Bacillus subtilis ypgA gene is fni, a nonessential gene encoding type 2 isopentenyl diphosphate isomerase

Article abstract of DOI:10.1271/bbb.68.132

We previously identified the fni gene of Streptomyces sp. strain CL190 as type 2 isopentenyl diphosphate (IPP) isomerase, which needs both FMN and NADPH for enzyme activity. An fni gene homolog, ypgA, was detected in the database of the Bacillus subtilis genome. However, the ypgA product was about 140 amino acids shorter in the N-terminal than the Streptomyces fni gene product. A database search found three new putative start codons in 129, 225, and 411 bases upstream of the original start codon of the ypgA gene. The longest gene product, which was named ypgA3, showed the most significant homology to the Streptomyces fni gene product. The ypgA3 gene was expressed with an N-terminal His-tag in Escherichia coli and the purified soluble protein was characterized in detail. The ypgA3 protein converted IPP to its isomer dimethylallyl diphosphate in the presence of both FMN and NADPH. The enzyme also catalyzed the reverse reaction in the presence of both the cofactors. Disruption of the ypgA3 gene was not lethal to B. subtilis. These results indicate that Bacillus ypgA3 gene is fni, a nonessential gene encoding type 2 IPP isomerase.

Full text of DOI:10.1271/bbb.68.132

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Bacillus subtilis ypgA Gene Is fni, a Nonessential
Gene Encoding Type 2 Isopentenyl Diphosphate
Isomerase
Motoki TAKAGIa, Kazuhide KANEDAa, Tomohiro SHIMIZUa, Yoichi HAYAKAWAa, Haruo SETOa
& Tomohisa KUZUYAMAa
a Institute of Molecular and Cellular Biosciences, University of Tokyo
Published online: 22 May 2014.
To cite this article: Motoki TAKAGI, Kazuhide KANEDA, Tomohiro SHIMIZU, Yoichi HAYAKAWA, Haruo SETO & Tomohisa
KUZUYAMA (2004) Bacillus subtilis ypgA Gene Is fni, a Nonessential Gene Encoding Type 2 Isopentenyl Diphosphate
Isomerase, Bioscience, Biotechnology, and Biochemistry, 68:1, 132-137, DOI: 10.1271/bbb.68.132
To link to this article: http://dx.doi.org/10.1271/bbb.68.132
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Biosci. Biotechnol. Biochem., 68 (1), 132–137, 2004
Bacillus subtilis ypgA Gene Is fni, a Nonessential Gene Encoding
Type 2 Isopentenyl Diphosphate Isomerase
*
Motoki TAKAGI, Kazuhide KANEDA, Tomohiro SHIMIZU, Yoichi HAYAKAWA,
y
**
Haruo SETO, and Tomohisa KUZUYAMA
Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
Received July 18, 2003; Accepted September 5, 2003
We previously identified the fni gene of Streptomyces
sp. strain CL190 as type 2 isopentenyl diphosphate (IPP)
isomerase, which needs both FMN and NADPH for
enzyme activity. An fni gene homolog, ypgA, was
detected in the database of the Bacillus subtilis genome.
However, the ypgA product was about 140 amino acids
shorter in the N-terminal than the Streptomyces fni gene
product. A database search found three new putative
start codons in 129, 225, and 411 bases upstream of the
original start codon of the ypgA gene. The longest gene
product, which was named ypgA3, showed the most
significant homology to the Streptomyces fni gene
product. The ypgA3 gene was expressed with an N-
terminal His-tag in Escherichia coli and the purified
soluble protein was characterized in detail. The ypgA3
protein converted IPP to its isomer dimethylallyl
diphosphate in the presence of both FMN and NADPH.
The enzyme also catalyzed the reverse reaction in the
presence of both the cofactors. Disruption of the ypgA3
gene was not lethal to B. subtilis. These results indicate
that Bacillus ypgA3 gene is fni, a nonessential gene
encoding type 2 IPP isomerase.
frame of Streptomyces sp. strain CL190 as a new type of
IPP isomerase that strictly needs both FMN and NADPH
for enzyme activity.7) The amino acid sequence of this
new IPP isomerase, designated as type 2 IPP isomerase,
shares no similarity to those of the idi gene products.
The fni gene homologs encoding type 2 IPP isomerase
are found in the gene cluster of the mevalonate pathway
not only from Streptomyces sp. strain CL190, but also
from Borrelia burgdorferi, and Gram-positive cocci
such as Staphylococcus aureus and Enterococcus fae-
calis. This fact implied that the fni gene is specific for
the mevalonate pathway.
Two distinct biosynthetic pathways for IPP and
DMAPP are known (Fig. 1).8–12) One is the well-known
mevalonate pathway (Fig. 1A), which operates in
eukaryotes, archaebacteria, Gram-positive cocci, and
cytosols of higher plants. The other is a recently
discovered pathway, the 2-C-methyl-D-erythritol 4-
phosphate (MEP) pathway (formerly called the non-
mevalonate pathway) (Fig. 1B), which is used by most
eubacteria, green algae, and the chloroplasts of higher
plants. In E. coli with the MEP pathway, IPP isomerase
is not essential, because IPP and DMAPP are biosyn-
thesized independently (Fig. 1B).
Bacillus subtilis has all the genes of the MEP pathway
and therefore uses this pathway for the biosynthesis of
IPP and DMAPP.13) In addition to these genes, an fni
gene homolog, ypgA, was detected in the database of the
B. subtilis genome.14) However, it was ambiguous that
the ypgA gene product actually had IPP isomerase
activity, because the amino acid sequence of the gene
product was about 140 amino acids shorter in the N-
terminal than the fni gene products from Streptomyces
sp. strain CL190 and S. aureus.
In these studies, we found an actual start codon for the
ypgA gene. In this paper, we describe the first unambig-
uous assignment of the ypgA gene product as type 2 IPP
isomerase and the enzymatic properties of the ypgA gene
products overexpressed in E. coli.
Key words: isoprenoid; biosynthesis; Bacillus subtilis;
isomerase; 2-C-methyl-D-erythritol 4-phos-
phate (MEP) pathway
All terpenoids are synthesized by consecutive con-
densations of a five-carbon-basic unit, isopentenyl
diphosphate (IPP) to its isomer, dimethylallyl diphos-
phate (DMAPP).1) IPP isomerase (EC 5.3.3.2) catalyzes
an important reaction in the biosynthesis of terpenoids
by converting IPP to DMAPP (Fig. 1).2) Many IPP
isomerase genes, which are encoded by idi genes, have
been cloned from various organisms such as humans,3)
Saccharomyces cerevisiae,4) Escherichia coli,5) and
Rhodobacter capsulatus.6) This enzyme needs only a
divalent cation such as Mg2þ for its enzyme activity.
However, we previously identified an open reading
y
To whom correspondence should be addressed. Fax: +81-3-5841-8485; E-mail: kuz@iam.u-tokyo.ac.jp
*
Present address: Shizuoka Eiwa Gakuin University Junior College, 1769 Ikeda, Shizuoka, Shizuoka 422-8005, Japan
Present address: Department of Applied Biology and Chemistry, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan
Abbreviations: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate
**

Characterization of the Bacillus subtilis ypgA Gene Product
133
Fig. 1. Mevalonate and MEP Pathways for IPP and DMAPP Biosyntheses.
In the MEP pathway, both IPP and DMAPP are formed from HMBDP by the action of the lytB gene product. HMG-CoA, 3-hydroxy-3-
methylglutaryl-CoA; DPMVA, diphosphomevalonate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; DXP, 1-deoxy-D-
xylulose 5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; MECDP, 2-C-methyl-D-erythritol 2,4-diphosphate; HMBDP, E-4-hydroxy-3-
methyl-2-butenyl diphosphate.
Materials and Methods
CACTATAGCTTGATGTATTG-30 (30 of ypgA3), in-
cluding a BamHI restriction site (underlined) were
synthesized (Sigma) and used together with total DNA
from B. subtilis 168 to amplify ypgA3. By using Taq
DNA polymerase (Roche Molecular Biochemicals) and
the protocol recommended by the supplier, a 1.1-kb
fragment was amplified. The PCR fragment was
digested with BamHI and cloned into the multi-cloning
site of the expression vector pQE30 to give pQEYpgA3.
Strains and vectors. B. subtilis 168 strain (wild-type)
and the ypgA gene-disruptant were kindly provided by
Prof. Ogasawara of the Nara Institute of Science and
Technology. This disruptant has the erythromycin-
resistance gene in the ypgA gene. pQE30, an expression
vector in E. coli, was purchased from Qiagen.
Growth conditions for B. subtilis. Fresh B. subtilis
cells were suspended in LB medium and the optical
density at 655 nm of this cell suspension was adjusted to
10ꢀ3. One hundred ꢀl of the cell suspension was
incubated in a well of a 96-well microplate at 37ꢁC with
shaking (700 rpm/min). The growth of the cell was
monitored at 655 nm with a Benchmark Microplate
Reader (BIORAD) adjusted at 37ꢁC.
Expression and Purification of the Recombinant
ypgA3 Product. E. coli M15 containing pREP4 [neo,
lacI] (Qiagen) was used as a host for the expression of
ypgA3. M15 (pREP4, pQEYpgA3) was cultured at 18ꢁC
in one liter of LB medium containing 25 ꢀg/ml
kanamycin (Nacalai Tesque, Kyoto) and 200 ꢀg/ml
ampicillin (Sigma) for 6 h with the addition of 0.1 mM
isopropyl-ꢁ-D-thiogalactopyranoside (IPTG) when an
optical density at 660 nm of 0.8 was reached. Cells
were harvested by centrifugation and resuspended in
Buffer A, composed of 100 ml of 0.1 M Tris-HCl (pH
8.0) and 5 mM DTT. After sonication, the lysate was
centrifuged at 10,000 ꢂ g for 20 min and the supernatant
was retained. The crude extract was put on a Ni-
nitrilotriacetic acid agarose column (Qiagen) previously
equilibrated with Buffer A. The resin was washed with
50 mM imidazole in Buffer A and then the protein bound
to the resin was eluted with 200 mM imidazole in
Buffer A.
DNA sequence analysis. The DNA sequence was
analyzed by the dideoxynucleotide chain-termination
method15) with an automated sequencer (model 4000L,
Li-cor) and the protocol of the supplier. The FASTA
program16) did homology searches with protein data-
bases of the DNA Data Bank of Japan (DDBJ) and the
National Center for Biotechnology Information (NCBI).
A GENETYX program ver. 11 (Software Development,
Tokyo, Japan) was used to search for putative start
codons and to align amino acid sequences.
Construction of the Plasmid for Overexpression of
ypgA3 in E. coli. On the basis of the nucleotide sequence
of ypgA3 from B. subtilis 168, two oligonucleotide
primers, 50-GGGGATCC ACTCGAGCAGAACGAAA-
AAGAC-30 (50 of ypgA3) and 50-GGGGATCC TCGCA-
Assay for type 2 IPP isomerase. An assay system for
type 2 IPP isomerase was based on the acid lability of
DMAPP. This assay mixture consisted of 0.1 M HEPES
buffer (pH 7.0), 5 mM MgCl2, 1 mM DTT, 10 ꢀM

134
M. TAKAGI et al.
[1-14C]IPP (2.0 GBq/mmol; American Radiolabeled
Chemicals, USA), 40 ꢀM IPP, 10 ꢀM FMN and 1 mM
NADPH in a final volume of 50 ꢀl. The reaction was
started by adding enzyme to the assay system followed
by incubation routinely at 37ꢁC for 1 min. Further
manipulations were done as described before.7)
Streptomyces sp. strain CL190, indicating that the
ypgA3 product is also a flavoprotein. SDS-PAGE
showed a subunit molecular mass of 41 kDa. The
apparent molecular mass of the ypgA3 product was
estimated to be 135 kDa by Superdex 200 gel filtration.
These data indicate that the ypgA3 product is most likely
to be a tetramer. When we attempted to overexpress the
ypgA, ypgA1, and ypgA2 genes in E. coli by the same
method as that for the ypgA3 gene, we were able to
obtain insoluble proteins but not soluble proteins.
pH-Dependent Activity of IPP isomerase. The IPP
isomerase assay was done as described above. Assay
solutions consisted of 0.1 M MES at pH 5.0–6.5, 0.1 M
HEPES at pH 6.5–7.5 or 0.1 M Tris-HCl at pH 7.0–9.5,
5 mM MgCl2, 10 ꢀM FMN, 5 mM NADPH and 1 mM
DTT.
FMN/NADPH-dependent IPP isomerase activity of
the recombinant ypgA3 product
We next investigated whether the recombinant ypgA3
product catalyzes conversion of IPP and DMAPP in the
presence of FMN and NADPH. As expected, the ypgA3
product showed IPP isomerase activity in the presence
of both the cofactors, confirming that the gene actually
encodes type 2 IPP isomerase (Table 1). The activity
was maximal when 10 ꢀM FMN and 1 mM NADPH were
added. The activity was slightly below maximal when
10 ꢀM FMN and 1 mM NADH was used. However,
replacement of FMN with FAD resulted in the loss of
IPP isomerase activity. Without addition of FMN or
NADPH, no activity was detected, demonstrating that
IPP isomerization catalyzed by the ypgA3 product was
strictly dependent on both FMN and NADPH. This
enzyme required Mg2þ, Mn2þ, or Ca2þ. The activity
was maximal with Mg2þ, but lower with Mn2þ and
Nucleotide sequence accession number. The nucleo-
tide sequence of the fni gene of B. subtilis has been
deposited in the DDBJ/EMBL/GenBank nucleotide
sequence databases with the accession number
AB047343.
Results
Identification of the fni gene homolog in the B. subtilis
genome
To identify fni gene homologs in the B. subtilis
genome, homology searches that use the sequence of the
fni gene product from Streptomyces sp. strain CL190 as
a query sequence was done using databases of DDBJ
and NCBI. This search identified a ypgA gene product
(accession no. L47648) with unknown function.14)
Figure 2 shows the multiple alignment of the amino
acid sequences of the ypgA gene product and the FNI
homologs from Streptomyces and Staphylococcus. The
Bacillus gene product showed significant homology to
these FNI homologs. However, the ypgA product was
about 140 amino acids shorter in the N-terminal than the
FNI homologs. This finding led us to assume that there
is another start codon upstream of the original one. As
expected, a database search found three new putative
start codons in 129, 225, and 411 nucleotides upstream
of the original start codon of the ypgA gene (Fig. 2). The
corresponding genes were named ypgA1, ypgA2, and
ypgA3, respectively. The longest gene product, YPGA3,
showed most significant homology to the Streptomyces
fni gene product, suggesting that the ypgA3 gene
actually encodes Bacillus FNI (Fig. 2). The stop codon
for ypfD gene flanking the ypgA gene was found 15
nucleotides upstream of the start codon for the ypgA3,
also supporting the above suggestion.
Ca2þ. No effects of other divalent cations such as Co2þ
,
Cu2þ, and Zn2þ at 5 mM were detected. Addition of
5 mM EDTA resulted in almost complete loss of the
enzyme activity.
The IPP isomerase encoded by ypgA3 was most active
in HEPES buffer. The optimum activity of the enzyme
occurred at pH 6.5–7.0. The effects of temperature on
the enzyme activity were investigated over the range of
15 to 55ꢁC, with the maximum activity being observed
at approximately 37ꢁC. Table 2 summarizes enzymatic
properties of type 2 IPP isomerases from B. subtilis,
Streptomyces sp. strain CL190,7) and S. aureus.7)
Phenotype of the Bacillus-ypgA3-disruptant
To investigate in vivo function of the ypgA3 gene, a
Bacillus-ypgA3-disruptant was cultivated in liquid LB
medium. The growth curve revealed that the disruption
of the gene had no apparent effects on growth.
Fosmidomycin (3.1 ꢀg/ml), a specific inhibitor of
DXP reductoisomerase of the MEP pathway,17) com-
pletely inhibited the growths of both B. subtilis 168 and
the ypgA3-disruptant, showing that enough IPP and
DMAPP to support growth were not supplied from LB
medium. These results demonstrate that ypgA3 is a
nonessential gene for B. subtilis growth.
Expression and Purification of the recombinant
ypgA3 product
To discover the in vitro function of the ypgA3
product, the ypgA3 was expressed with an N-terminal
His-tag in E. coli using the T7 promoter system and the
product was purified to homogeneity as a soluble
protein. After purification, the concentrated protein
solution was yellow, as was the fni protein from
Discussion
In this study, we have demonstrated that the B.

Characterization of the Bacillus subtilis ypgA Gene Product
135
Fig. 2. Amino Acid Alignment of Type 2 IPP Isomerases and (S)-2-Hydroxy-acid Oxidase.
The putative first methionines of YPGA are marked by arrows. The type 2 IPP isomerases from three origins were aligned with (S)-2-hydroxy-
acid oxidase from A. thaliana using the GENETYX program. Amino acid residues conserved among 3 or 4 proteins are boxed. Dashes indicate
gaps introduced for the optimization of the alignment. The highly conserved His residues are marked by an asterisk and the His254 in the active
site of (S)-2-hydroxy-acid oxidase is marked by a sharp. Bsub, B. subtilis (accession no. AB047343); CL190, Streptomyces sp. strain CL190
(accession no. AB046667); Saur, S aureus (accession no. AB047344); HAO, A. thaliana (S)-2-hydroxy-acid oxidase (accession no. Q9LRR9).
subtilis ypgA3 gene is a nonessential gene encoding type
2 IPP isomerase and compared the kinetic parameters of
the B. subtilis enzyme to those of Streptomyces and
Staphylococcus homologs. B. subtilis enzyme was also a
flavoprotein and had FMN- and NAD(P)H-dependent
IPP isomerase activity. The enzymatic properties of the
B. subtilis enzyme were almost the same as those of the
Streptomyces enzyme (Table 2). However, the Km value
for IPP of the B. subtilis enzyme was about 30-fold
higher than that of the S. aureus enzyme. This higher
value might be explained by the non-essentiality of type
2 IPP isomerase for B. subtilis growth. On the other
hand, type 2 IPP isomerase is essential for the growth of
S. aureus. Therefore, the Km value of type 2 IPP
isomerase of S. aureus could be lower than those of IPP
isomerase from Streptomyces sp. strain CL190 and B.
subtilis, both of which use the MEP pathway for growth.
It should be noted that two types of IPP isomerases
are widespread in microorganisms.12) To the best of our
knowledge, all archaebacteria, which use the mevalo-
nate pathway, have type 2 IPP isomerase. In eubacteria,
Gram-positive cocci such as Staphylococcus, Strepto-
coccus, and Enterococcus, which also use the mevalo-
nate pathway, have type 2 enzyme. E. coli with the MEP
pathway has type 1 IPP isomerase, as do all eukaryotes.
Interestingly, B. subtilis and Synechocystis sp. strain
PCC6803 using the MEP pathway have type 2 IPP
isomerases, which are nonessential for their growth.

136
M. TAKAGI et al.
Table 1. FMN and NAD(P)H Dependence of the Activity of the
Bacillus IPP Isomerase
acid alignment (Fig. 2) identified two conserved regions,
258GG(L/V)(R/Q) and 265D(V/A)X(K/R)(A/S)(I/L)-
ALGA in the B. subtilis FNI. These conserved regions
could in part associate with FMN binding in the enzyme
molecule, because both the enzymes strictly need FMN
as the common cofactor. In addition, His254 in (S)-2-
hydroxy-acid oxidase has been proposed to remove the
ꢂ-proton of the substrate as the first step in catalysis
(Fig. 2).21) In analogy with this enzyme, the highly
conserved His147 in B. subtilis IPP isomerase might be
involved in the removal of 2-H of the IPP molecule
(Fig. 2).
Additions to reaction buffera
FMN or FAD, ꢀM
NAD(P)H, mM
relative activity, %
FMN
0
NADPH
0
ND
2.7
0
1
10
10
0
4.1
1
NADH
1
100
10
FAD
10
97.8
ND
NADPH
1
During preparation of this paper, Steinbacher et al.
reported the crystal structure of type 2 IPP isomerase
from B. subtilis.22) The structural analysis revealed that
the phosphate function of FMN was hydrogen bonded to
the amide of Gly259 of the glycine-rich motif 258GG-
(L/V)(R/Q) mentioned above and to the amides of
Ala280 and Gly28. Also, His147 in the crystal structure
is located near the FMN molecule, suggesting that this
residue could play an important role in catalysis, as
discussed above. This crystal structure will help to
clarify the mechanism of the unusual isomerase reaction.
a
The basal reaction buffer consisted of 0.1 M HEPES (pH 7.0), 5.0 mM
MgCl2, 1 mM DTT, 5 ꢀg enzyme. ND, not detected.
Table 2. Comparison of Enzymatic Properties of Type
Isomerases
2 IPP
B. subtilis CL190* S. aureus*
Km for IPP (ꢀM) 670
Vmax (mmol/min/mg of protein) 400
Optimum temperature (ꢁC)
Optimum pH
Divalent cations
450
490
35
19
270
NA
37
6.5–7.0
Mg2þ
7.0
Mg2þ
NA
Mg2þ
Acknowledgments
Molecular mass
SDS-PAGE (kDa)
Superdex-200 (kDa)
Multimeric form
40
135
42
135
41
155
We sincerely thank Dr. K. Kobayashi, Graduate
School of Information Science, Nara Institute of Science
and Technology, and Dr. Hiromi Nishida, Institute of
Molecular and Cellular Biosciences, The University of
Tokyo, for fruitful discussions. This work was supported
by a Grant-in-Aid for Scientific Research on Priority
Areas, ‘‘Genome Biology’’, to T.K. from the Ministry of
Education, Culture, Sports, Science, and Technology of
Japan.
tetramer tetramer tetramer
*These data were cited from reference number 7.
NA, not available.
Expression analysis with DNA microarray of B. subtilis
has revealed that the ypgA gene is actually expressed in
the exponential growth period (Kobayashi, K., personal
communication), demonstrating that B. subtilis uses the
ypgA gene to supply DMAPP from IPP. On the other
hand, it has been demonstrated that Synechocystis IPP
isomerase had no function in vivo.18,19) Therefore, B.
subtilis represents the first example of a microorganism
that uses both the MEP pathway and type 2 IPP
isomerase for the biosynthesis of the isoprene units, IPP
and DMAPP.
It is also very interesting that two types of the
enzymes showing no sequence homology catalyze the
same reaction. How does nature evolve these two types
of IPP isomerases? What are the roles of FMN and
NADPH in the reaction catalyzed by type 2 IPP
isomerase? These are the next questions to be answered.
In order to gain insight into these questions, homol-
ogy searches that use the sequence of Streptomyces FNI
as a query were done using the FASTA program and the
SWISS-PROT database. Interestingly, this search
identified (S)-2-hydroxy-acid oxidase (accession no.
Q9LRR9) from Arabidopsis thaliana (22.1% identity
in 339 amino acids overlap).20) This enzyme is a
flavoprotein and belongs to the FMN-dependent ꢂ-
hydroxy acid dehydrogenases family. Multiple amino
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