(Received for publication, July 19, 1994; and in revised form, October 24, 1994)
From the
Saccharomyces cerevisiae has a monofunctional
riboflavin synthase that catalyzes the formation of riboflavin from
6,7-dimethyl-8-ribityllumazine. We have isolated the gene encoding this
enzyme from a yeast genomic library by functional complementation of a
mutant, rib5-10, lacking riboflavin synthase activity.
Deletion of the chromosomal copy of RIB5 led to riboflavin
auxotrophy and loss of enzyme activity. Intragenic complementation
between point and deletion mutant alleles suggested that the encoded
protein (Rib5p) assembles into a multimeric complex and predicted the
existence of a discrete functional domain located at the N terminus.
Nucleotide sequencing revealed a 714-base pair open reading frame
encoding a 25-kDa protein. Rib5p was purified to apparent homogeneity
by a simple procedure. The specific activity of the enzyme was enriched
8500-fold. The N-terminal sequence of the purified enzyme was identical
to the sequence predicted from the nucleotide sequence of the RIB5 gene. Initial structural characterization of riboflavin synthase
by gel filtration chromatography and both nondenaturing pore limit and
SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a
trimer of identical 25-kDa subunits. The derived amino acid sequence of RIB5 shows extensive homology to the sequences of the
subunits of riboflavin synthase from Bacillus subtilis and
other prokaryotes. In addition, the sequence also shows internal
homology between the N-terminal and the C-terminal halves of the
protein. Taken together, these results suggest that the Rib5p subunit
contains two structurally related (substrate-binding) but catalytically
different (acceptor and donator) domains.
Riboflavin, vitamin B, is commercially important as
an additive in food industries. The yeast Saccharomyces cerevisiae has been widely used to analyze flavinogenesis biochemically and
genetically(1) , and some yeast species have been developed for
the biotechnological production of riboflavin(2) . Six
complementation groups of S. cerevisiae mutants (rib1, rib2, rib3, rib4, rib5, and rib7) with different defects in riboflavin
biosynthesis have been identified, and their normal genetic functions
have been correlated with specific metabolic steps (3, 4) .
The last step in riboflavin biosynthesis is catalyzed by riboflavin synthase (EC 2.5.1.9). The enzyme catalyzes the dismutation of two molecules of the substrate 6,7-dimethyl-8-ribityllumazine to yield one molecule each of riboflavin and 5-amino-2,6-dihydroxy-4-ribitylamino-pyrimidine. Riboflavin synthase activity has been observed in many microorganisms and in spinach (for a review see (1) ). The protein from bakers' yeast has been partially purified, and a molecular weight in the range of 80,000 has been estimated; but its structure has not been further investigated(5, 6) .
Bacillus subtilis contains two forms of riboflavin synthase characterized by the
subunit structures (light enzyme) and
(heavy enzyme)(7) .
Riboflavin synthase activity resides in the
subunit, whereas the
subunit catalyzes the synthesis of the substrate
6,7-dimethyl-8-ribityllumazine(8, 9) . The complete
amino acid sequence of the
subunit was obtained by direct
sequencing of overlapping peptides. The sequence shows marked internal
homology between the N-terminal and C-terminal halves, thus suggesting
that the monomer forms two structurally similar, substrate-binding,
domains(8) . Sequences have subsequently been reported for the
genes encoding riboflavin synthase from the bacteria Photobacterium
leiognathi(10) and Escherichia coli, on the
basis of homology to B. subtilis sequence data.
Although
early genetic studies provide useful information suggesting the role of RIB5, no further evidence has elucidated the function of the RIB5 gene and its gene product. In this paper we report the
isolation and characterization of the RIB5 gene and the
evidence supporting that RIB5 is the structural gene for
riboflavin synthase in yeast. The gene appears to be expressed
constitutively at very low levels. We also describe the purification of
the enzyme to apparent homogeneity. The M of the
enzyme is about 75,000 with three subunits of M
25,000 each. Our results support the model suggesting that the
monomer would contain two structurally similar, although functionally
different, domains.
The rib514 allele, which lacks
nucleotides 4-15 of the RIB5 ORF, was constructed by the
counter polymerase chain reaction method as described by Hemsley et
al.(20) . pUC19-RIB5, which contains a 2.9-kb KpnI fragment bearing RIB5, was used as the template,
and oligonucleotides RIB56 and RIB57 were used as forward and reverse
primers, respectively.
Transformants were confirmed for the
correctly mutated rib514 allele by sequencing. The 2.9-kb KpnI fragment containing the rib5
14 allele was
cloned into YEp352, yielding pJR932. Cell-free extracts of RIB5-disrupted yeast cells transformed with pJR932 showed the
presence of a catalytically inactive, anti-Rib5p immunoreactive,
protein of the expected size.
The pJR465 plasmid was constructed by inserting a 0.5-kb fragment, encompassing the 5`-half of RIB5, in the KpnI-EcoRI sites of the URA3 CEN4 plasmid YCplac33(21) .
RNA
was extracted from yeast as described by Schmitt et al.(25) . When appropriate, poly(A) RNA was
purified by oligo(dT)-Sepharose chromatography. In Northern blot
analyses, RNA was electrophoresed on 1% agarose-formaldehyde gels and
transferred to nylon membranes. DNA probes were
P-labeled
by the random priming method. Hybridization was carried out at 42
°C in 5
SSC and in the presence of 50% formamide. Washing
was carried out at 65 °C in 0.1
SSC, 0.1% SDS. The levels
of mRNA were estimated quantitatively by densitometry of
autoradiographs on a Bio-Image image analyzer (Millipore Corp.).
DNA sequencing reactions were performed using the dideoxy chain reaction (26) using the T7 sequencing system (Pharmacia).
Primer extension analysis were done as described (22) using the synthetic oligonucleotide RIB52 that is complementary to nucleotides 104-130 of the RIB5 ORF. The reactions in formamide loading buffer were loaded adjacent to the dideoxy sequencing reactions of the RIB5 DNA using the same primer.
Riboflavin synthase activity was determined by published methods(7) . Enzyme activity is expressed as nanomoles of riboflavin formed per hour.
The amino-terminal sequence of purified riboflavin synthase (approximately 5 µg) was determined on an Applied Biosystems 470A sequencer, with an Applied Biosystems 120A phenylthiohydantoin-derivative analyzer as an on-line detection system.
Figure 1:
Characterization and deletion
of the RIB5 locus. A, restriction map of the isolated
13-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 AJ17 (rib5-10). B, RIB5 gene deletion. A
2.3-kb NcoI/PstI fragment encompassing the RIB5 ORF was replaced by a 1.1-kb HindIII fragment URA3 gene (left). Genomic DNA was extracted from wild type
(JC2a) and deleted rib511::URA3 mutant (AJ53)
strains and digested with KpnI. The digestion products were
separated on a 1% agarose gel, transferred to a nylon membrane, and
probed with a
P-labeled, 2.9-kb KpnI fragment (right).
To
verify the identity of the RIB5 gene, a one-step gene
disruption (13) was performed. A deletion of the putative RIB5 gene was performed by excision of a 2.3-kb NcoI-PstI fragment encompassing the RIB5-complementing sequences. These two sites were filled, a HindIII linker was added, and the HindIII cassette
bearing the yeast URA3 gene (isolated from YEp24) was
inserted. Two independent ura3 haploid strains, JC2a and TD28,
were transformed with a linear fragment containing the 1.9-kb KpnI rib511::URA3 deletion construct, selecting
for Ura
prototrophy (Fig. 1B). Genomic
integration and deletion of the RIB5 locus within
Ura
transformants was verified by Southern blot
analysis using the 2.9-kb,
P-labeled KpnI
fragment as probe (Fig. 1B). Deletion of RIB5 in JC2a or TD28 strains led to riboflavin auxotrophy and
accumulation of the intermediate compound
6,7-dimethyl-8-ribityllumazine. Genetic complementation analysis was
used to verify that the rib5
11::URA3 deletion
mutation in AJ53, a JC2a-derived strain, was allelic to rib5-10 . Strain AJ53 was crossed with strain AJ17. The
resulting diploids had an Rib
phenotype, suggesting
allelism of the cloned gene and rib5-10. Strong support
for allelism was obtained by sporulating these diploids and showing
that all 12 tetrads analyzed gave four Rib
spores. We
conclude that the RIB5 was cloned.
The cloned KpnI-EcoRI fragment, which complements the rib5-10 mutation, hybridized in a yeast chromosome blot to chromosome II (data not shown). This result is in agreement with the previously reported location of the rib5 locus at 3.7 centimorgans proximal to the his7 locus on the right arm of chromosome II(32) .
Figure 2: Nucleotide sequence and predicted amino acid sequence of the RIB5 gene. Amino acids are shown in single-letter code. The numbering adopted for both nucleotides and amino acids is initiated at the first ATG of the RIB5 ORF. Restriction sites referred to in the text are boxed. In the 5`-flanking region, the consensus TATA element is underlined, and a pyrimidine-rich tract is double underlined. A downward arrowhead indicates the major transcription initiation site. In the 3`-flanking region, sequences matching the transcription termination and polyadenylation signals are indicated by open circles and solid dots, respectively.
The predicted encoded product is a moderately charged, acidic protein with a calculated pI of 4.96. Hydropathy analysis (34) predicts a moderately hydrophilic profile with no obvious membrane-spanning domains. From the distribution of the codons used, a codon bias index of 0.175 can be calculated(35) , which suggest a poorly expressed gene.
Analysis of the putative 5`-promoter region demonstrated a characteristic AT-rich (63%) yeast promoter with a 12-base pair-long poly(dT) stretch. Such sequence elements have been shown to serve as promoter elements for constitutive expression(36, 37) . One TATA element obeying the consensus (38, 39) occurs relatively close upstream between positions -66 and -59.
The 3`-untranslated region shows two copies of the classical polyadenylation signal, AATAAA, at positions 717-722 and 1139-1143, whereas sequences matching a yeast consensus termination motif, TAG . . . TATGT . . . TTT, were identified at a region starting at 787 and ending at 915(40) . The yeast intron splice signal, TACTAAC (41) , was not found.
Figure 3:
Analysis of the RIB5 transcript. A, Northern analysis. Total RNA (50 µg) from yeast was
purified, electrophoresed, transferred to a nylon membrane, and probed
with a P-labeled fragment containing part of the RIB5 ORF. Lane 1, strain JC2a (wild type RIB5); lane 2, strain AJ53 (rib5
11::URA3); lane 3, strain AJ17 (rib5-10). The positions of
RNA markers (RNA ladder, Life Technologies, Inc.) are shown at the left. B, Primer extension analysis. A synthetic
30-mer oligonucleotide (RIB52), complementary to nucleotides
104-133 of the RIB5 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 position of the major extension product is indicated on the right.
We determined the transcriptional initiation sites by primer extension of an oligonucleotide complementary to the sense strand of the ORF between positions 132 and 104. One major extension product 142 nucleotides long (Fig. 3B) was detected, indicating that transcription predominantly starts at the cytosine residue at position -10 with respect to the ATG initiation codon. This predicts a 10-nucleotide untranslated leader sequence that is shorter than that normally found in yeast transcripts. In addition, several minor initiation points, lying downstream of the TATA sequence, were also detected. This finding is not unusual since heterogeneity in transcript 5`-ends is common for yeast mRNA(43) .
Figure 4:
Immunochemical identification of the RIB5 gene product. Extracts (25 µg of protein) from wild
type X2180-1A (lane 1), mutant
AJ17(rib5-10) (lane 2), deletion mutant AJ53 (rib511::URA3) (lane 3) cells, or AJ53
cells harboring the plasmid pJR235 (lane 4) were
electrophoresed, transferred to membranes, and analyzed by
immunoblotting with antiserum against Rib5p. The migration of
prestained molecular mass markers is shown on the right.
Riboflavin synthase was purified from soluble extracts of yeast cells harboring the RIB5 gene on a multicopy plasmid (pJR235) by successive methanol fractionation and chromatography on a Mono Q column (Table 1). The majority of the enzyme was recovered from the Mono Q column as a sharp peak when 250 mM NaCl was applied (Fig. 5). An increment in total enzyme activity was consistently observed after Mono Q chromatography, suggesting the removal of inhibitory compounds at this purification step (Table 1). On average, a 8500-fold increase in specific activity could be achieved with a recovery of about 167% as compared with the total cell lysate. The final preparation had a specific activity of 18.9 mmol/h/mg of protein and was used as the source of enzyme for further studies. As revealed by silver staining after SDS-PAGE, the final preparation migrated as a single protein band with an apparent molecular mass of 24,5 kDa (Fig. 6A). This value is in good agreement with the predicted molecular mass of Rib5p based on its deduced amino acid composition. The identity of the band was confirmed by immunoblotting using anti-Rib5p polyclonal antiserum (Fig. 6B). Purity of the Mono Q-purified enzyme was also assessed by analyzing riboflavin synthase-active fractions eluting from the Mono Q column by SDS-polyacrylamide gel electrophoresis followed by silver staining, which also revealed a single band, suggesting the homogeneity of the final preparation (data not shown). The authenticity of the 24.5-kDa immunoreactive band was further confirmed by N-terminal amino acid sequencing. The amino acids determined in 8 cycles matched the sequence predicted for Rib5p from its DNA sequence beginning at the initiator Met.
Figure 5: Elution profile of the riboflavin synthase activity from Mono Q column. The riboflavin synthase active fraction from the methanol precipitation step was applied and eluted from the Mono Q column as described under ``Experimental Procedures.'' Inset, Fractions containing enzyme activity were analyzed by immunoblotting with anti-Rib5p antiserum.
Figure 6: SDS-PAGE and immunoblot analysis of fractions collected during purification of riboflavin synthase. Proteins were resolved on 14% SDS-polyacrylamide gels and were visualized either by silver staining (A) or by immunoblotting using anti-Rib5p polyclonal antibodies (B). Molecular masses of standards (kDa) are indicated on the left. The position of Rib5p is marked with an arrow. T, total cell lysate; M, 50% methanol-soluble fraction; Q, Mono Q.
To determine the
relative molecular mass (M) of the native enzyme,
purified riboflavin synthase was chromatographed by FPLC on a Superose
12 column. From the elution profile of riboflavin synthase and of a
number of protein standards a molecular mass of 79 kDa was estimated (Fig. 7A). A similar value (72 kDa) was obtained when
the native enzyme was analyzed by nondenaturing pore limit PAGE (Fig. 7B). The implication is that the native enzyme
consists of three polypeptides of identical M
values.
Figure 7:
Characterization of native riboflavin
synthase. A, Superose 12 filtration of riboflavin synthase,
with ferritin, catalase, aldolase, bovine serum albumin, ovalbumin, and
chymotripsinogen A as inclusive standards. The calculated value for
riboflavin synthase was 79. B, relative migration of native
riboflavin synthase in nondenaturing polyacrylamide (4-20%)
gradient gel. Standard proteins used as markers are shown in the
illustration, with riboflavin synthase M estimated
as 72. (Inset: left lane, markers; right lane,
purified riboflavin synthase).
Figure 8: A, sequence comparison among riboflavin synthases from S. cerevisiae, B. subtilis, E. coli, and P. leiognathi. The amino acid sequence of yeast riboflavin synthase was aligned to the sequences from B. subtilis, E. coli, and P. leiognathi. Amino acids identical in at least two sequences are boxed. Regions showing a higher degree of homology are designated a-f. The 6,7-dimethyl-8-ribityllumazine-binding sites and the cysteinyl residue of the putative catalytic site are highlighted by a black background. B, internal homology of the N and C termini of yeast riboflavin synthase. Evolutionary conservative amino acid substitutions are highlighted by a gray shaded background, in contrast to the black background for identical residues.
Six regions, a-f (Fig. 8A), appeared highly
conserved in both eukaryotic and prokaryotic enzymes. The four proteins
shared a completely conserved 5-amino acid sequence at their amino
termini, region a, suggesting a functional role for this
region. Direct evidence of the functional significance of the
N-terminal region of yeast riboflavin synthase was obtained by
constructing a mutant allele, rib514, which expressed a
protein lacking amino acids Phe
to Ile
(see
``Experimental Procedures''). Protein extracts of AJ53 (rib5
11::URA3) cells containing the rib5
14 allele on a multicopy plasmid showed no enzyme
activity although a high amount of Rib5 protein was detected by
immunoblotting (data not shown).
Ligand binding studies have shown
that the mechanism for the riboflavin synthase reaction involves the
formation of a ternary complex consisting of the binding of two
molecules of the substrate 6,7-dimethyl-8-ribityllumazine to two sites
in the enzyme(5, 47, 48) . Moreover, the
subunit of prokaryotic riboflavin synthases and other proteins
involved in bacterial bioluminescence, which also bind
6,7-dimethyl-8-ribityllumazine, show a marked internal sequence
homology between the N-terminal and C-terminal halves. Therefore, it
was suggested that the monomer forms two structurally similar domains,
each contributing the binding site for one substrate molecule. As shown
in Fig. 8B, the eukaryotic enzyme also showed a
considerable internal sequence homology between the two protein halves.
Furthermore, a short sequence motif
(Gly-X-X-Val-Asn-Leu-Glu) proposed as the ligand
binding site(8, 44) , was doubly represented in the
amino acid sequence, regions c and f, one within each
proposed domain.
We have cloned and characterized a gene, RIB5, which encodes riboflavin synthase in the yeast S. cerevisiae. The accumulation of the substrate 6,7-dimethyl-8-ribityllumazine and the absence of detectable enzyme activity in a strain disrupted for RIB5 confirmed the identity of the gene. RIB5 is present as a single copy gene located on chromosome II.
The RIB5 ORF consists of 714 nucleotides beginning at the first in-frame methionine, predicting a protein of 238 amino acids. Primer extension analysis indicates that there is one major transcription and several minor transcription start sites within 70 nucleotides upstream of the start of translation. Yeast often has multiple sites for transcription initiation that vary depending on the nutritional condition of the cell. The 5`-untranslated sequence shows the presence of a poly(dA-dT) region suggested to be an upstream promoter element for constitutive expression(36) . Analysis of RIB5 transcript levels on different culture conditions indicates that RIB5 behaves as a housekeeping gene, which is constitutively expressed at low level. This pattern of gene expression is in accordance with the scarce (0.06-0.72 µmol of riboflavin/g of protein)(2) , though indispensable, necessity of riboflavin for the cell.
The complementation between the original rib5-10 mutant allele used to clone the gene and the deleted allele expressing only the amino-terminal half of the encoded polypeptide is consistent with the classical interpretations of intragenic complementation, suggesting not only that Rib5p assembles into a multimeric complex but also that there is a discrete functional domain located at the amino terminus.
The hypothesis that Rib5p forms multimers has been demonstrated by
purifying the enzyme. Published methods of purification of yeast
riboflavin synthase are rather cumbersome and inefficient because of
the low riboflavin synthase level in cell extracts and the absence of
sensitive enzymatic assays(47) . We have now developed a simple
method of purification that relies on RIB5 overexpression by
using a multicopy plasmid and the use of Rib5p-specific antibodies.
With this approach we were able to purify the enzyme to apparent
homogeneity using a three-step purification protocol, which achieved an
overall purification of 8500-fold with a recovery of 167%. This fairly
high yield made it possible to analyze some of its properties. From
comparison of either the elution volume or the R of
pure native riboflavin synthase with those of standard proteins by gel
filtration analysis or nondenaturing pore limit PAGE, respectively, a
molecular mass of approximately 75,000 daltons was estimated for the
native enzyme. This value is in reasonable agreement with the molecular
mass reported by Harvey and Plaut (5) for a partially purified
riboflavin synthase preparation on the basis of its sedimentation
coefficient. 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 Rib5p based on its
deduced amino acid composition. These findings indicate that the native
riboflavin synthase of S. cerevisiae exists in the trimer form
of identical subunits. Riboflavin synthase appears in B. subtilis as two different species characterized by its subunit composition.
The so-called light enzyme is a trimer of
, riboflavin synthase,
identical subunits. The heavy enzyme consists of an
subunit
trimer enclosed within an icosahedral structure composed of 60
,
6,7-dimethyl-8-ribityllumazine synthase,
subunits(7, 49, 50) . Unlike Bacillus, we found no evidence of a similar heavy multienzyme
complex in Saccharomyces.
Significant internal homology was
found between the N-terminal and C-terminal halves of the Saccharomyces Rib5p, as described previously for the B.
subtilis subunit(8) . Since the enzyme binds two
molecules of the substrate 6,7-dimethyl-8-ribityllumazine, it was
suggested that the protomer forms two structurally similar,
substrate-binding domains. In this context, the intragenic
complementation effect observed reinforces this hypothesis by showing
that a discrete functional domain exists in the N-terminal half of
Rib5p. The existence of a second functional domain in the C-terminal
half of Rib5p can also be predicted by homology to the N-terminal
domain. Research is currently under way at our laboratory to determine
whether the separate N-terminal and C-terminal domains of Rib5p are
able to bind the substrate and whether the interaction of both types of
separate domains can restore the enzyme activity.
Although
structurally similar as a consequence of the probable common origin, by
gene duplication, and endowed with the same substrate binding function,
both domains should not be strictly identical because of their
different catalytic properties. In the enzymatic formation of
riboflavin one molecule of substrate acts as the donor and other as the
acceptor of the four-carbon moiety that transforms the xylene ring of
6,7-dimethyl-8-ribityllumazine into the isoalloxazine ring of
riboflavin. The respective donor and acceptor sites can be
distinguished by their affinities for different
ligands(5, 48) , i.e. only the acceptor site
binds the product riboflavin, whereas the donor site has high affinity
for analogs of the second product of the reaction,
5-amino-2,6-dihydroxy-4-ribitylamino-pyrimidine. The temporary binding
of 5-amino-2,6-dihydroxy-4-ribitylamino-pyrimidine to the donor domain
in the course of the reaction prompted us to compare the sequences of
both N-terminal and C-terminal domains of Rib5p with that of the
6,7-dimethyl-8-ribityllumazine synthase from S. cerevisiae. This enzyme also binds
5-amino-2,6-dihydroxy-4-ribitylamino-pyrimidine as substrate, and the
gene encoding the enzyme, RIB4, has recently been cloned and
its nucleotide sequence determined. ()A substantially higher
degree of similarity of the deduced amino acid sequence of RIB4 with the C-terminal domain (38.8%) than with the N-terminal domain
(27.6%) of Rib5p was found. Therefore, it is tempting to speculate
about the existence of an N-terminal domain binding the acceptor
substrate molecule and a C-terminal binding domain binding the donor
substrate molecule. Binding assays with the products of the reaction or
structural analogs and the split N-terminal and C-terminal domains
would be necessary to answer this question.
In the course of our
analysis of the RIB5 gene, we compared the deduced amino acid
sequence of the yeast enzyme with the riboflavin synthases from B.
subtilis, E. coli, and P. leiognathi. Although
the four proteins shared significant similarity along their entire
lengths, a higher degree of similarity in the amino acid sequences from
these four proteins was identified in six regions, a-f. Because of the common function of these
proteins, it seems likely that these regions are of functional
importance. Experimental evidence for the functional relevance of the
strictly conserved amino end, region a, was obtained for the
yeast enzyme by deleting part of the sequence. In this respect, it is
also significant that the N terminus of Rib5p is not modified
post-translationally (i.e. signal sequence processing or
N-terminal amino acid modification), as deduced from the N-terminal
amino acid sequencing data. Since a homologous region is symmetrically
located on the C-terminal domain, region d, a role in the
formation of the substrate binding site rather than in catalysis may be
predicted for both conserved regions. Similarly, substrate binding
function may be also attributed to the homologous symmetrical regions c and f. Moreover, both regions contain the sequence
motif (Gly-X-X-Val-Asn-Leu-Glu) proposed by
O'Kane et al.(44) to be involved in the binding
of 6,7-dimethyl-8-ribityllumazine. Early studies by Plaut et al.(6) with sulfhydryl group-binding reagents have showed
that a thiol group is involved in the enzyme action. The presence of
only one cysteinyl residue conserved in all four known amino acid
sequences, Cys in Rib5p, makes this residue a good
candidate for proton abstraction from the 7-methyl group of the
acceptor substrate molecule, which initiates the proposed reaction
mechanism(6) . In agreement with this assumption, it has
recently been found that mutants of Rib5p with amino acid substitutions
at Cys
are inactive. (
)Since Cys
is located in region b, it thus seems likely that this
conserved region would participate in the formation of the catalytic
center. At present, no role can be directly extrapolated for region e, although other functions such as trimerization of the
enzyme or the presence of other site-active residues should be
considered.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z21621[GenBank].