Catalytic activity of the anaerobic tyrosine lyase required for thiamine biosynthesis in Escherichia coli

Article abstract of DOI:10.1074/jbc.M109.056606

Thiazole synthase in Escherichia coli is an αβ heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the Cα-Cβ bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. This reactive intermediate acts as one of three substrates for the thiazole cyclization reaction catalyzed by ThiG. ThiH is a radical S-adenosylmethionine (AdoMet) enzyme that utilizes a [4Fe-4S]⁺ cluster to reductively cleave AdoMet, forming methionine and a 5′-deoxyadenosyl radical. Analysis of the time-dependent formation of the reaction products 5′- deoxyadenosine (DOA) and p-cresol has demonstrated catalytic behavior of the tyrosine lyase. The kinetics of product formation showed a pre-steady state burst phase, and the involvement of DOA in product inhibition was identified by the addition of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase to activity assays. This hydrolyzed the DOA and changed the rate-determining step but, in addition, substantially increased the uncoupled turnover of AdoMet. Addition of glyoxylate and ammonium inhibited the tyrosine cleavage reaction, but the reductive cleavage of AdoMet continued in an uncoupled manner. Tyrosine analogues were incubated with ThiGH, which showed a strong preference for phenolic substrates. 4-Hydroxyphenylpropionic acid analogues allowed uncoupled AdoMet cleavage but did not result in further reaction (Cα-Cβ bond cleavage). The results of the substrate analogue studies and the product inhibition can be explained by a mechanistic hypothesis involving two reaction pathways, a product-forming pathway and a futile cycle.

Full text of DOI:10.1074/jbc.M109.056606

Enzymology:
Catalytic Activity of the Anaerobic
Tyrosine Lyase Required for Thiamine
Biosynthesis in Escherichia coli
Martin R. Challand, Filipa T. Martins and
Peter L. Roach
J. Biol. Chem. 2010, 285:5240-5248.
doi: 10.1074/jbc.M109.056606 originally published online November 18, 2009
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 8, pp. 5240–5248, February 19, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Catalytic Activity of the Anaerobic Tyrosine Lyase Required
□
S
for Thiamine Biosynthesis in Escherichia coli*
Received for publication, August 18, 2009, and in revised form, November 18, 2009 Published, JBC Papers in Press, November 18, 2009, DOI 10.1074/jbc.M109.056606
Martin R. Challand, Filipa T. Martins, and Peter L. Roach1
From the School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, United Kingdom
Thiazole synthase in Escherichia coli is an ␣␤ heterodimer of
ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the C␣–C␤
bond of tyrosine, generating p-cresol as a by-product, to form
dehydroglycine. This reactive intermediate acts as one of three
substrates for the thiazole cyclization reaction catalyzed by
ThiG. ThiH is a radical S-adenosylmethionine (AdoMet)
ylpyrimidine pyrophosphate 6 and the recently characterized
(4) 2-carboxy-4-methyl-5-(␤-hydroxyethyl)-thiazole phos-
phate (Fig. 1A, Thz-P carboxylate 4). Phosphorylation to TPP 7
by ThiL completes the biosynthesis of the catalytically active
vitamin (5). The 4-amino-5-hydroxymethylpyrimidine moiety
is formed in a complex radical mediated rearrangement of
5-aminoimidazole ribotide 5, in a reaction that requires ThiC, a
recent addition to the rapidly expanding radical S-adenosylme-
thionine (AdoMet) superfamily (6, 7). The formation of
4-methyl-5-(␤-hydroxyethyl) thiazole phosphate carboxylate
(Fig. 1B) requires the precursor 1-deoxyxyulose 5-phosphate 3,
a sulfur atom from cysteine, which is transferred via a thiocar-
boxyl terminus of the small protein, ThiS 12 (8, 9), and dehy-
droglycine 10, which serves as the first common intermediate
between aerobic and anaerobic thiazole biosynthetic pathways.
The aerobe Bacillus subtilis has been the model bacterium for
probing the mechanism of the thiazole ring-forming reaction,
catalyzed by ThiG (8, 10, 11). B. subtilis, like all other aerobic
prokaryotes, uses dioxygen to oxidize glycine to dehydroglycine
in a reaction catalyzed by ThiO (12). The pathway to this elec-
tron-deficient intermediate differs significantly in anaerobes
such as E. coli, which cannot oxidize glycine. As an alternative,
anaerobes use ThiH to produce dehydroglycine by the cleavage
of the C␣–C␤ bond of tyrosine. ThiH has been shown to belong
to the “radical AdoMet” family, with the characteristic iron-
sulfur cluster-binding motif (13–15), and in vitro studies on
Escherichia coli ThiGH have demonstrated the requirement for
AdoMet and a reductant for activity (16, 17).
؉
enzyme that utilizes a [4Fe-4S] cluster to reductively cleave
AdoMet, forming methionine and a 5ꢀ-deoxyadenosyl radical.
Analysis of the time-dependent formation of the reaction prod-
ucts 5ꢀ-deoxyadenosine (DOA) and p-cresol has demonstrated
catalytic behavior of the tyrosine lyase. The kinetics of product
formation showed a pre-steady state burst phase, and the
involvement of DOA in product inhibition was identified by the
addition of 5ꢀ-methylthioadenosine/S-adenosylhomocysteine
nucleosidase to activity assays. This hydrolyzed the DOA and
changed the rate-determining step but, in addition, substan-
tially increased the uncoupled turnover of AdoMet. Addition of
glyoxylate and ammonium inhibited the tyrosine cleavage reac-
tion, but the reductive cleavage of AdoMet continued in an
uncoupled manner. Tyrosine analogues were incubated with
ThiGH, which showed a strong preference for phenolic sub-
strates. 4-Hydroxyphenylpropionic acid analogues allowed
uncoupled AdoMet cleavage but did not result in further reac-
tion (C␣–C␤ bond cleavage). The results of the substrate ana-
logue studies and the product inhibition can be explained by a
mechanistic hypothesis involving two reaction pathways, a
product-forming pathway and a futile cycle.
The products of tyrosine cleavage in vitro have been charac-
terized as glyoxylate and p-cresol. The glyoxylate is proposed to
form as a result of the hydrolysis of dehydroglycine (18). The
reactivity of the intermediate dehydroglycine presents a poten-
tial problem, especially during its transfer from ThiH to ThiG
as the release of dehydroglycine into aqueous solution would
result in hydrolysis. The consequence of this wasteful outcome
is the net loss of 1 eq of AdoMet, tyrosine, and NADPH, with no
overall benefit. Product inhibition of ThiGH has recently been
observed with the products of AdoMet cleavage, DOA and
methionine. This product inhibition can be overcome by the
addition of 5Ј-methylthioadenosine/S-adenosylhomocysteine
nucleosidase (MTAN), which catalyzes the hydrolysis of DOA
to adenine and 5Ј-deoxyribose (19).
Thiamine pyrophosphate (TPP)2 is an essential cofactor for
several enzymes, including pyruvate decarboxylase, pyruvate
dehydrogenase, and transketolase (1). Prokaryotes and some
eukaryotes, such as yeast and plants, can biosynthesize TPP (2).
In prokaryotes, ThiE catalyzes the formation of thiamine
monophosphate by covalently linking two independently
formed heterocyclic precursors (3), 4-amino-5-hydroxymeth-
* This work was supported by the Biotechnology and Biological Sciences
Research Council, the Royal Society, and the University of Southampton.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S4 and Tables S1 and S2.
1 To whom correspondence should be addressed: School of Chemistry, Uni-
versity of Southampton, Highfield, Southampton SO17 1BJ, United King-
dom. Tel.: 44-2380-595919; E-mail: plr2@soton.ac.uk.
Here, we report the application of an in vitro activity assay for
ThiH-mediated tyrosine cleavage to demonstrate that ThiH
can undergo more than a single turnover when provided with
sufficient substrates and reductant. Tyrosine lyase activity of
ThiGH was found to exhibit a “burst phase” kinetic profile due
to release of the products becoming rate-limiting when under-
2 The abbreviations used are: TPP, thiamine pyrophosphate; AdoMet,
S-adenosylmethionine; HPLC, high pressure liquid chromatography; DOA,
5Ј-deoxyadenosine; MTAN, 5Ј-methylthioadenosine/S-adenosylhomocys-
teine nucleosidase; 4-HPPA, hydroxyphenylpropionic acid; 4-HPHPA, 4-hy-
droxyphenyl-␣-hydroxypropionic acid; MOPS, 4-morpholinepropanesul-
fonic acid.
5240 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 8•FEBRUARY 19, 2010

Catalytic Activity of ThiH
Methods—All proteins were puri-
fied and reconstituted, and activities
were assayed in an anaerobic glove
box maintained at less than 1 ppm
O2 (Belle Technology, Portesham,
UK). HPLC analysis and UV spectra
were recorded as described previ-
ously (17). Proteins were expressed
and purified as reported previously
(17, 20). ThiGH and ThiH were
further purified by Superdex 200 gel
filtration chromatography as fol-
lows; the protein was chemically
reconstituted as described previ-
ously (17) and concentrated to a vol-
ume of less than 5 ml using a 10-kDa
molecular mass cutoff filter (Milli-
pore). The concentrated protein
was then applied to a Superdex 200
gel filtration column (2.6 ϫ 60 cm)
pre-equilibrated in anaerobic buffer
A (50 mM MOPS (pH 7.7), 100 mM
NaCl, 12.5% (w/v) glycerol, 5 mM di-
thiothreitol). The column was
eluted with buffer A (ϳ100 ml) until
the A280 was observed to rise, at
which point 7.5-ml fractions were
collected. The ThiGH complex typ-
ically eluted in the first six fractions
and the monomeric ThiH in the fol-
lowing six fractions (supplemental
Fig. S1). An aliquot of each fraction
(150 ␮l) was retained for SDS-PAGE
and iron analysis, and the remainder
was stored at Ϫ80 °C in sealed 15-ml
FIGURE1. Biosynthesisofthiaminepyrophosphateinprokaryotes. A, biosyntheticenzymesandprecursors
of TPP in E. coli. B, proposed mechanism for formation of the thiazole moiety. The formation of 13 has been
characterized using ThiG from B. subtilis (8, 10, 11) and is proposed to be, along with dehydroglycine, a com-
mon intermediate in the biosynthetic pathway in all prokaryotes. The generation of dehydroglycine varies
between anaerobic and aerobic organisms. The thiazole moiety is coupled with 4-amino-5-hydroxymeth-
ylpyprimidine (HMP) pyrophosphate and phosphorylated to TPP in the later steps of the biosynthesis of TPP.
going multiple turnovers. The addition of MTAN was found to Falcon tubes for further investigation. Iron content of protein
alter this rate-limiting step in subsequent turnovers and samples was determined by the method of Fish (21).
changed the profile of the time course. Addition of MTAN also
In Vitro Assay for Tyrosine Lyase Activity—The two most
resulted in a significant increase in the amount of uncoupled concentrated fractions of either the ThiGH complex or mono-
AdoMet cleavage. Experiments with tyrosine analogues or meric ThiH (1–3 mg/ml, 15 ml) were thawed inside an anaero-
combinations of products indicated that AdoMet cleavage is bic glove box. To ensure a full complement of 4Fe-4S cluster,
coupled to the generation of a substrate radical, most likely by the protein was carefully reconstituted, first by the addition of
abstraction of the phenolic hydrogen atom. The experiments aqueous dithiothreitol solution (to a final concentration of 5
also suggest a potential mechanism for uncoupled turnover of mM). After 20 min of gentle mixing, 2.5 mol eq of FeCl3 (from a
AdoMet. These results clarify the likely site of substrate radical freshly prepared anaerobic 10 mM aqueous stock solution) were
formation and indicate the complex regulation required to effi- added dropwise, and after a further 15 min, 2.5 mol eq of Na2S
ciently form and successfully utilize dehydroglycine during thi- was added. The resulting solution was stirred at room temper-
amine biosynthesis.
ature for 1 h before concentrating the protein to a volume of 1
ml. The protein was then exchanged into buffer B (50 mM
MOPS (pH 7.5), 100 mM NaCl, and 5% (w/v) glycerol) via a
EXPERIMENTAL PROCEDURES
Materials—Reagents and materials were obtained from the Sephadex G-25 pre-packed column. Assays (150 ␮l) were pre-
following suppliers: far-UV HPLC-grade acetonitrile from pared in 1.6-ml microcentrifuge tubes by addition of compo-
Fisher; protein chromatography media from GE Healthcare; nents to the following final concentrations: ThiGH or ThiH
arabinose from Alfa-Aesar (Heysham, UK); dithiothreitol, (35–95 ␮M; the exact concentration of ThiH or ThiGH for time
NADPH, and antibiotics from Melford Laboratories (Ipswich, course experiments is given in the figure legends), AdoMet
UK). All other chemicals used were of the highest quality avail- (1 mM), and tyrosine (1 mM). The assay solutions were equili-
able and were purchased from Sigma or Fluka. AdoMet was brated at 37 °C by incubating in a water bath contained within
purchased from Sigma and used without further purification.
an anaerobic glove box for 5 min. A stock solution of the reduc-
FEBRUARY 19, 2010•VOLUME 285•NUMBER 8
JOURNAL OF BIOLOGICAL CHEMISTRY 5241

Catalytic Activity of ThiH
tant system was prepared containing flavodoxin 1 (280 ␮M),
flavoprotein:NADPH oxidoreductase (70 ␮M), and NADPH (15
mM) and incubated at room temperature for 15 min to permit
the formation of the blue-colored semiquinone (22). The assays
were initiated by the addition (20 ␮l) of the reductant system
(final concentrations of flavodoxin 1 (37 ␮M), flavoprotein:
NADPH oxidoreductase (9 ␮M), and NADPH (2 mM)). Each
time point (1–60 min) was stopped by protein precipitation
with 20% perchloric acid (10 ␮l) and then cleared by centrifu-
gation (Eppendorf 5415D microcentrifuge, maximum speed).
Supernatants were analyzed by HPLC using a Gemini C18,
5-␮m, 110-Å reverse phase column (Phenomenex). The mobile
phase solvents were 0.1% AcOH in water (solvent A) or 0.1%
AcOH in acetonitrile (solvent B), and the flow rate was 0.8
ml/min. An initial isocratic phase of 100% solvent A for 8 min
was followed by a linear gradient to 50% solvent B over 32 min,
followed by an increase to 100% solvent B over 3 min, which was
held for 5 min before returning to 100% solvent A over 2 min
and re-equilibration for 10 min (total time of 60 min). To inves-
k͓P͔
max
kcat0 ϭ
(Eq. 3)
͓E͔
where [E] is the ThiGH concentration as estimated by the Brad-
ford assay (24). For the product inhibition experiment (Fig. 4),
data were fitted to a 4-parameter logistic sigmoid as shown in
Equation 4,
⌬A
A ϭ A0 ϩ
(Eq. 4)
h
͓I͔
1 ϩ
ͩ ͪ
IC50
where A is the observed activity; A0 is the minimum activity; ⌬A
is the difference between the maximum and minimum
observed activity; [I] is the concentration of inhibitor (glyoxy-
late and ammonium), and h is the Hill coefficient.
RESULTS
Isolation of Monomeric ThiH and ThiGH Complex—Experi-
tigate the effect of removing DOA formed in situ during the ments with tyrosine lyase purified by nickel-affinity chroma-
experiment, the reaction assays were supplemented with tography have permitted much initial characterization of the
MTAN (10 ␮M) that had been previously exchanged into anaer- protein (17, 18). In addition, analytical gel filtration chromatog-
obic buffer B. To investigate inhibition by glyoxylate and raphy (14) has shown that the protein isolated from the initial
ammonia, these compounds (10–2000 ␮M) were added to the nickel-charged chelating Sepharose column contained a mix-
assay.
ture of two forms of ThiH as follows: as part of a large multi-
Experiments with Substrate Analogues—Stock solutions (33 meric complex with at least six ThiGH heterodimers (apparent
mM) of substrate analogues were prepared in 100 mM ammo- mass ϳ440 kDa) or as a monomer (apparent mass ϳ42 kDa). To
nium bicarbonate and deoxygenated in an anaerobic glove box simplify the interpretation of subsequent experiments, it was
overnight. ThiGH/ThiH isolated by nickel-affinity chromatog- preferable to isolate these species so that their properties could
raphy was chemically reconstituted as described previously be compared. Resolution of these two species was achieved by
(17), concentrated, and exchanged into buffer B. Assays were preparative gel filtration chromatography using Superdex 200
prepared with chemically reconstituted ThiGH/ThiH mixture resin. The protein isolated after nickel-charged chelating
(60–90 ␮M), AdoMet (1 mM), substrate (1 mM) and analyzed as Sepharose chromatography (ThiGH/ThiH mixture) was chem-
described above.
ically reconstituted to improve the stability of ThiH, allowing it
Data Analysis—Data were analyzed and graphs prepared to be concentrated to ϳ25 mg/ml for application to the prepar-
using SigmaPlot (Systat Software Inc., London). Data from ative gel filtration column. Typically, a 1:1 ratio of ThiGH:
experiments where a pre-steady state burst phase was observed ThiH, with ϳ1 ␮mol of each, was successfully isolated (supple-
(Fig. 2 and Fig. 6, A and B) were fitted to a function that contains mental Fig. S1). Despite working under anaerobic conditions
a single exponential and a linear component (23) as shown in (less than 1 ppm O2), the labile 4Fe-4S cluster did not remain
Equation 1,
intact during this purification step, and the iron content of puri-
fied protein fractions was reduced to 3.1 Ϯ 0.2 mol eq of iron
per ThiGH and 2.5 Ϯ 0.3 mol eq of iron per ThiH (supplemental
Table S1). The 4Fe-4S cluster of ThiH shows a broad absorb-
ance at 400 nm (17). UV-visible spectroscopy was used to mon-
itor the loss of the labile 4Fe-4S cluster during chromatography
and its efficient reconstitution (supplemental Fig. S2). To
ensure optimal activity of protein samples, the 4Fe-4S cluster
was reconstituted immediately before activity measurements.
Activity of the ThiGH Complex—The time dependence of
product formation was studied with ThiGH complex isolated
by gel filtration chromatography. In previous experiments,
tyrosine lyase activity had been limited to less than one turn-
over (18), but this may have resulted from insufficient reduc-
tant or substrate in the assay. ThiGH assays were therefore
͑1 Ϫ kburstt͒
͓P͔ ϭ ͓E͔͑1 Ϫ e
͒ ϩ Lt
(Eq. 1)
where [P] is the observed concentration of product; [E] is the
amplitude of the burst phase and is equal to the concentration
of enzyme active sites in the reaction; kburst is the observed
single exponential rate constant for the burst phase; L is the
observed linear rate; and t is time. The rate constant for the
steady state phase (kss) was derived by dividing the observed
linear rate by the burst amplitude. For experiments where the
reaction displayed first order kinetics (Fig. 3), data were fitted to
Equation 2,
͓P͔ ϭ ͓P͔ ͑1 Ϫ eϪkt
͒
(Eq. 2)
max
where [P] is the observed concentration of product; [P]max is the prepared with greater than 10 mol eq of tyrosine and AdoMet
maximum observed product concentration, and k is the and a large excess of reductant (2 mM NADPH) to ensure the
observed first order rate constant. To derive the initial turnover [4Fe-4S] cluster could repeatedly access the ϩ1 oxidation state
number (kcat0), Equation 3 was used,
5242 JOURNAL OF BIOLOGICAL CHEMISTRY
during catalytic turnover. Analytical HPLC was used to quan-
VOLUME 285•NUMBER 8•FEBRUARY 19, 2010

Catalytic Activity of ThiH
FIGURE 2. In vitro time course of tyrosine lyase activity by ThiGH. Forma-
tion of DOA (E) and p-cresol (F) in a ThiGH activity assay (containing 80 ␮M
ThiGH) was monitored by HPLC. Data are the average of experiments carried
out in duplicate, shown with the standard error, and were fitted to a pre-
steady state burst phase function (Equation 1) to give the results shown in
Table 1.
FIGURE 3. In vitro time course of tyrosine lyase activity by ThiGH coupled
with MTAN-mediated hydrolysis of DOA. Formation of adenine (E) and
p-cresol (F) in a ThiGH activity assay (containing 95 ␮M of ThiGH), coupled
with MTAN-mediated hydrolysis of DOA, was monitored by HPLC. Data are
the average of experiments carried out in duplicate, shown with standard
errors, and were fitted to a first order exponential function (Equation 2) to
give the results shown in Table 2.
TABLE 1
Kinetic analysis of tyrosine lyase activity showing a burst phase of
product formation (Figs. 2 and 6)
of DOA was 79 Ϯ 6 ␮M, and the concentration of p-cresol was
71 Ϯ 2 ␮M, giving a DOA to p-cresol ratio of 1.1:1. However,
after the first turnover, when the release of products may
become rate-limiting, the reaction slows down, and uncoupled
turnover of AdoMet becomes more significant. In the steady
state phase (after about 15 min, Fig. 2), the ratio of the rate of
formation of DOA to the rate of p-cresol formation increases to
1.7:1
These results were obtained by fitting data to Equation 1. ͓P͔ is the observed con-
centration of product; ͓E͔ is the observed burst amplitude; kburst is the observed
single exponential rate constant for the burst phase, and kss is the steady state rate
constant, derived by dividing the observed linear rate by the burst amplitude. Data
are presented with standard errors, and R2 is a measure of the goodness of fit.
Form of ThiH
͓P͔
͓E͔
␮M
kburst
kss
R2
Ϫ1
Ϫ1
ϫ10Ϫ4 s
ϫ10Ϫ4 s
ThiGH complex
p-Cresol
89 Ϯ 4
90 Ϯ 4
53 Ϯ 6
1.6 Ϯ 0.2 0.99
DOA
63 Ϯ 10
2.9 Ϯ 0.2 0.99
Monomeric ThiH p-Cresol
38 Ϯ 4
32 Ϯ 8
5.9 Ϯ 0.2 0.99
10.7 Ϯ 1.0 0.99
Addition of MTAN to in Vitro Assays Changes the Rate-lim-
iting Step—The addition of MTAN to time course experiments
with ThiGH resulted in the rapid hydrolysis of DOA to 5Ј-de-
oxyribose and adenine (19). Adenine could be readily quanti-
DOA
42 Ϯ 9
27 Ϯ 11
Monomeric ThiH p-Cresol
92 Ϯ 16
32 Ϯ 10
4.3 Ϯ 0.1 0.98
with MTAN
Adenine 180 Ϯ 75
32 Ϯ 23
16.0 Ϯ 3.3 0.95
tify the formation of the products DOA and p-cresol. Reactions fied using the same analytical HPLC protocol (Rt ϭ 6.0 min, see
were stopped at a range of time points up to 1 h, and the time supplemental Fig. S2). During the quantification of adenine
course showed a pre-steady state burst phase for product for- produced in these time course experiments, particular care was
mation (Fig. 2), followed by a slower reaction once steady state required with negative control samples (lacking reductant), as
was reached. In a typical experiment, after 1 h ThiGH had gen- commercial supplies of AdoMet contain a small proportion of
erated 1.8 mol eq of p-cresol and 2.3 mol eq of DOA, with an 5Ј-methylthioadenosine. 5Ј-Methylthioadenosine also yields
overall DOA:p-cresol ratio of 1.3:1, indicating that there was adenine upon hydrolysis by MTAN (20), and this background
some uncoupled turnover of AdoMet to DOA. The time-de- was subtracted from activity assays.
pendent product formation could be fitted to Equation 1. This
The addition of MTAN changed the profile of the ThiGH
analysis gave the rate constant for p-cresol formation in the activity time course (Fig. 3). In situ hydrolysis of the DOA elim-
pre-steady state exponential phase of 53 Ϯ 6 ϫ 10Ϫ4 sϪ1, and inated the burst phase and extended the period of relatively
during steady state phase, kcat was calculated as 1.6 Ϯ 0.2 ϫ rapid turnover (where kcat for the formation of cresol was in the
Ϫ4
10
s
Ϫ1. The rate constant for formation of 5Ј-deoxyade- range 30–50 ϫ 10Ϫ4 sϪ1) beyond a single turnover; however,
nosine was 63 Ϯ 10 ϫ 10Ϫ4 sϪ1 during the burst phase, but the rate of product formation slowly declined and could be
this slowed to 2.9 Ϯ 0.2 ϫ 10Ϫ4 sϪ1 during the steady state fitted as a first order process (Equation 2) with a final p-cresol
phase. The fitting of the data gave values of 89 Ϯ 4 and 90 Ϯ concentration of 220 Ϯ 5 ␮M (ϳ2.3 turnovers). Subsequent
4 ␮M for burst phase amplitude, which corresponds to the experiments with lower ThiGH concentrations demonstrated
concentration of ThiH active sites and is in good agreement that after 1 h, ThiGH could produce up to 7 eq of p-cresol
0
with the ThiGH concentration estimated by the Bradford (supplemental Table S2). The initial turnover numbers (kcat
)
Ϫ4
assay (24) of 80 ␮M.
for ThiGH calculated from the initial rates are 37 Ϯ 3 ϫ 10
The pre-steady state burst phase (up to ϳ10 min, Fig. 2 and and 51 Ϯ 6 ϫ 10Ϫ4 sϪ1 for the formation of p-cresol and ade-
Table 1) reflects the catalytic rate for the chemical reaction, and nine, respectively. These initial rates are similar to those
during this phase there is efficient coupling of DOA formation observed in the absence of MTAN (Fig. 2 and Tables 1 and 2).
to tyrosine cleavage; for example, after 5 min, the concentration At time points beyond 5 min, a further increase in uncoupled
FEBRUARY 19, 2010•VOLUME 285•NUMBER 8
JOURNAL OF BIOLOGICAL CHEMISTRY 5243

Catalytic Activity of ThiH
TABLE 2
Kinetic analysis of tyrosine lyase activity showing an exponential
profile of product formation (Fig. 3)
These results were obtained by fitting data to Equation 2. ͓P͔ is the observed con-
centration of product; ͓P͔max is the maximum observed product concentration; k is
0
the observed first order rate constant, and kcat is the initial turnover number
obtained from Equation 3. Data are presented with standard errors, and R2 is a
measure of the goodness of fit.
0
Form of ThiH
͓P͔
͓P͔
␮M
k
kcat
R2
max
ϫ10Ϫ4 s
ϫ10Ϫ4 s
Ϫ1
Ϫ1
ThiGH complex p-Cresol 220 Ϯ 5
with MTAN Adenine 590 Ϯ 24
16 Ϯ 1
37 Ϯ 3
0.99
0.99
8 Ϯ 1
51 Ϯ 6
turnover of AdoMet was observed, and the final concentration
of adenine reached 590 Ϯ 24 ␮M, corresponding to a ratio of
adenine:p-cresol of 2.7:1.
Glyoxylate and Ammonia Inhibit the Tyrosine Cleavage
Reaction—A different strategy was required for investigating
the potential of dehydroglycine to inhibit tyrosine lyase. The
hydrolysis of dehydroglycine is a rapidly reversible equilibrium,
and the addition of relatively high concentrations of glyoxylate
and ammonium yields a low concentration of dehydroglycine
in the solution. This method was used advantageously by Begley
and co-workers (11) during studies in which they demonstrated
that the addition of glyoxylate and ammonium provided suffi-
cient dehydroglycine to reconstitute the ThiG-dependent
cyclization reaction.
Using this approach to examine the effect of dehydroglycine
on tyrosine lyase activity, increasing concentrations of glyoxy-
late and ammonium ions were added to activity assays. To
ensure product inhibition by DOA did not disguise any effects
of glyoxylate and ammonium, these assays also contained suf-
ficient MTAN to hydrolyze the DOA produced in the assay to
adenine and 5Ј-deoxyribose. The effects of glyoxylate and
ammonium were measured using HPLC analysis, measuring
tyrosine cleavage by detecting the production of p-cresol and
reductive cleavage of AdoMet by detecting the production of
adenine. The combination of ammonium and glyoxylate
inhibited the cleavage of tyrosine (Fig. 4A) with an apparent
IC50 of 440 Ϯ 55 ␮M and a Hill coefficient of 1.1 Ϯ 0.1,
FIGURE 4. Inhibition of tyrosine lyase activity by glyoxylate and ammo-
nium. Assays were coupled with MTAN-mediated hydrolysis of DOA to pre-
clude the possibility of inhibition by DOA and methionine. A, tyrosine C␣–C␤
bond cleavage was measured by the formation of p-cresol and was fitted to
Equation 4. B, AdoMet cleavage was measured by the formation of adenine.
Assays were incubated for 1 h and analyzed by HPLC, and values are relative
to standard assays with no additions. Data are the average of experiments
carried out in duplicate, shown with the standard errors, and were fitted to
Equation 4.
suggesting a 1:1 complex of the inhibitor and the enzyme. AdoMet, and the formation of p-cresol or other aromatic prod-
Subsequent experiments in which 1 mM of the individual ucts allowed the measurement of the C␣–C␤ bond cleavage
species was added to activity assays showed this inhibition step. The negative control samples (which contained ThiGH,
was due solely to glyoxylate and that ammonium had no AdoMet, and reductant but no tyrosine) were particularly
effect (supplemental Fig. S4).
important in this experiment, as very little uncoupled AdoMet
In contrast, no inhibition of AdoMet cleavage was observed cleavage occurred in the absence of tyrosine (less than 10%
over the whole range of glyoxylate and ammonium concentra- relative to a positive control sample containing tyrosine). Sev-
tions (Fig. 4B). At high glyoxylate and ammonium concentra- eral tyrosine analogues were investigated using this strategy
tions (2 mM), greater than 90% of the reductive cleavage was (Fig. 5 and Table 3). Tyramine 17 and L-phenylalanine 18 were
uncoupled, resulting in the accumulation of up to 600 ␮M ade- not substrates and did not show an increase in the cleavage of
nine but only 40 ␮M p-cresol. Similar results were obtained in AdoMet relative to the negative control. However, it was found
experiments using either ThiGH complex or monomeric ThiH, that 4-hydroxyphenylpropionic acid (4-HPPA, 16) and 4-hy-
suggesting the presence of ThiG alone does not influence this droxyphenyl-␣-hydroxypropionic acid (4-HPHPA, 15) lead to
inhibition.
the formation of ϳ50% of the amount of DOA formed com-
Activity Assays with Tyrosine Analogues—To obtain further pared with the positive control (with tyrosine as the substrate).
insight into the mechanism of tyrosine cleavage, the reactivity Careful examination of the HPLC trace confirmed that little or
of structural analogues of tyrosine in ThiGH activity assays was no p-cresol was formed (less than 5% relative to positive control
investigated. Analysis of these analogue assays by HPLC samples). These results suggest 4-HPPA and 4-HPHPA did not
allowed two steps in the mechanism to be monitored; the for- undergo C␣–C␤ bond cleavage but are able to support uncou-
mation of DOA indicated the extent of the reductive cleavage of pled AdoMet cleavage.
5244 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 8•FEBRUARY 19, 2010

Catalytic Activity of ThiH
investigated, specifically 4-amino-,
4-methyl-, and 4-fluoro-L-pheny-
lalanines. None of these tyrosine
analogues increased AdoMet cleav-
age or yielded detectable aromatic
products. Assays with D-tyrosine
showed relatively slow uncoupled
AdoMet cleavage, but no p-cresol
formation was observed. Further
experiments in which reaction
products or potential substrate
mimics were added to activity
assays are shown in Table 4. They
show that combinations of reaction
products do not increase uncoupled
AdoMet cleavage unless L-tyrosine
is present in the mixture.
Activity of Monomeric ThiH—
Size exclusion chromatography
allowed the purification of ThiGH
complex and monomeric ThiH. The
FIGURE 5. HPLC analysis of ThiH assays with substrate analogues. A, structure of substrate analogues.
B, analytical HPLC traces of tyrosine lyase activity assays with tyrosine analogues, displaced upwards for clarity.
The detector measures the absorbance at 280 nm. The traces are for assays with the following (from top to natural pathway for thiazole biosyn-
bottom): L-tyrosine 1; 4-HPHPA 15; 4-HPPA 16; tyramine 17; L-phenylalanine 18 and with no substrate (nega-
thesis includes efficient transfer of
tivecontrol). Comparisonwithauthenticsamplesidentifiedthefollowingpeaks: a, tyrosine;b, DOA;c, 5Ј-meth-
ylthioadenosine (MTA); d, 4-HPHPA; e, 4-HPPA; f, p-cresol.
dehydroglycine from ThiH to ThiG
(Fig. 1B), and it was therefore of
TABLE 3
interest to determine the kinetic properties of ThiH in the
Amount of turnover from substrate analogues
absence of ThiG. The time courses obtained using HPLC anal-
AdoMet cleavage was assessed by monitoring the amount of DOA produced. Values
ysis of activity assays with monomeric ThiH are shown in Fig.
are given as a percentage with respect to the amount of activity observed in a
standard assay (including tyrosine as the substrate) that was measured in parallel.
6A. The kinetics of product formation from ThiH showed burst
Standard errors are given where the results have been replicated.
phase similar to that observed with ThiGH. Fitting of the data
Compound
AdoMet cleavage
from Fig. 6A to Equation 1 gave a burst phase rate constant
(32 Ϯ 8 ϫ 10Ϫ4 sϪ1 for p-cresol) that is marginally slower than
for ThiGH but a substantially faster steady state rate (5.9 Ϯ
0.2 ϫ 10Ϫ4 sϪ1 for p-cresol), which is consistent with a faster
(but still rate-limiting) product release step. The addition of
MTAN to the ThiH assays did not abolish the burst phase but
did increase the degree of uncoupled turnover (Table 1). This
suggests that for monomeric ThiH, a step other than release of
DOA is rate-limiting.
%
L-Tyrosine 1
100
50.9 Ϯ 3.0
50.9 Ϯ 6.8
14.3
4-Hydroxyphenylpropionic acid 16
4-Hydroxyphenyl-␣-hydroxypropionic acid 15
D-Tyrosine
4-Hydroxyphenylcinnamic acid
Tyramine 17
7.2 Ϯ 2.2
5.9 Ϯ 3.0
5.4 Ϯ 1.3
4.8 Ϯ 0.6
3.0
L-Phenylalanine 18
4-Amino-L-phenylalanine
4-Methyl-L-phenylalanine
4-Fluoro-L-phenylalanine
No substrate (negative control)
2.6
6.6 Ϯ 1.7
TABLE 4
DISCUSSION
Amount of turnover measured from combinations of in vitro
products or potential substrate mimics
Dehydroglycine is a precursor of the vitamin thiamine pyro-
phosphate in a wide range of prokaryotes (2). It is derived from
(at least) two sources. Aerobes use dioxygen as an electron
acceptor to facilitate the oxidation of glycine in a reaction cat-
alyzed by ThiO (11, 12). Anaerobes cannot use this pathway,
but instead they make use of a radical AdoMet enzyme, tyrosine
lyase (ThiH), to break the C␣–C␤ bond of tyrosine to yield
p-cresol and dehydroglycine (17, 18). The energetic challenge
of using AdoMet to generate and control the deoxyadenosyl
radical has been highlighted previously (25, 26). Not only does
the reaction need to be isolated in the active site to permit the
radical reaction to proceed, the reaction product dehydrogly-
cine must also be protected from the aqueous environment as it
is hydrolytically unstable. To use dehydroglycine for the forma-
tion of thiazole carboxylate 4, the protection of dehydroglycine
AdoMet cleavage was assessed by monitoring the amount of DOA generated, and
C␣—C␤ bond cleavage was assessed by monitoring the amount of p-cresol formed.
Values are given as a percentage with respect to the amount of activity observed in
a standard assay (including tyrosine as the substrate) that was measured in parallel.
Standard errors are given where the results have been replicated. NA means not
applicable.
Compound(s)
AdoMet cleavage C␣–C␤ bond cleavage
%
%
L-Tyrosine
100
117 Ϯ 7
8.4
100
25.2 Ϯ 5.1
NA
NHϩ4 ϩ glyoxylate ϩ L-tyrosine
Glycine ϩ p-cresol
p-Cresol
7.8
NA
NHϩ4 ϩ glyoxylate ϩ p-cresol
Glycine
6.9
NA
6.8
NA
NHϩ4 ϩ glyoxylate
4.8
NA
This strategy of analyzing for DOA formation was used to
screen further tyrosine analogues (see Table 3). Analogues with
different functional groups at the phenol position were must extend to its transfer from ThiH to ThiG. This require-
FEBRUARY 19, 2010•VOLUME 285•NUMBER 8
JOURNAL OF BIOLOGICAL CHEMISTRY 5245

Catalytic Activity of ThiH
constant across the whole time course. The turnover number
derived from this rate constant was similar to that of the burst
phase when MTAN was not present (Tables 1 and 2). The rate-
limiting step for ThiH reaction catalysis is unknown, but for
anaerobic sulfatase-maturating enzyme, another member of
the radical AdoMet family, the observed deuterium isotope
effect indicates that hydrogen atom abstraction by the 5Ј-de-
oxyadenosyl radical is the rate-limiting step (28). The burst
phase rate constant obtained for ThiH reaction catalysis (53 Ϯ
6 ϫ 10Ϫ4 sϪ1) is comparable with other members of the radical
AdoMet family; for example, apparent rate constants have been
reported as 60 ϫ 10Ϫ4 and 12 ϫ 10Ϫ4 sϪ1 for the AtsB (formyl-
glycine-generating enzyme) (29) and biotin synthase (30),
respectively.
The hydrolytic instability of dehydroglycine implies that a
tight interaction with ThiGH would be advantageous, helping
to sequester the imine away from the aqueous medium. The
observed inhibition of tyrosine cleavage by glyoxylate may indi-
cate competitive binding to the active site, but further rational-
ization of this observation is difficult in the absence of struc-
tural data. A more surprising observation was the almost
complete (Ͼ90%) uncoupling of AdoMet turnover from the
tyrosine cleavage reaction in the presence of relatively high
concentrations of glyoxylate and ammonium ions (2 mM each).
This apparently wasteful process is unlikely to occur in a cellu-
lar context, and the effect was maximized under specific in vitro
conditions with the addition of MTAN plus high concentra-
tions of glyoxylate and ammonium. However, the experiment
does indicate that in cells where ThiGH is not actively synthe-
sizing the thiazole carboxylate 4, one of the functions of prod-
uct inhibition by DOA may be to reduce the uncoupled turn-
over of AdoMet. The release of dehydroglycine observed in
vitro is unlikely to occur during cellular thiamine biosynthesis,
when transfer of the dehydroglycine probably occurs directly
between ThiH and ThiG. The full biosynthetic reaction path-
way for ThiGH (which additionally requires ThiS thiocarboxy-
late 12 and 1-deoxyxyulose 5-phosphate 3, see Fig. 1) may be
faster than the steady state rate observed in vitro for the partial
reaction, making it difficult to assess the physiological rele-
FIGURE 6. In vitro time course of monomeric ThiH activity. A, formation of
DOA (E) and p-cresol (F) in a monomeric ThiH assay (containing 35 ␮M ThiH).
B, formation of adenine (ꢁ) and p-cresol (F) in a monomeric ThiH assay (con-
taining 90 ␮M of ThiH) coupled with MTAN-mediated hydrolysis of DOA. To
facilitate a comparison, the data are presented in number of equivalents of
product formed per enzyme. Data are the average of experiments carried out
in duplicate, shown with the standard error, and were fitted to a pre-steady
state burst phase function (Equation 1) to give the results shown in Table 1.
ment may explain the functional role of complex formation vance of the slow steady state rate. Although evidence has been
between ThiG and ThiH.
found in these studies for product inhibition of tyrosine lyase by
The time course of product formation (Fig. 2) showed that both DOA and dehydroglycine, the complexity of the reaction
ThiGH is capable of more than one cycle of tyrosine cleavage, catalyzed by ThiGH does not allow the description of a defini-
but the enzyme exhibited burst phase kinetics. The observation tive kinetic model for product release at this stage.
of burst phase kinetics indicates a rate-limiting step that occurs
The problem of uncoupled AdoMet cleavage in radical
after chemical catalysis and can be due to product release or a AdoMet proteins is well documented (26). The reactivity of the
conformational change (27). ThiGH yields four reaction prod- primary 5Ј-deoxyadenosyl radical makes its uncontrolled for-
ucts, methionine, DOA, p-cresol, and dehydroglycine, provid- mation potentially hazardous for the cell. There are several
ing potential for a complex pattern of product inhibition. Ear- mechanisms by which this family of enzymes regulate uncou-
lier in vitro studies have determined that tyrosine lyase activity pled AdoMet cleavage, including modifying the redox potential
was inhibited by accumulation of DOA and methionine (19) of the 4Fe-4S cluster in response to substrate binding (25) or by
and that this could be overcome by the addition of MTAN, a cooperative substrate binding (31). The nature of the reductant
nucleosidase that hydrolyzes DOA. The observed changes in has been proposed to have a role in the degree of uncoupled
the time course of product formation in the presence of MTAN AdoMet cleavage with less uncoupling being observed when
(Fig. 3) indicates a change in the rate-determining step during using the natural electron donor systems. Our studies made use
subsequent turnovers and suggests an ordered sequence of of the NADPH, flavodoxin, and flavoprotein:NADPH oxi-
product release from ThiGH. The addition of MTAN gave a doreductase system, which is assumed to be the natural intra-
profile of product formation that could be fitted to a single rate cellular reductant for E. coli ThiGH.
5246 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 8•FEBRUARY 19, 2010

Catalytic Activity of ThiH
dence for concerted AdoMet cleav-
age and phenolic hydrogen atom
abstraction, but it does highlight the
importance of a phenolic O–H
bond in the active site as a prerequi-
site for AdoMet cleavage. The for-
mation of a tyrosine radical is ther-
modynamically favorable as a result
of the reactivity of the primary
deoxyadenosyl radical, the strength
of the C–H bond formed in 5Ј-de-
oxyadenosine and relative weakness
of the phenolic O–H bond. The
model proposes two possible fates
for the phenolic radical as follows:
either turnover leading to product
formation or completion of a futile
cycle leading back to tyrosine. Dur-
FIGURE 7. Proposed mechanism for tyrosine lyase. The reductive cleavage of AdoMet yields the 5Ј-deoxya-
denosyl radical that can abstract a hydrogen atom from the phenolic O–H bond. The resultant phenolic radical
19 can follow two possible reaction pathways, and the nature of the group R can affect which of these two
pathways is favored. Immediate reduction, followed by protonation, completes an unproductive futile cycle
back to the substrate. For L-tyrosine (R ϭ NH2), cleavage of the C␣–C␤ bond yields dehydroglycine and the
resonance-stabilized radical anion 21a7 21b, which requires the addition of two protons and an electron to
give p-cresol 8.
ing turnover leading to product for-
mation, the electron-deficient phe-
nolic radical can undergo C␣–C␤
bond cleavage with the assistance of
the lone pair of electrons on the ␣-a-
mine of tyrosine. Such a mechanism
The experiments with tyrosine analogues and reaction prod-
ucts (Fig. 5 and Tables 3 and 4) had two related objectives as
follows: to identify the structural features that were essential for
the turnover of tyrosine and to elucidate the factors that pro-
moted uncoupled turnover of AdoMet. With regard to under-
standing the uncoupled turnover, several negative controls
were important as follows: incubation of ThiGH with the
reductant and AdoMet in the absence of tyrosine resulted in
very little uncoupled turnover (less than 10% relative to assays
with tyrosine present), and the addition of reaction products,
again in the absence of tyrosine, did not increase the uncoupled
cleavage of AdoMet (Table 4). Comparing the tyrosine struc-
tural analogues, all of the analogues that replaced the phenol
functional group were inactive, and it appears that ThiH is very
sensitive to modifications at this position. Fluorine has 97% of
the van der Waals radius of oxygen, but 4-fluoro-L-phenylala-
nine was not sufficiently similar to L-tyrosine to increase
AdoMet cleavage above the background level. Although the
ArO–H bond is weak in phenols such as tyrosine, a comparison
of bond dissociation energy of phenol (ArO–H, 360 kJ molϪ1),
aniline (ArNH–H, 388 kJ molϪ1), and toluene (ArCH2–H, 355
kJ molϪ1) (32) indicate that 4-amino- and 4-methyl-L-pheny-
lalanine ought to be susceptible to hydrogen atom abstraction.
As neither of these analogues resulted in AdoMet cleavage, the
subtle substrate selectivity of tyrosine lyase cannot be based
purely on the bond strength. The substrates that permitted
AdoMet cleavage, L- and D-tyrosine, 4-HPPA 16, and 4-HPHPA
15 (Fig. 5 and Table 3) share a common structural motif of
being 4-hydroxyphenylpropionic acids, and the phenol appears
to be required for AdoMet cleavage.
provides a direct route to dehydroglycine and the resonance
stabilized radical anion 21a7 21b that requires reduction and
protonation to form p-cresol 8. To achieve uncoupled turnover,
the phenolic radical 19 is proposed to undergo immediate
reduction followed by protonation of the phenoxide 20, giving
a direct route back to tyrosine. In this model, the difference
between the uncoupled and product-forming pathways
depends on the timing of the reduction step (reduction of rad-
ical 19 or 21). In the case of D-tyrosine and the analogues
4-HPPA and 4-HPHPA, the C␣–C␤ bond does not undergo
cleavage, and these substrates are limited to futile cycling with
all of the AdoMet cleavage being uncoupled. A precise stereo-
electronic explanation for the observed substrate selectivity will
require a structural model of the ThiH-active site. The forma-
tion of p-cresol from L-tyrosine requires a reduction step, in
addition to the reductive cleavage of AdoMet, which is unusual
for a radical AdoMet protein (33). The immediate source of the
additional reducing equivalent is unknown, but the [4Fe-4S]
cluster that is used to accelerate the reductive cleavage of
AdoMet is a possible candidate.
During the time course with L-tyrosine as a substrate, the
burst phase of the reaction is efficiently coupled (up to 90%), but
during the steady state phase, the ratio of coupled to uncoupled
reaction pathways is 1.7:1. Uncoupled turnover is further
increased by the addition of MTAN and becomes dominant
when glyoxylate and ammonia are added to the assay. One
interpretation of these data is that dehydroglycine is able to
bind to the ThiGH complex, and this suppresses the C␣–C␤
bond cleavage step. The mechanism by which dehydroglycine
and/or DOA might modulate the ratio of coupled to uncoupled
activities is not clear but may result from dehydroglycine
remaining bound at the interface between ThiG and ThiH. This
The observed product inhibition, uncoupled turnover, and
studies with these tyrosine analogues can be integrated with a
mechanistic model (Fig. 7). The strong preference shown by
tyrosine lyase for a phenolic substrate does not provide evi- provides a convenient mechanism to ensure tyrosine cleavage
FEBRUARY 19, 2010•VOLUME 285•NUMBER 8
JOURNAL OF BIOLOGICAL CHEMISTRY 5247

Catalytic Activity of ThiH
F. W., Begley, T. P., and Ealick, S. E. (2004) Biochemistry 43, 11647–11657
10. Chatterjee, A., Han, X., McLafferty, F. W., and Begley, T. P. (2006) Angew.
Chem. Int. Ed. Engl. 45, 3507–3510
catalyzed by ThiH is synchronized with the incorporation of
dehydroglycine by ThiG into the thiazole carboxylate 14.
In conclusion, catalytic turnover of the tyrosine lyase activity
of E. coli ThiGH complex has been demonstrated. However,
tyrosine cleavage is strictly controlled by the accumulation of
the products, which results in a burst phase kinetic profile. The
function of this product inhibition in cells actively synthesizing
thiamine may be to coordinate the rate of formation of dehy-
droglycine by ThiH with its utilization by ThiG for thiazole
formation. Structural analogues of tyrosine have been used to
define the substrate requirements of the enzyme and in partic-
ular the need for a phenolic O–H bond. Furthermore, phenolic
tyrosine analogues that lacked a correctly positioned amine
functional group were able to undergo a partial reaction leading
to uncoupled AdoMet cleavage but with no evidence for
C␣–C␤ bond cleavage.
11. Park, J. H., Dorrestein, P. C., Zhai, H., Kinsland, C., McLafferty, F. W., and
Begley, T. P. (2003) Biochemistry 42, 12430–12438
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T. P., and Ealick, S. E. (2003) Biochemistry 42, 2971–2981
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