A tomato enzyme catalyzing the phosphorylation of 3,4-dihydroxy-2-butanone.

Article abstract of DOI:10.1016/S0031-9422(02)00056-0

A riboflavin biosynthesis ribB mutant of Escherichia coli deficient of 3,4-dihydroxy-2-butanone 4-phosphate synthase was complemented with a cDNA library from Lycopersicon esculentum. The complementing gene was isolated and expressed in E. coli. The resulting protein was shown to specify a 62 kDa protein which phosphorylates dihydroxyacetone, both enantiomers of 3,4-dihydroxy-2-butanone, and several other aldoses and ketoses. Sequence analysis revealed homology to dihydroacetone kinases (dak) genes from plants, animals, fungi and some eubacteria. Genes with similarity to the 5' part of the dak gene from tomato were found in many other eubacteria. The physiological role of the dak gene is still incompletely known.

Full text of DOI:10.1016/S0031-9422(02)00056-0

Phytochemistry 60 (2002) 3–11
www.elsevier.com/locate/phytochem
A tomato enzyme catalyzing the phosphorylation of
3,4-dihydroxy-2-butanone
Stefan Herz, Klaus Kis, Adelbert Bacher, Felix Rohdich*
Lehrstuhl fur Organische Chemie und Biochemie, Technische Universitat Munchen, Lichtenbergstr. 4, D-85747 Garching, Germany
¨
¨
¨
Received 14 November 2001; received in revised form 31 January 2002
Abstract
A riboflavin biosynthesis ribB mutant of Escherichia coli deficient of 3,4-dihydroxy-2-butanone 4-phosphate synthase was com-
plemented with a cDNA library from Lycopersicon esculentum. The complementing gene was isolated and expressed in E. coli. The
resulting protein was shown to specify a 62 kDa protein which phosphorylates dihydroxyacetone, both enantiomers of 3,4-di-
hydroxy-2-butanone, and several other aldoses and ketoses. Sequence analysis revealed homology to dihydroacetone kinases (dak)
genes from plants, animals, fungi and some eubacteria. Genes with similarity to the 50 part of the dak gene from tomato were found
in many other eubacteria. The physiological role of the dak gene is still incompletely known. # 2002 Elsevier Science Ltd. All rights
reserved.
Keywords: 3,4-Dihydroxy-2-butanone 4-phosphate; Riboflavin; 3,4-Dihydroxy-2-butanone; Dihydroxyacetone; Lycopersicon esculentum; Solana-
ceae; Tomato; Kinase; Phosphorylation
1. Introduction
experiments using a ribB mutant of E. coli (Katzen-
meier, 1991) and a cDNA library from tomato (Lycopersi-
con esculentum). The ribB mutant is deficient of
3,4-dihydroxy-2-butanone 4-phosphate synthase and
requires riboflavin in very high concentration for
growth. A plant gene was cloned that could complement
the 3,4-dihydroxy-2-butanone 4-phosphate synthase
deficiency of the E. coli ribB mutant. Strangely, how-
ever, complementation required the presence of yeast
extract in the culture medium.
The cloned plant gene was subsequently found to
specify a kinase which is not involved in the riboflavin
biosynthetic pathway. Putative orthologs of that
enzyme are present in plants, animals and certain
microorganisms.
The riboflavin precursor, l-3,4-dihydroxy-2-butanone
4-phosphate (2), is biosynthesized from ribulose
5-phosphate (1) by a rearrangement reaction under
release of carbon atom 4 as formate (Fig. 1) (Volk and
Bacher, 1988, 1990; Bacher, 1991; Bacher et al., 1997a,
b). In Escherichia coli, this complex reaction is catalyzed
by the homodimeric 3,4-dihydroxy-2-butanone 4-phos-
phate synthase specified by the ribB gene (Richter et al.,
1992; Bacher et al., 1996). The bacterial enzyme has
been studied in some detail (Richter et al., 1992, 1999;
Kelly et al., 1999, 2001; Liao et al., 2001). A bifunc-
tional enzyme with 3,4-dihydroxy-2-butanone 4-phos-
phate synthase and GTP cyclohydrolase II activity has
been cloned more recently from Arabidopsis thaliana
(Herz et al., 2000). This enzyme is similar to bifunctional
orthologs from various bacteria and catalyzes both initial
reactions of the convergent riboflavin pathway.
2. Results
Prior to the cloning of the bifunctional plant enzyme
(Herz et al., 2000), we performed complementation
In an attempt to clone 3,4-dihydroxy-2-butanone
4-phosphate synthase genes from plants, we performed
complementation experiments using a ribB mutant of E.
coli deficient of 3,4-dihydroxy-2-butanone 4-phosphate
synthase (Katzenmeier, 1991). A cDNA library from
tomato (Schmid et al., 1992) in the plasmid pBlueskript
* *Corresponding author. Tel.: +49-89-289-13364; fax +49-89-
289-13363.
E-mail address: felix.rohdich@ch.tum.de (F. Rohdich).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00056-0

4
S. Herz et al. / Phytochemistry 60 (2002) 3–11
Fig. 1. Biosynthesis of riboflavin.
SK IIꢀ was transformed into E. coli ribB cells which
were plated on Luria Bertani (LB) medium. The ribo-
flavin content of this medium is well below the con-
centration level required for growth of riboflavin
deficient E. coli mutants (Shavlovskii et al., 1982; Kat-
zenmeier, 1991). Colonies growing on this medium were
found to harbor a plasmid designated p10–20 contain-
ing an insert of 2.2 kbp. Retransformation of plasmid
p10–20 into two different ribB mutants (rib5 and rib10)
of E. coli reproducibly afforded clones growing on LB
medium without added riboflavin.
The growth rate of the recombinant E. coli strains on
LB medium was relatively low and could be increased
by the addition of riboflavin in high concentration (400
mg lꢀ1). We concluded tentatively that the plasmid p10–
20 contained a tomato gene which is capable of restoring
the function of the defective ribB gene, albeit with lim-
ited efficiency.
The DNA insert of plasmid p10–20 was sequenced
(EMBL Acc. No. Y12090) and was shown to contain a
putative open reading frame of 1782 bp specifying a
predicted peptide of 594 amino acid residues. Moreover,
the insert contained 138 untranslated nucleotides at the
50 end and 300 untranslated bp including a polyA
sequence of 18 residues at the 30 end. This gene is sub-
sequently designated dak.
Sequence comparison of the predicted tomato protein
showed similarity to putative ATP-dependent dihy-
droxyacetone kinases from eukaryotes and several
eubacteria (Figs. 2 and 3). Dihydroxyacetone kinases
are required for glycerol and methanol metabolism in
eubacteria and yeast (Kato et al., 1988; Daniel and

S. Herz et al. / Phytochemistry 60 (2002) 3–11
5
Fig. 2. Comparison of protein sequences specified by putative dak orthologs. A, Homo sapiens; B, Lycopersicon esculentum; C, Citrobacter freundi;
D, Caenorhabditis elegans; E, Saccharomyces cerevisiae (DAK2). The sequences were aligned with the PILEUP program using the default para-
meters (GCG, Madison, WI, USA). Identical residues conserved in at least 60% of the sequences were shown in inverse contrast.
Gottschalk, 1992; Kimura et al., 1998). In order to rule
out the possibility that the isolated gene could originate
from a microbial DNA contamination in the tomato
cDNA library, PCR experiments were performed with
chromosomal tomato DNA. Several segments of the
dak gene could be amplified from the chromosomal
tomato DNA. These experiments leave no doubt that
the dak gene is an authentic tomato gene.
The open reading frame of the tomato dak gene was
cloned into the plasmid vector pNCO113 under control
of a T5 promoter and lac operator affording plasmid
pDAK. A recombinant E. coli strain carrying this plas-
mid was shown to express a 62 kDa peptide at an esti-
mated yield of 5% based on total cell protein. The
recombinant protein was purified to about 95%
homogeneity as judged by SDS-PAGE (Fig. 4) by two
chromatographic steps as described under Experimental
(Table 1).
The recombinant tomato protein was shown to
phosphorylate dihydroxyacetone at a rate of 2.6 mmol
minꢀ1 mgꢀ1 (Table 2). Moreover, both enantiomers
of 3,4-dihydroxy-2-butanone can serve as substrate
for the tomato kinase, but the d enantiomer is phos-
phorylated at a considerably higher rate. Several
other hydroxyketones and hydroxyaldehydes were
phosphorylated at low rates. Polyalcohols are unable
to serve as substrates. Mg2+ is required for catalytic
activity.
These findings suggested that the complementation
experiment conducive to the isolation of the tomato
clone p10–20 had involved the formation of the
riboflavin
intermediate,
3,4-dihydroxy-2-butanone

6
S. Herz et al. / Phytochemistry 60 (2002) 3–11
4-phosphate (2), by phosphorylation of the correspond-
ing alcohol, 3,4-dihydroxy-2-butanone (3), which might
have been present in the LB medium used for the selec-
tion of colonies. In line with this hypothesis, we found
that ribB mutants of E. coli carrying the plasmid
p10–20 could not grow on mineral salt medium,
whereas growth could be restored by the addition of
3,4-dihydroxy-2-butanone to the minimal medium (data
not shown). RibB mutants without the plasmid were
unable to grow on LB medium but could use diacetyl
Fig. 3. Sequence comparison of proteins or predicted proteins with similarity to 3,4-dihydroxyacetone kinases from tomato. The dendrogram was con-
structed with the PILEUP program using the default parameters. The GenBank and Swissprot accession numbers, respectively are indicated in parentheses.

S. Herz et al. / Phytochemistry 60 (2002) 3–11
7
Table 1
Purification of recombinant dihydroxyacetone kinase
Procedure
Protein
(mg)
Specific activitya
(mmol minꢀ1 mgꢀ1
)
Cell extract
QA52 cellulose
Mono Q
73
17
2.0
0.06
0.28
0.78
a
The activity was determined with d-3,4-dihydroxy-2-butanone as
substrate.
Table 2
Phosphorylation of different substrates by dihydroxyacetone kinase
from tomato at 37 ꢁ
Substrate
C
Specific activity
(mmol minꢀ1 mgꢀ1
)
Dihydroxyacetone
2.6
0.78
d-3,4-Dihydroxy-2-butanone
l-3,4-Dihydroxy-2-butanone
d,l-Glyceraldehyde
Hydroxyacetone
d-Erythrose
0.04
0.13
0.06
0.12
Fig. 4. Purification of the DAK protein from tomato. Lane A, crude
cell extract of XL1-pDAK; lane B, DAK protein after anion exchange
chromatography with Sepharose Q; lane C, DAK protein after anion
exchange chromatography with Mono Q; lane D, molecular weight
markers.
l-Erythrulose
d-Ribulose
d-Ribose
d-Xylulose
Glycerol
0.07
0.03
0.31
0.24
<0.01
<0.01
<0.01
<0.01
Erythritol
d-Threitol
l-Threitol
instead of riboflavin for growth since diacetyl 9 con-
denses spontaneously with 5 under formation of 6
(Fig. 5) (Plaut and Harvey, 1971; Bacher and Mai-
lander, 1978). Quantitative growth experiments sug-
gested that the yeast extract used for the preparation of
LB medium used in this study contained approximately
1 mg of 3,4-dihydroxy-2-butanone per g of dry powder.
Enzyme activity was determined with the photometric assay described
under Experimental.
and Streptomyces coelicolor (Fig. 3). The two DAK
proteins of the eubacteria Citrobacter freundii and
Mesorhizobium loti are surprisingly similar to the
orthologous proteins of the plants L. esculentum and
A. thaliana (about 43% identity). This may reflect a
horizontal transfer of plant genes to plant-associated
microorganisms.
The DAK protein of tomato shows a significant
degree of internal sequence similarity between the N-
terminal and C-terminal parts (34 identical amino acids
equivalent to about 17% identity) (Fig. 6). This suggests
tentatively that the peptide folds into two domains with
similar topology. This hypothesis is well in line with the
finding that substantially shorter genes with similarity
to the 50 part of the plant dak genes are found in
numerous eubacteria where they have been annotated as
putative dihydroxy acetone kinases. The second puta-
tive domain of the eukaryotic dak kinases is a putative
phosphatase (see Conserved Domain Database at
http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).
The expression of dihydroxyacetone kinase from S.
cerevisiae specified by dak1 increases fourfold under
3. Discussion
The dak gene of tomato specifies a kinase accepting
a variety of simple carbohydrates as substrate. The
presence of a carbonyl group and a chain length of
3–5 appears to be required for substrate activity. The
recombinant gene could be expressed efficiently in a
bacterial host strain.
Two putative orthologs of this were found on chromo-
somes 1 and 3 of Arabidopsis thaliana, and a putative
ortholog fragment was reported from Glycine maxima.
Putative orthologs were also found in the human genome
and in Caenorhabiditis elegans but not in Drosophila
melanogaster. The yeasts Saccharomyces cerevisiae and
Schizosaccharomyces pombe each contain two putative
orthologs. Genes with similarity to the tomato dak
gene over its full length were also found in the
eubacteria Citrobacter freundii, Mesorhizobium loti

8
S. Herz et al. / Phytochemistry 60 (2002) 3–11
Fig. 5. Formation of 3,4-dihydroxy-2-butanone 4-phosphate (2) from 3,4-dihydroxy-2-butanone (3) under catalysis of dihydroxyacetone kinase and
detection of 2 as lumazine 6.
Fig. 6. Internal sequence homology of the kinase (N-terminus) and putative phosphatase (C-terminus) domain of dihydroxyacetone kinase from
tomato. The sequences were aligned with the PILEUP program using the default parameters. Identical residues were shown in inverse contrast,
similar residues were shaded in grey.
osmotic stress (Norbeck and Blomberg, 1997). It was
also reported that the accumulation of glycerol is the
main response to osmotic stress in S. cerevisiae (Olz et
al., 1993). Therefore, it has been proposed that the
enzyme is required for glycerol catabolism and thus
regulates the glycerol concentration in yeast cells (Nor-
beck and Blomberg, 1997). On the hypothesis, that the
mechanism for osmotolerance is conserved in eukar-
yotes it can be suggested, that the plant homolgs have
similar functions (Lee et al., 1999).
The outcome of the complementation experiment
reported in this paper is best explained by the hypoth-
esis that the yeast extract used for the preparation of the
culture medium contained an appreciable amount of
3,4-dihydroxy-2-butanone as estimated by bioassay with
the recombinant E. coli strain XL1-pDAK. 3,4-Di-
hydroxy-2-butanone did not originate during heat ster-
ilization of the medium since similar results were
obtained with autoclaved and sterile-filtered medium,
respectively.

S. Herz et al. / Phytochemistry 60 (2002) 3–11
9
Table 3
Bacterial strains and plasmids used
Strain/plasmid
Relevant characteristics
Reference
Strains
E. coli XL1-Blue
recA1,endA1,gyrA96,thi-1,hsdR17,supE44,relA1,
lac [F0,proAB,lacIqZM15,Tn10(tetr]
Stratagene, La Jolla, USA
Rib 5, Rib 10
Fꢀ, thiꢀ, leuꢀ proꢀ, lacꢀ, galꢀ, araꢀ, mtlꢀ, xylꢀ,
supE44, endAꢀ, hsd(rꢀ,mꢀ), pheS, recAꢀ, ribB
Richter et al., 1993; Katzenmeier, 1991
Plasmids
pBluescript II SK-
p10–20
High-copy-number phagemid vector
pBluescriptIISK- with 2.2 kb fragment of tomato DNA
pNCO113 with dak-gene (tomato)
Expression vector
Stratagene, La Jolla, USA
This study
This study
pDAK
pNCO113
Richter et al., 1997
Table 4
Oligonucleotides used in this study
Designation
Sequence
MU
MR
50-CGCAGCTGCTCTGTACCAC-30
50-CTGACTGCAGCTATCTGTGGG-30
MU2
MR2
50-CATGGTCTTCAAGTATAGCCAC-30
50-CACGCTTCTTGTTCACAAGGTTG-30
MU3
MR3
50-CATTAATCAGGAGCACGACTCTAC-30
50-GGCTTTTCAATTTCGGTAATGAAGG-30
50-GCAGTCAAACCAAAATTCAGTCG-30
MU4
MR4
50-GCGGTATCTTATACAGTATATTCTG-30
50-GAGGAGAAATTAACTATGGATTTTCAGAGCAAAAAAC-30
50-TATTATGGATCCTTATGCTGTAATTATACTTCTCC-30
50-CAATTTGAATTCATTAAAGAGGAGAAATTAACTATG-30
DHK1
DHK2
BSECORI
A literature search for the occurrence of 3,4-di-
hydroxy-2-butanone in natural materials retrieved no
results except for cigarette smoke condensate (Schu-
macher et al., 1977), and the origin of 3,4-dihydroxy-
2-butanone which appears to be present in yeast extract
remains unknown. An anaerobic fermentation process
similar to those affording butanediol, 3-hydroxy-
2-butanone and propanediol can be considered. The
recombinant E. coli ribB mutant carrying the pDAK
plasmid could be used to screen for the formation of
3,4-dihydroxy-2-butanone by a simple bioassay.
Recombinant 6,7-dimethyl-8-ribityllumazine synthase
of Bacillus subtilis was prepared by published proce-
dures (Bacher et al., 1997b). Oligonucleotides were
custom-synthesized by MWG Biotech, Ebersberg, Ger-
many. Polyols and carbohydrates were purchased from
Fluka (Buchs, Switzerland) and Sigma (Deisenhofen,
Germany).
4.2. Bacterial strains, media and plasmids
Bacterial strains and plasmids used in this study are
summarized in Table 3. E. coli strains were cultured in
Luria Bertani (LB) medium or minimal medium (Sam-
brook et al., 1989) containing 10 mg lꢀ1 thiamine, 50 mg
4. Experimental
ꢁ
lꢀ1 leucine and 50 mg lꢀ1 proline at 37 C. Ampicillin
4.1. Materials
was added at a concentration of 150 mg lꢀ1 and ribo-
flavin at a concentration of 400 mg lꢀ1 (dissolved at
65 C) as required. A cDNA library of tomato flowers
(Schmid et al., 1992) was a gift of J. Schmid.
ꢁ
5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
(5) was freshly prepared by catalytic hydrogenation
of 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
(Cresswell and Wood, 1960). dl-, d- and l-3,4-di-
hydroxy-2-butanone (3) and 6,7-dimethyl-8-ribityllu-
mazine (6) were prepared as described (Fischer et al.,
1937; Kis et al., 1995; Plaut and Harvey, 1971).
4.3. Isolation of tomato DNA
Lycopersicon esculentum pulp (5 g) was powdered in
liquid nitrogen. The frozen powder was suspended in

10
S. Herz et al. / Phytochemistry 60 (2002) 3–11
30 ml of 100 mM Tris–hydrochloride, pH 8.0, con-
taining 50 mM EDTA, 500 mM NaCl and 10 mM
2-mercaptoethanol. SDS-solution (20%, 2 ml) was
added, and the mixture was incubated for 10 min at
methanesulfonyl fluoride and 1 mg of lꢁysozyme. The
suspension was incubated for 1 h at 37 C, cooled on
ice, disrupted by sonication, and centrifuged. The
supernatant was applied to an anion exchange column
Sepharose Q (Amersham Pharmacia Biotech; volume,
30 ml) which had been equilibrated with 20 mM Tris–
hydrochloride, pH 8.5. The column was developed with
a gradient of 0 to 1 M NaCl containing 20 mM Tris–
hydrochloride, pH 8.5. Fractions were collected, desal-
ted with a Highprep 26/10-column (Amersham Phar-
macia Biotech) and applied to a Mono Q column
(Amersham Pharmacia Biotech; volume, 35 ml) which
had been equilibrated with 20 mM Tris–hydrochloride,
pH 8.5. The column was developed with a gradient of 0
to 1 M KCl containing 20 mM Tris–hydrochloride, pH
8.5. Fractions of the protein which appeared at a gra-
dient concentration of about 250 mM were collected
and concentrated by ultrafiltration using 30 kDa mem-
branes (Pall, Ann Arbor, MI, USA).
ꢁ
65 C. After the addition of 10 ml of 5 M potassiꢁum
acetate, the mixture was incubated for 20 min at 0 C.
The suspension was centrifuged and passed through a
0.2 mm filter. DNA was precipitated with 20 ml iso-
propanol and redissolved in 1.4 ml of 50 mM Tris–
hydrochloride containing 10 mM EDTA. The solution
was centrifuged, and DNA was precipitated by the
addition of 0.15 ml of 3 M sodium acetate and 1 ml of
iso-propanol to the supernatant. The pellet was washed
twice with 70% ethanol and redissolved in 0.2 ml redis-
tilled H2O.
4.4. Plasmid isolation
Plasmid DNA was isolated from 5 ml of fresh over-
night culture using the mini plasmid isolation kit from
Quiagen (Hilden, Germany).
4.8. Electrophoresis
4.5. DNA sequence determination
Sodium dodecyl sulfate polyacrylamide gel electro-
phoresis was performed as described by Lammli (1970).
Sequencing was performed by the automated dideoxy-
nucleotide method (Sanger et al., 1977) using an ABI
Prism 377 DNA sequencer from Perkin Elmer (Foster
City, USA) with the ABI Prism Sequencing Analysis
Software.
4.9. Fluorimetric assay of 3,4-dihydroxy-2-butanone
kinase activity of dihydroxyacetone kinase
Assay mixtures contained 250 mM K2HPO4, pH 7.5,
10 mM MgCl2, 16 mM ATP, 20 mM DTT, 0.9 mM 5,
6.7 mM 3,4-dihydroxy-2-butanone, and 90 mg of
recombinant lumazine synthase from B. subtilis in a
total volume of 75 ml. The assay was started by the
addition of protein solution (25 ml), and the mixture was
4.6. Construction of an expression clone
A segment of plasmid p10–20 was amplified by PCR
using the primers DHK1 and DKH2 (Table 4). The
amplificate served as template in a second PCR ampli-
fication using the oligonucleotides BSECORI and
DHK2 as primers. The amplificate was treated with
BamH1 and EcoRI. The DNA fragment was purified
using the PCR purification kit from Qiagen and was
then ligated into the expression vector pNCO113 which
had been pretreated with the same restriction enzymes.
The resulting plasmid pDAK was transformed into E.
coli XL1-Blue yielding strain XL1-pDAK.
ꢁ
incubated at 37 C. Aliquots (10 ml) were retrieved at
intervals, and 15% trichloroacetic acid was added to a
final volume of 100 ml. The samples were analyzed by
reversed-phase HPLC using a column of Nucleosil
RP18 (4ꢂ300 mm). The eluent contained 30 mM formic
acid and 10% methanol. The effluent was monitored
fluorimetrically (excitation, 408 nm; emission, 487 nm);
synthetic 6 was used as standard.
4.10. Photometric assay of polyol kinase activity of
dihydroxyacetone kinase
4.7. Protein purification
The recombinant E. coli strain XL1-Blue harboring
plasmid pDAK was grown at 37 ꢁC in 2 l shaking
flasks containing 0.5 l of LB medium containing 75 mg
of ampicillin. At an optical density of 0.8 (600 nm),
isopropylthiogalactopyranoside was added to a final
concentration of 1 mM. The suspension was incubated
Assay mixtures contained 50 mM Tris–hydrochloride,
pH 8.0, 50 mM KCl, 1 mM EDTA, 4 mM MgCl2, 1
mM phosphoenol pyruvate, 0.5 mM ATP, 0.12 mM
NADH, 5 U (mmol minꢀ1) lactate dehydrogenase, 5 U
pyruvate kinaꢁse and 2 mM substrate in a total volume
of 1 ml at 37 C. The assay was started by the addition
of 10 ml of protein solution (18 mg) and the absorbance
at 340 nm was recorded (eNADH=6,220 Mꢀ1 cmꢀ1).
Control experiments were performed without addition
of substrate.
ꢁ
with shaking at 37 C for 5 h. The cells were harvested
by centrifugation and were washed with 0.9% NaCl.
Wet cell mass (2 g) was suspended in 10 ml of 20 mM
Tris–hydrochloride, pH 8.5, containing 6 mM phenyl-

S. Herz et al. / Phytochemistry 60 (2002) 3–11
11
characterization of two genes encoding dihydroxyacetone kinase
from Schizosaccharomyces pombe IFO 0354. Biochim. Biophys.
Acta 1442, 361–368.
Acknowledgements
This work was supported by grants from the
Deutsche Forschungsgemeinschaft and the Fonds der
Chemischen Industrie. We thank J. Schmid, ETH Zur-
ich, for a cDNA library from tomato. We thank Sabine
Eberhardt for support during the early phase of the
study. Expert help by Angelika Werner with the pre-
paration of the manuscript is gratefully acknowledged.
Kis, K., Volk, R., Bacher, A., 1995. Biosynthesis of riboflavin. Studies
on the reaction mechanism of 6,7-dimethyl-8-ribityllumazine syn-
thase. Biochemistry 34, 2883–2892.
Lammli, U.K., 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lee, J.H., Van Montagu, M., Verbruggen, N., 1999. A highly con-
served kinase is an essential component for stress tolerance in yeast
and plant cells. Proc. Natl. Acad. Sci. USA 96, 5873–5877.
Liao, D.-I., Calabrese, J.C., Wawrzak, Z., Viitanen, P.V., Jordan,
D.B., 2001. Crystal structure of riboflavin synthase. Structure 9,
11–18.
References
Norbeck, J., Blomberg, A., 1997. Metabolic and regulatory changes
associated with growth of Saccharomyces cerevisiae in 1.4 M NaCl.
Evidence for osmotic induction of glycerol dissimilation via the
dihydroxyacetone pathway. J. Biol. Chem. 272, 5544–5554.
Olz, R., Larsson, K., Adler, L., Gustafsson, L., 1993. Energy flux and
osmoregulation of Saccharomyces cerevisiae grown in chemostats
under NaCl stress. J. Bacteriol. 175, 2205–2213.
Bacher, A., 1991. Biosynthesis of flavins. In: Muller, F. (Ed.), Chem-
istry and Biochemistry of Flavoenzymes, Vol. 1. CRC Press, Boca
Raton, pp. 215–259.
Bacher, A., Eberhardt, S., Richter, G., 1996. Biosynthesis of ribo-
flavin. In: Neidhardt, F. (Ed.), Escherichia coli and Salmonella.
American Society for Microbiology, Washington DC, pp. 657–664.
Bacher, A., Mailander, B., 1978. Biosynthesis of riboflavin in Bacillus
subtilis: function and genetic control of the riboflavin synthase
complex. J. Bacteriol. 134, 476–482.
Plaut, G.W.E., Harvey, R.H., 1971. The enzymatic synthesis of ribo-
flavin. Flavin and derivatives. Methods Enzymol. 18b, 515–538.
Richter, G., Fischer, M., Krieger, C., Eberhardt, S., Luttgen, H.,
Gerstenschlager, I., Bacher, A., 1997. Biosynthesis of riboflavin:
characterization of the bifunctional deaminase-reductase of Escher-
ichia coli and Bacillus subtilis. J. Bacteriol. 179, 2022–2028.
Richter, G., Kelly, M.J.S., Krieger, C., Yu, Y., Bermel, W., Karlsson,
G., Bacher, A., Oschkinat, H., 1999. NMR studies on the 46-kDa
dimeric protein, 3,4-dihydroxy-2-butanone 4-phosphate synthase,
using 2H, 13C, and 15N-labelling. Eur. J. Biochem. 261, 57–65.
Richter, G., Volk, R., Krieger, C., Lahm, H., Rothlisberger, U.,
Bacher, A., 1992. Biosynthesis of riboflavin: cloning, sequencing, and
expression of the gene coding for 3,4-dihydroxy-2-butanone 4-phos-
phate synthase of Escherichia coli. J. Bacteriol. 174, 4050–4056.
Richter, G., Ritz, H., Katzenmeier, G., Volk, R., Kohnle, A., Lott-
speich, F., Allendorf, D., Bacher, A., 1993. Biosynthesis of ribo-
flavin: cloning, sequencing, mapping, and expression of the gene
coding for GTP cyclohydrolase II in Escherichia coli. J. Bacteriol.
175, 4045–4051.
Bacher, A., Richter, G., Ritz, H., Fischer, M., Krieger, C., 1997a.
Biosynthesis of riboflavin: GTP cyclohydrolase II, deaminase, and
reductase. Methods Enzymol. 280, 382–389.
Bacher, A., Eberhardt, S., Fischer, M., Mortl, S., Kis, K., Kugelbrey,
K., Scheuring, J., Schott, K., 1997b. Biosynthesis of riboflavin:
lumazine synthase and riboflavin synthase. Methods Enzymol. 280,
389–399.
Cresswell, R.M., Wood, H.C.S., 1960. The biosynthesis of pteridines.
I. The synthesis of riboflavin. J. Chem. Soc., 4768–4775.
Daniel, R., Gottschalk, G., 1992. Growth temperature-dependent
activity of glycerol dehydratase in Escherichia coli expressing the
Citrobacter freundii dha regulon. FEMS Microbiol. Lett. 100, 281–
286.
Fischer, H.O.L., Baer, E., Pollok, H., Niedecker, J., 1937. Synthesen
niederer Methylzucker. Helv. Chim. Acta 20, 1213–1226.
Herz, S., Eberhardt, S., Bacher, A., 200. Biosynthesis of riboflavin in
plants. The ribA gene of Arabidopsis thaliana specifies a bifunctional
GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate syn-
thase. Phytochemistry 53, 723–731.
Sambrook, J., Fritsch, E.F., Maniatis, M.. In: Sambrook, J. (Ed.),
Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY.
Kato, N., Yoshikawa, H., Tanaka, K., Shimao, M., Sakazawa, C.,
1988. Dihydroxyacetone kinase from the methylotrophic yeast;
Hansenula polymorpha CBS 4732. Arch. Microbiol. 150, 155–159.
Katzenmeier, G., 1991. Klonierung und Molekularbiologische Char-
akterisierung des Gens fur die GTP-Cyclohydrolase I aus Escher-
ichia coli. PhD thesis, Technische Universitat Munchen, Germany.
Kelly, M.J.S., Krieger, C., Ball, L.J., Yu, Y., Richter, G., Schmieder,
P., Bacher, A., Oschkinat, H., 1999. Application of amino acid type-
Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with
chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–
5468.
Schmid, J., Schaller, A., Leibinger, U., Boll, W., Amrhein, N., 1992.
The in-vitro synthesized tomato shikimate kinase precursor is enzy-
matically active and is imported and processed to the mature
enzyme by chloroplasts. Plant Journal 2, 375–383.
Schumacher, J.N., Green, C.R., Best, F.W., Newell, M.P., 1977.
Smoke composition. An extensive investigation of the water-soluble
portion of cigarette smoke. J. Agric. Food Chem. 25, 310–320.
Shavlovskii, G.M., Tesliar, G.E., Strugovshchikova, L.P., 1982. Fla-
vinogenesis regulation in riboflavin-dependent Escherichia coli
mutants. Mikrobiologiia 51, 986–992.
1
specific H- and 14N-labeling in a 2H-, 15N-labeled background to a
47 kDa homodimer: potential for NMR structure determination of
large proteins. J. Biomol. NMR 14, 79–83.
Kelly, M.J.S., Ball, L.J., Krieger, C., Yu, Y., Fischer, M., Schiffmann,
S., Schmieder, P., Kuhne, R., Bermel, W., Bacher, A., Richter, G.,
Oschkinat, H., 2001. The NMR structure of the 47-kDa dimeric
enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase and ligand
binding studies reveal the location of the active site. Proc. Natl.
Acad. Sci. USA 98, 13025–13030.
Volk, R., Bacher, A., 1988. Biosynthesis of riboflavin. The structure of
the four-carbon precursor. J. Am. Chem. Soc. 110, 3651–3653.
Volk, R., Bacher, A., 1990. Studies on the 4-carbon precursor in the
biosynthesis of riboflavin. Purification and properties of
L-3,4-dihydroxy-2-butanone-4-phosphate synthase. J. Biol. Chem.
265, 19479–19485.
Kimura, T., Takahashi, M., Yoshihara, K., Furuichi, T., Suzuki, K.,
Imai, K., Karita, S., Sakka, K., Ohmiya, K., 1998. Cloning and