(Received for publication, May 2, 1995; and in revised form, July 24, 1995)
From the
6,7-Dimethyl-8-ribityllumazine, the immediate biosynthetic
precursor of riboflavin, is synthesized by condensation of
5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
with 3,4-dihydroxy-2-butanone 4-phosphate. The gene coding for
6,7-dimethyl-8-ribityllumazine synthase in Saccharomyces cerevisiae (RIB4) has been cloned by functional complementation of a
mutant accumulating
5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione,
which can grow on riboflavin- or diacetyl- but not on
3,4-dihydroxy-2-butanone-supplemented media. Gene disruption of the
chromosomal copy of RIB4 led to riboflavin auxotrophy and loss
of enzyme activity. Nucleotide sequencing revealed a 169-base pair open
reading frame encoding a 18.6-kDa protein. Hybridization analysis
indicated that RIB4 is a single copy gene located on the left
arm of chromosome XV. Overexpression of the RIB4 coding
sequence in yeast cells under the control of the strong TEF1 promoter allowed ready purification of
6,7-dimethyl-8-ribityllumazine synthase to apparent homogeneity by a
simple procedure. Initial structural characterization of
6,7-dimethyl-8-ribityllumazine synthase by gel filtration
chromatography and both nondenaturing pore limit and SDS-polyacrylamide
gel electrophoresis showed that the enzyme forms a pentamer of
identical 16.8-kDa subunits. The derived amino acid sequence of RIB4 shows extensive homology to the sequences of the
subunits of riboflavin synthase from Bacillus subtilis and
other prokaryotes.
Riboflavin, vitamin B, is the precursor of flavin
mononucleotide and flavin adenine dinucleotide, which function as
coenzymes for a wide variety of enzymes in intermediate metabolism.
Whereas lower organisms are able to biosynthesize riboflavin, mammals
have lost this capacity and, therefore, rely on its dietary ingestion
to meet their metabolic needs. As a consequence, this compound is
commercially important as an additive in food industries, and several
flavinogenic microorganisms, including some yeast species, are used in
industry to produce riboflavin by fermentation(1) .
The yeast Saccharomyces cerevisiae has been thoroughly used to analyze flavinogenesis biochemically and genetically (2) and some yeast species have been developed for the biotechnological production of riboflavin(1) . Six complementation groups of S. cerevisiae riboflavin auxotrophs (rib1 to rib5 and rib7) have been identified(3, 4) . The immediate precursor of riboflavin, 6,7-dimethyl-8-ribityllumazine (see Fig. 1, 4), is formed by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (see Fig. 1, 3) with 3,4-dihydroxy-2-butanone 4-phosphate (see Fig. 1, 2). Two types of mutants, rib3 and rib4, have been shown to be impaired in the synthesis of the immediate precursor of riboflavin, 6,7-dimethyl-8-ribityllumazine, but they have not been correlated with specific defects in the synthesis of riboflavin. We report here that rib4 mutants are defective in 6,7-dimethyl-8-ribityllumazine synthase.
Figure 1: Biosynthesis of 6,7-dimethyl-8-ribityllumazine. 1, ribulose-5-phosphate; 2, 3,4-dihydroxy-2-butanone 4-phosphate; 3, 5amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione; 4, 6,7-dimethyl-8-ribityllumazine.
To construct the TEF1-RIB4 gene fusion, appropriately designed oligonucleotides and polymerase chain reactions were used. Firstly, a fragment containing the 5` region of the TEF1 gene (nucleotides -635 to 3) (17) was amplified using oligonucleotides Tef1 and Tef2 and genomic DNA from strain X2180-1A as template. Secondly, another fragment containing nucleotides -18 to 3 of TEF1 joined to the coding and terminator sequences of RIB4 (nucleotides 3-726) was amplified using oligonucleotides RIB41 and RIB42 and DNA of pJR633 as template. Finally, the two partially overlapping fragments generated in the two previous reactions were joined by polymerase chain reaction in a contiguous fragment (containing nucleotides -635 to -1 of TEF1 fused to nucleotides 1-726 of RIB4) using oligonucleotides Tef1 and RIB42 and the two previously amplified fragments as templates. The resulting fragment was verified by sequencing and cloned into YEp352 to yield pJR627.
DNA sequencing reactions were performed using the dideoxy chain reaction (21) using the T7 sequencing system (Pharmacia). Primer extension analyses were done as described (18) using the synthetic oligonucleotide RIB43, which is complementary to nucleotides 57-80 of the RIB4 ORF.
Figure 2:
Characterization and deletion of the RIB4 locus. A, restriction map of the isolated 6.6-kb
fragment and complementation analysis of different subclones in the
multicopy plasmid YEp352. Plus and minus signs indicate whether this fragment complements (+) or does not
complement(-) the riboflavin auxotrophy of strain AJ21 (rib4-1). B, RIB4 gene deletion. A
0.5-kb SspI-PstI fragment encompassing most of
the RIB4 ORF was replaced by a 1.1-kb URA3 gene (left). Genomic DNA was extracted from wild-type (TD30) and
deleted rib411::URA3 mutant (AJ106) strains and
digested with ClaI. The digestion products were separated on a
1% agarose gel, transferred to a nylon membrane, and probed with a
P-labeled, 1.5-kb ClaI fragment (right).
Based on the phenotype of the rib4-1 mutation, disruption of the RIB4 locus
was not expected to be lethal but should result in riboflavin
auxotrophy. To inactivate RIB4 function, the RIB4 gene of the haploid, wild-type TD30 strain was disrupted by the
one-step gene disruption method(13) . A deletion/substitution
of the 0.5-kb SspI-PstI fragment, spanning the
cluster of restriction sites, by a 1.1-kb fragment containing the URA3 gene was created as described under ``Experimental
Procedures'' (Fig. 2B). Uracil prototroph
(Ura) transformants were riboflavin auxotrophs
(Rib
) as determined by replica-plating onto complete
media lacking riboflavin. Moreover, these transformants showed the rib4 phenotype (i.e. accumulation of
5-amino-6-ribitylamino-2, 4(1H,3H)-pyrimidinedione
and the ability to grow on diacetyl but not on
3,4-dihydroxy-2-butanone-supplemented minimal medium). A Southern
hybridization analysis of genomic DNA of the parental strain TD30 and
one Rib
Ura
transformant, designated
AJ106 (rib4
11::URA3), were used to confirm
genomic integration and deletion at the correct site (Fig. 2B). Strain AJ106 was crossed with strain AJ22;
the resulting diploid had an Rib
phenotype,
suggesting that the two mutations are allelic. Finally, the diploid was
sporulated, and 15 tetrads were dissected. As expected, all tetrads
analyzed segregated four Rib
spores, showing the
identity of the cloned gene and RIB4.
Figure 3: Nucleotide sequence and predicted amino acid sequence of the RIB4 gene. Amino acids are shown in three-letter codes. The numbering adopted for both nucleotides and amino acids is initiated at the first ATG of the RIB4 ORF. The consensus TATA element is underlined, and the four repeats are underlined with arrows. Two downwardarrowheads indicate the major transcription initiation sites. In the 3`-flanking region, sequences matching the transcription termination and polyadenylation signals are indicated by open circles and filled circles, respectively.
The predicted encoded product is a slightly charged protein with a calculated pI of 6.61. Hydropathy analysis (32, 33) predicts a moderately hydrophilic, globular protein with no sequence of sufficient length and hydrophobicity to be considered a membrane-spanning domain. From the distribution of the codons used, a codon adaptation index of 0.271 can be calculated(34) , which suggests a weak or moderate level of gene expression.
Direct evidence for the expression of the RIB4 open reading frame came from Northern blot analysis of S. cerevisiae mRNA using the 0.3-kb BamHI-PstI containing part of RIB4 as a probe. In the experiments of Fig. 4A, mRNA transcripts of the anticipated size for RIB4, approximately 700 nucleotides, were readily detected even in the presence of saturating concentrations of riboflavin in the culture medium, suggesting that the endogenous RIB4 gene is constitutively expressed in yeast. We have also determined the initiation sites for RIB4 transcription by primer extension analyses (Fig. 4B). Two major initiation sites occur roughly 60 nucleotides downstream from the TATA box-like sequence CATATAAA and therefore occur well within the region predicted for a S. cerevisiae TATA box(35, 36, 37) .
Figure 4:
Analysis of the RIB4 transcript. A, Northern analysis. Total RNA (50 µg) from wild-type
strain X2180-1A grown on different culture media was purified,
electrophoresed, transferred to a nylon membrane, and probed with a P-labeled fragment containing part of the RIB4 ORF. Lane 1, minimal medium; lane 2, minimal
medium supplemented with 200 µg/ml riboflavin; lane 3,
rich medium supplemented with 200 µg/ml riboflavin. The ACT1 transcripts serve as an internal control. B, Primer
extension analysis. A synthetic 24-mer oligonucleotide (RIB43),
complementary to nucleotides 57-80 of the RIB4 ORF, was
annealed to 25 µg of poly(A)
RNA from the
wild-type strain X2180-1A (lane 1) or 25 µg of yeast
tRNA (lane 2) and extended with avian myeloblastosis virus
reverse transcriptase. The extension products were run on a denaturing
polyacrylamide gel next to a sequencing reaction using the same
oligonucleotide as sequencing primer. The positions of the major
extension products are indicated on the right.
Figure 5: SDS-PAGE analysis of fractions collected during purification of lumazine synthase. Proteins were resolved on 14% SDS-polyacrylamide gels and were visualized by silver staining. Molecular masses of standards (kDa) are indicated on the left. The position of Rib4p is marked with an arrow. T, total cell lysate; M, 50% methanol-soluble fraction; Q, MonoQ; S, SuperDex 200.
Two methods were used to estimate the native subunit structure of lumazine synthase (Fig. 6). Gel filtration chromatography indicated that the molecular mass of the native protein was 95 kDa using a column calibrated with typical globular proteins (Fig. 6A). Nondenaturing pore limit polyacrylamide gels were used to verify the molecular mass determined by gel filtration chromatography. The migration of lumazine synthase in 4-20% gradient nondenaturing polyacrylamide gel compared with the position of standards proteins indicated a molecular mass of 91 kDa (Fig. 6B). Based on these two results, we concluded that lumazine synthase was a homopentamer.
Figure 6:
Characterization of native riboflavin
synthase. A, SuperDex 200 filtration of riboflavin synthase,
with thyroglobulin (a), ferritin (b), catalase (c), lactate dehydrogenase (d), bovine serum albumin (e), ovalbumin (f), carbonic anhydrase (g),
and -lactalbumin (h) as inclusive standards. The
calculated value for lumazine synthase was 95. B, relative
migration of native lumazine synthase in nondenaturing polyacrylamide
(4-20%) gradient gel. Standard proteins used as markers are shown
in the illustration with lumazine synthase M
estimated as 91. Inset, lane 1, markers; lane 2, purified lumazine
synthase.
Figure 7: Sequence comparison among lumazine synthases from S. cerevisiae, E. coli, P. leiognathi, and B. subtilis. The amino acid sequence of yeast lumazine synthase was aligned to the sequences from E. coli, P. leiognathi, and B. subtilis. Amino acids identical in at least two sequences are boxed and highlighted by a gray shaded background. Amino acids identical in all four sequences are boxed and highlighted by a black background.
In this report we describe the isolation, sequence, and expression of a S. cerevisiae gene, RIB4, which encodes lumazine synthase. Nutritional tests using chemically synthesized 3,4-dihydroxy-2-butanone suggested that rib4 mutants would be defective in lumazine synthase, whereas rib3 mutants would be affected in 3,4-dihydroxy-2-butanone synthase. The fact that the 4-carbon unit 3,4-dihydroxy-2-butanone supported the growth of rib3 mutants implies that in vivo this compound is converted into a biologically active precursor molecule of riboflavin, thus compensating for the absence of the 3,4-dihydroxy-2-butanone synthase activity of these mutants. However, the nonphosphorylated form of the 4-carbon unit cannot be used as a substrate in vitro by lumazine synthase (data not shown). Thus, 3,4-dihydroxy-2-butanone must be phosphorylated in vivo in the rib3 mutant yeast cells prior its use as substrate by lumazine synthase.
Analysis of RIB4 transcript levels under different culture conditions indicates that RIB4 is constitutively expressed at moderate levels. Overexpression of the gene resulted in an approximately 90-fold increase in lumazine synthase activity but only in a slight stimulatory effect on the accumulation of free riboflavin in the cells. This suggests that lumazine synthase is not a limiting step in the metabolic flux of riboflavin formation in yeast.
Since the data presented here were obtained, we have
developed a simple method of purification of Rib4p that relies on RIB4 overexpression in yeast cells by using a multicopy
plasmid and placing the RIB4 coding sequence under the control
of the TEF strong promoter. With this approach we were able to
purify the enzyme to apparent homogeneity using a three-step
purification protocol that achieves an overall purification of 120-fold
with a recovery of about 4%. From comparison of either the elution
volume or the R of pure native lumazine synthase
with those of standard proteins by gel filtration analysis or
nondenaturing pore limit PAGE, respectively, a molecular mass of
approximately 90,000 daltons was estimated for the native enzyme.
However, an apparent subunit molecular mass of 24,500 daltons was
obtained for the pure protein by SDS-PAGE analysis, in good agreement
with the predicted molecular mass of Rib4p based on its deduced amino
acid composition. These findings indicate that the native lumazine
synthase of S. cerevisiae exists in the pentamer form of
identical subunits.
Riboflavin synthase appears in B. subtilis as two different species characterized by their subunit
composition. The so-called light enzyme is a trimer of identical subunits of riboflavin synthase. The heavy enzyme,
characterized by a molecular mass of 1 MDa, consists of an
subunit trimer enclosed within an icosahedral structure composed of 60
subunits of 6,7-dimethyl-8-ribityllumazine
synthase(5, 6, 8, 41) .
The
complete sequence of the B. subtilis subunit was first
established by direct sequencing of the protein(42) .
Subsequently, the amino acid sequence of lumazine synthase was
confirmed by sequencing the ribH open reading frame of the B. subtilis riboflavin operon(43) . A computer
prediction of the secondary structure indicated the presence of
approximately 34%
helix, 30%
sheet, and 18%
turn in
the
subunit. Most
helices and
strands predicted by a
modified Chou-Fasman/Robson algorithm (44) in yeast lumazine
synthase (data not shown) match the observed secondary elements in the Bacillus enzyme surprisingly well, thus suggesting a similar
chain folding for the enzyme in both organisms. However, completely
different quaternary structures, pentamers in yeast and icosahedral
capsids in Bacillus, are formed.
The atomic structure of
the Bacillus subunit has been deduced from electron
density maps to 3.3 Å resolution by the group of
Bacher(9) . The main chain of the
subunit folds into a
structure of four parallel
-sheet flanked on both sides by two
-helical segments. Due to the symmetry relations occurring in the
-60 icosahedral capsid, intersubunit contacts are those of dimers,
trimers, and pentamers. In this context, it should be noted that most
amino acid residues contributing to the stability of pentamers in the Bacillus lumazine synthase (Met
, Ile
,
Arg
, Met
, Val
,
Pro
, Phe
, Glu
,
Arg
, Tyr
, Lys
, Pro
,
Gln
, and Thr
) are conserved in the yeast
enzyme. By contrast, a large proportion of amino acids involved in
dimer (Arg
, His
, Gln
,
Asp
, and Val
) or trimer (Asp
,
Lys
, Asp
, Glu
,
Phe
, Phe
, and Thr
) contacts in
the icosahedral Bacillus enzyme are not conserved in yeast.
Therefore, it is tempting to speculate that the discrepancy in the
quaternary structures shown by the Bacillus and yeast enzymes
are the result of different levels of complexity in the same pathway of
protein assembly, the pentamer form corresponding to an intermediate
level and the
-60 icosahedral enzyme being the most complex stage
of organization. Finally, it is assumed that in Bacillus the
His
may take part in the catalytic reaction as a
proton-abstracting base. In agreement with this assumption, this
residue is conserved in all four organisms considered.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z21620[GenBank].