

To investigate the kinetics and stoichiometry of product

formation, the p-cresol and glyoxylate formed in a ThiH

reaction were quantified using HPLC over a two-hour period

(Figure 3). The reaction produced p-cresol and glyoxylate in

an approximate 1:1 ratio over the time course and approached

completion after 1 h (Table 1). In a subsequent experiment,

Figure 3. Time course of product formation in a ThiH assay. The graph

shows the increase in the concentration of p-cresol ( , dashed line)

and glyoxylate ( , solid line) with time. The assays, which contained

reconstituted ThiGH complex, tyrosine, AdoMet, and a reductant, were

incubated for the indicated time at 378C.

Table 1: Kinetic parameters for product formation. Data from Figure 3

were fitted to first-order processes to determine the calculated final

concentration of products and the apparent first-order rate constant.

Results are shown with standard errors.

Figure 4. Evidence for the ThiH-dependent formation of p-cresol in

vivo. A) Section of the GCMS chromatogram of an extract of medium

from E. coli cultures that express ThiH (solid line, displaced upwards

for clarity) and the negative control sample (no ThiH, dashed line).

The p-cresol peak at 13.38 min is marked. B) The mass spectrum

corresponding to the new peak at 13.38 min showing the expected M⁺

signal for p-cresol (m/z 108.1).

Product

Rate constant [10^ꢀ2min^ꢀ1

]

Final conc. [mm]

p-cresol

glyoxylate

4.6ꢁ0.7

6.3ꢁ1.1

176.2ꢁ7.8

171.6ꢁ7.9

Angew. Chem. Int. Ed. 2007, 46, 9223 –9226

ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.angewandte.org

9225

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Communications

[6] E. C. Settembre, P. C. Dorrestein, J. H. Park, A. M. Augustine,

T. P. Begley, S. E. Ealick, Biochemistry 2003 , 42 , 2971.

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Biol. 2004, 11, 1373.

[10] M. Kriek, F. Martins, R. Leonardi, S. A. Fairhurst, D. J. Lowe,

P. L. Roach, J. Biol. Chem. 2007, 282, 17413.

[11] R. H. White, F. B. Rudolph, Biochim. Biophys. Acta Gen. Subj.

1978, 542, 340.

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E. coli B strains (but not K strains) express an oxidase that

can accept p-cresol as a substrate.^[24]It is therefore probable

that tyrosine cleavage in E. coli produces p-cresol, but those

strains with a suitable oxidase convert p-cresol to p-hydroxy-

benzyl alcohol.

In conclusion, we have characterized the products of

tyrosine cleavage in the presence of the “radical Adomet”

protein ThiH, AdoMet, and a reductant. The side chain of

tyrosine is converted to p-cresol during this reaction. The

remaining fragment of tyrosine likely forms the intermediate

dehydroglycine, which can be combined with Dxp and a sulfur

donor during thiazole biosynthesis in vivo. During in vitro

experiments that lacked these other thiazole precursors,

glyoxylate was observed to accumulate from the hydrolysis

of the dehydroglycine.

[15] R. Leonardi, S. A. Fairhurst, M. Kriek, D. J. Lowe, P. L. Roach,

FEBS Lett. 2003 , 539 , 95.

[16] R. Leonardi, P. L. Roach, J. Biol. Chem. 2004 , 279 , 17054.

[17] N. B. Ugulava, C. J. Sacanell, J. T. Jarrett, Biochemistry 2001 , 40 ,

8352.

[18] R. M. Cicchillo, D. F. Iwig, A. D. Jones, N. M. Nesbitt, C.

Baleanu-Gogonea, M. G. Souder, L. Tu, S. J. Booker, Biochem-

istry 2004 , 43 , 6378.

[19] L. Yu, M. Blaser, P. I. Andrei, A. J. Pierik, T. Selmer, Biochem-

istry 2006 , 45 , 9584.

Received: June 12, 2007

Published online: October 30, 2007

Keywords: biosynthesis · enzyme mechanisms · radicals ·

thiamine

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9226 www.angewandte.org

ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Angew. Chem. Int. Ed. 2007, 46, 9223 –9226

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Article Doi

Thiamine biosynthesis in Escherichia coli: Identification of the intermediate and by-product derived from tyrosine

DOI: 10.1002/anie.200702554

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Authors:

Article abstract of DOI:10.1002/anie.200702554

Full text of DOI:10.1002/anie.200702554

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