(Received for publication, July 24, 1995; and in revised form, October 23, 1995)
From the
The pur cluster which encodes the puromycin
biosynthetic pathway from Streptomyces alboniger was subcloned
as a 13-kilobase fragment in plasmid pIJ702 and expressed in an
apparently regulated manner in the heterologous host Streptomyces
lividans. The sequencing of a 9.1-kilobase DNA fragment completed
the sequence of pur. This permitted identification of seven
new open reading frames in the order: napH, pur7, pur10,
pur6, pur4, pur5, and pur3. The latter is followed by the
known pac, dmpM, and pur8 genes. Nine open reading
frames are transcribed rightward as a unit in opposite direction to
that of the pur8 gene which is expressed as a monocistronic
transcript from the rightmost end. napH encodes the known N-acetylpuromycin N-acetylhydrolase. The deduced
products from other open reading frames present similarities to: NTP
pyrophosphohydrolases (pur7), several oxidoreductases (pur10), the putative LmbC protein of the lincomycin
biosynthetic pathway from Streptomyces lincolnensis (pur6), S-adenosylmethionine-dependent
methyltransferases (pur5), a variety of presumed
aminotransferases (pur4), and several monophosphatases (pur3). According to these similarities and to previous
biochemical work, a puromycin biosynthetic pathway has been deduced. No
cluster-associated regulatory gene was found. However, both pur10 and pur6 genes contain a TTA codon, which suggests that
they are translationally controlled by the bldA gene product,
a specific tRNA.
Nucleoside antibiotics constitute an important group of microbial secondary metabolites some of which are effective agents against plant and human diseases. Examples are ribavarin (antiviral), polyoxins (herbicides), and mildiomycin (plant antifungal antibiotic). Given the key role accomplished by nucleosides and nucleotides in biochemical processes, the nucleoside antibiotics have found a fundamental application as specific inhibitors of a high variety of biochemical reactions (for a review, see (1) ). These features rise up the question of how the producing organisms defend themselves against the toxic effects of their products and, in most cases, of the relevant biosynthetic precursors.
Puromycin is an aminoacyl
nucleoside antibiotic produced by Streptomyces alboniger. It
is a broad spectrum secondary metabolite active against Gram-positive
bacteria, protozoans, and mammalian cells, including tumor cells. It
has been a key compound in various cell-free systems directed to
elucidate the mechanism of protein synthesis and the mode of action of
other inhibitors of this process(2, 3) . Concerning
the biosynthetic pathway of puromycin, some data are available.
Adenosine is known to be a direct precursor for the
3`-amino-3`-deoxyadenosine moiety of puromycin(4) . Moreover, a
commercial sample of puromycin was found to be contaminated by small
amounts of N,N
,O-tridemethylpuromycin, N
,O-didemethylpuromycin, and O-demethylpuromycin. This led to the proposal that puromycin
biosynthesis probably proceeds through these intermediates and in that
order(5) . Three enzymes of the pathway, an O-demethylpuromycin O-methyltransferase (DmpM), (
)a puromycin N-acetyltransferase (Pac), and an N-acetylpuromycin N-acetylhydrolase (NapH) have been
characterized(6, 7, 8) . Biochemical studies
suggest that Pac inactivates the intermediate N
,N
,O-tridemethylpuromycin
by acetylation, and that DmpM methylates N-acetyl-O-demethylpuromycin to form N-acetylpuromycin(7) . The latter would be excluded
from the cells and then hydrolyzed by the extracellular NapH activity (8) yielding the biologically active puromycin antibiotic. In
addition to Pac, the putative transmembrane protein Pur8 was shown to
confer resistance to puromycin in Streptomyces lividans and,
consequently, in S. alboniger, possibly by promoting an active
efflux energized by a proton-dependent electrochemical
gradient(9) . The complete set of genes (15 kb) encoding the
puromycin biosynthetic pathway (pur cluster) from S.
alboniger has been cloned in low-copy number cosmids and expressed
in a regulated pattern in heterologous hosts, S. lividans and Streptomyces griseofuscus(10) . Three genes of pur have been sequenced, pac, dmpM, and pur8, which encode Pac, DmpM, and Pur8,
respectively(9, 11, 12) . The three genes are
contiguously located at the right end of the cluster and in this order.
In addition, the napH gene, located at the left end of pur, was isolated and its product (NapH)
characterized(8) . Here, we report the completion of the
nucleotide sequence of pur, an analysis of its transcriptional
organization, and a novel proposal for the puromycin biosynthetic
pathway.
DNA sequencing was by the dideoxy chain termination method (23) using Sequenase 2.0 (U. S. Biochemical Corp.) or Taq DNA polymerase (Promega Biotech) following the manufacturer's protocols. 7-Deaza-dGTP was used instead of dGTP. 4, 5, and 6% polyacrylamide gels containing 7 M urea were used. Occasionally, to resolve band compressions, 6% polyacrylamide gels containing 40% formamide were used. Appropriate DNA restriction fragments were cloned previously in M13 mp18 and M13 mp19. Universal primer and custom made oligonucleotides (Isogen Bioscience, Amsterdam, The Netherlands) were used as required.
Figure 1: Restriction maps of the DNA inserts from several constructs, puromycin production phenotype of S. lividans transformants and organization of the ORFs of pur. A, the restriction maps of cosmids pPB5.13, pPB4.6, and pPB11.40 and plasmid pCXS were modified from(10) . Plasmid pRCP11 was obtained by subcloning the 13-kb ClaI-EcoRI (labeled E* in other restriction maps as well) fragment from pCXS into Bluescript. From the resulting plasmid, this fragment was isolated as a KpnI-PstI piece, which was finally inserted in the KpnI-PstI replicon fragment of pIJ702. + and - indicate production or no production of puromycin by the relevant S. lividans transformants, respectively. Enzymes: C, ClaI; E, EcoRI; K, KpnI; N, NcoI; P, PstI; S, SpeI; X, XhoI; Xb, XbaI. B, ORFs and transcriptional organization of the pur cluster. The incomplete orfA and orf1 are interrupted by the ClaI and EcoRI sites, respectively. The gray region indicates the sequence obtained in this work. Only relevant restriction sites are indicated. Size of the ORFs is indicated by small arrows drawn immediately below. Lines a, b, c, and d indicate the DNA fragments used as probes in low resolution S1-protection assays. Above them, continuous arrows indicate the size and direction of clearly identified transcripts, whereas dotted arrows indicate possible transcripts.
Figure 2: Nucleotide sequence of a 9.12-kb DNA fragment from the pur cluster. The deduced gene products are indicated in the one-letter code under the DNA sequence. Possible ribosome binding sites (rbs) are indicated by dotted lines. Putative translation initiation codons are in bold letters. The start and direction of each of the ORFs are indicated by up arrows and named accordingly. A presumptive signal peptide of NapH is underlined and its putative cleavage site is indicated by a vertical arrow. A proposed motif for Pur10 and similar oxidoreductases is underlined. An inverted repeat located 3` to pur5, which could form a stem-loop for transcription termination is indicated by horizontal arrows. Putative -10 and -35 regions of napH are indicated. Possible transcription initiation and termination sites are indicated by bold triangles and an open triangle, respectively. Restriction sites with an asterisk are not unique in the sequence DNA; they are referred to in the text. The 5` region of the previously reported pac sequence(11) , which has been revised in this work, is indicated by small letters.
Previously, it was shown that the napH gene is part of a 2.5-kb SphI-NcoI fragment from the left end of pur(8) . The single complete ORF of 1458 nucleotides found in this fragment (Fig. 2) was, therefore, attributed to napH. Its deduced product has 485 amino acids (Table 1). Both upstream and downstream of the chosen initiator codon there are other putative initiator codons (Fig. 2). They were rejected as probable initiators because, for those located downstream, destruction of a BamHI site at position 730 ( Fig. 1and Fig. 2) prevented expression of NapH activity (8) and, for those located upstream, only the selected initiator codon was preceded by a good ribosomal binding site (Table 1; Fig. 2). NapH is an extracellular enzyme (8) and, in agreement with this, its N-terminal region presents the characteristics of a signal peptide(29) . Thus, it contains a positively charged leading region (MLHRIQRKR), followed by a hydrophobic central region (AMTAGAVGVLFLAQLVI) and a polar region (SSSSAAA). The putative signal peptide contains 33 residues, a value close to the mean size of 35 for the putative signal peptides from Streptomyces(30) . The presence of this signal peptide also helps to select the corresponding DNA sequence as a coding region, despite the fact that its codon usage deviates from that typical of Streptomyces. Comparisons of the NapH sequence with those in data banks disclosed significant similarities (28.9% identity; 49.3% similarity) with the aminopeptidase Y from Saccharomyces cerevisiae(31) .
The ORF corresponding to pur7 comprises 459 nucleotides, of which the first eight overlap with
the 3` end of the napH coding sequence. A similar situation
was previously found for other Streptomyces genes(32) . pur7 encodes a hydrophilic protein
(Pur7) of 152 residues (Table 1; Fig. 2) which contains a
highly conserved domain present in proteins from a wide variety of
organisms. Besides this, no generally significant similarities are
present in this group of proteins. This domain was defined by Koonin (33) as a region of approximately 40 residues, generally
located at the N terminus. Its consensus sequence is
GXEX
-(STAGC)-(LIVMA)-XRE-(LIVMF)-XEE,
where X represents any amino acid and residues in parentheses
indicate the most frequent ones in those positions (Fig. 3). It
has been proposed that this domain is the catalytic center of
NTP-pyrophosphohydrolases which produce PP
and the relevant
NMP. Indeed, such activity has been found in several of these proteins
of which the most thoroughly studied are MutT from E. coli,
which names the
family(34, 35, 36, 37) , MutX from Streptococcus pneumoniae(38) , MutT from Proteus
vulgaris(39) , and MutT from humans(40) . MutT
hydrolyses 8-oxo dGTP and, less efficiently, other
nucleotides(41) . Curiously, dATP is a better substrate for
MutX, which presents the highest similarity to Pur7, than for other
members of the MutT family(42) . Moreover, Orf17 from E.
coli(42) , which does not complement the mutT mutation in E. coli, hydrolyses all dNTPs. These data
suggest a nucleotide-pyrophosphohydrolase activity for Pur7.
Figure 3: Alignment of Pur7 with several proteins which contain the domain of the MutT family. The alignment corresponds to the different protein regions where the domain is localized and some of the relevant flanking amino acids. Identical residues in all proteins are in white letters with a black background. Residues which are identical in Pur7 and several of the other proteins, and conservative replacements are in bold letters. Orf257 is a NADH pyrophosphatase from E. coli(87) . Accession numbers of Orf154 from Streptomyces ambofaciens, InvA from Bartonella bacilliformis and of D250R from porcine African swine fever virus (Asfv) are Z19590, L25276, and L07263, respectively. References of the other proteins are indicated in the text.
The pur10 gene would encode a hydrophilic protein of 338 amino
acids (Table 1; Fig. 2). Curiously, a TTA codon is present
at position 2797 of the ORF (Fig. 2). This is a very rare codon
in Streptomyces. It encodes leucine 29. The matching
tRNA is encoded in the bldA gene, which has been
implicated in translational regulation(43) . Pur10 showed
similarities, mainly at the N-terminal region, with proteins implicated
in oxidoreduction activities, including the mammalian biliverdin
reductase (22.6% identity; 50.5% similarity)(44) ; the
glucose-fructose oxidoreductase from Zymomonas mobilis (20.5%
identity; 43.2% similarity)(45) ; a galactose dehydrogenase
from Pseudomonas fluorescens (22.8% identity; 50.2%
similarity)(46) ; and LmbZ from S. lincolnensis (24%
identity; 46.7% similarity), for which an oxidoreductase activity has
been proposed in the lincomycin biosynthetic pathway(47) . It
is worth mentioning the presence of an invariant aspartic acid (residue
73 in Pur10) in all these proteins. Moreover, a highly conserved region
localized between residues 95 and 107 could define a motif
(GKH-(IVLM)-(IVLM)-XEkPX
(TS)) which would
represent these proteins (Fig. 2). Therefore, we propose an
oxidoreductase activity for Pur10.
The pur6 gene would encode a hydrophilic protein of 772 amino acids (Table 1; Fig. 2). Similarly to pur10, it possesses a TTA codon which encodes leucine 3 (Fig. 2). Sequence comparisons only showed certain similarities between the N-terminal halves of Pur6 and the LmbC deduced protein of the lincomycin biosynthetic pathway from S. lincolnensis. Thus, the first 300 amino acids of these two proteins share 24.6% identity and 48.1% similarity. The precise function of LmbC is still unknown. However, it presents significant similarities with members of the peptide synthetase family of enzymes as well as to a subfamily of aminoacyl adenylate-forming domains of aromatic amino acid-activating enzymes (47) . LmbC has been proposed as an L-tyrosine-activating enzyme, besides other putative activating functions. All these proteins contain an AMP-binding domain, which could not be detected in Pur6. Given the low similarity of Pur6 with peptide synthetases, i.e. with TycA (tyrocidine synthetase from Bacillus subtilis, accession number P09095) the values are 19.6% identity and 40.2% similarity, it is difficult to assign an amino acid (i.e. tyrosine, a moiety of puromycin) activating activity for Pur6, although there are some hints to suggest that it might be implicated in the transfer of tyrosine to 3`-amino-3`-deoxyadenosine (see ``Discussion'').
The pur4 gene would determine a protein (Pur4) of 429 amino
acids (Table 1; Fig. 2). Although this structure is
chiefly hydrophilic, it contains some hydrophobic stretches in its
N-terminal half. A partial sequence of Pur4 was previously
reported(10) . It presented significant similarities with the
deduced amino acid sequences of eryC1, from the erythromycin
biosynthetic pathway of Saccharopolyspora
erythraea(48) , orf10.4 from the rfb cluster of Salmonella typhimurium(49) , and the
pleiotropic, putative regulatory gene degT of B.
subtilis(50) . Also, significant similarities of Pur4 were
found with the products of the dnrJ, strS, tylB, and lmbS genes, which are implicated in the
biosynthetic pathways of several antibiotics from Streptomyces(47, 51, 52, 53) .
Based on the clear similarities of these genes with the degT gene, a regulatory role in the relevant biosynthetic pathways was
proposed for some of them(10, 51, 52) . More
recently, however, it was suggested that these genes including pur4 may encode aminotransferases (54, 55, 56) . Indeed, all of them have a
conserved lysine residue (lysine 204 in Pur4; Fig. 2) which
could be used to bind the presumptive aminotransfer coenzyme pyridoxal
phosphate by forming a Schiff base. Moreover, this conserved lysine is
located in a motif
(GXDX
AX
EDX
GX
KX
geGGX
G;
residues 153-229 in Pur4; Fig. 2) according to
Piepersberg(56) . Therefore, Pur4 may display an
aminotransferase activity.
The pur5 gene encodes a protein
(Pur5) of 228 amino acids (Table 1; Fig. 2). This product
is mainly hydrophilic with a hydrophobic stretch between residues 40
and 80. Sequence comparison of Pur5 with proteins from data banks
detected a small glycine-rich region between amino acids 64 and 80
which is highly conserved, usually at the N-terminal region, in a
variety of methyltransferases and other enzymes which use AdoMet as a
cosubstrate (Fig. 4). This domain corresponds to region I as
defined by Ingrosso et al.(57) . Its consensus
sequence was later slightly modified (58) as
hhD/EhGXGXGXhXXXhh, where h is
a hydrophobic amino acid, X is any amino acid, and
is
generally a charged amino acid. The presence of this charged amino acid
in the last position of the domain could be questioned, according to
the alignment presented in Fig. 4. Two other regions (II and
III), which were proposed elsewhere (57, 59) are not
rigorously detected in several AdoMet-dependent methyltransferases
including Pur5 (58; not shown). The presence of region I in Pur5
suggests that it may have a AdoMet-dependent methyltransferase
activity.
Figure 4: Alignment of Pur5 and several proteins which carry a methyltransferase domain. Alignment was performed as in Fig. 3. Asterisks represent glycine residues which could conform a turn at the center of the domain, according to the secondary structure prediction. Dots indicate the putative position of hydrophobic amino acids, according to Wu et al.(58) . Aligned proteins are: UbiG (E. coli, M87509); EryG (Saccharopolyspora erythraea, X60379); ErmE (S. erythraea, X51891); TehB (Klebsiella aerogenes, M74072); PRMA (E. coli, P28637); HNMT (human, U08092); RdmB (Streptomyces purpuracens, U10405); PmtA (Rhodobacter sphaeroides, L07247); LmbG and LmbW (S. lincolnensis, X79146); and dnr-ORF5 (S. peucetius, L35560), where figures in brackets represent the relevant accession number.
Finally, the pur3 gene would encode a protein
(Pur3) of 273 amino acids (Table 1; Fig. 2). Although this
product is mainly hydrophilic it contains two significant hydrophobic
regions between residues 90-120 and 190-230. Pur3 has
significant similarity with a family of proteins(60) , which
includes a variety of inositol monophosphatases and other proteins of
different origins (Fig. 5), including the bovine IMP (28.3%
identity and 44.8% similarity), a key enzyme of the inositol phosphate
second messenger pathway(61) . This enzyme hydrolyses D-inositol phosphates producing inositol. It is also
significant the similarity of Pur3 with the Hal2 protein from S. cerevisiae (27.9% identity; 47.9% similarity),
which is involved in salt tolerance and methionine
biosynthesis(62) , and CysQ from E. coli (27.7%
identity; 48.3% similarity), which is required for cysteine
biosynthesis and could help control the pool of 3`-phosphoadenosine
5`-phosphosulfate(63) . All these proteins share two highly
conserved domains(60) , as well as certain identical residues
mainly at the N-terminal half (Fig. 5). The tridimensional
structure of IMP (64) indicates that these two domains
participate in the binding of phosphate and Mg, which
are essential for enzymic activity. The two domains are also present in
the bovine inositol polyphosphate phosphatase(65) , which has
scarce similarity in other parts of the sequence with other proteins of
the family. As indicated by Neuwald et al.(60) , most
proteins carrying these two domains could be phosphatases. This
activity could, therefore, be attributed to Pur3.
Figure 5:
Alignment of Pur3 with several proteins.
The bovine brain inositol monophosphatase (IMP) was introduced
as an example of proteins displaying this enzymic activity. QutG from Aspergillus nidulans (28.3% identity; 49.4% similarity; 88)
and Qa-X from Neurospora crassa (27% identity; 49.6%
similarity; 89) are implicated in the utilization of quinic acid in
these organisms. SuhB from E. coli (30.6 identity; 50.6
similarity) is implicated in the regulation of a transcriptional
-factor of genes encoding heat-shock proteins(90) .
Yhr046C is an ORF from S. cerevisiae which pertains
to this family of proteins (25.5% identity; 47.4%
similarity)(91) . The values of identity and similarity refer
to comparisons of the different proteins with Pur3. Alignments and
features are as in Fig. 3. The final amino acids of several
proteins are not presented.
A higher resolution study was also carried out using S1 protection experiments to confirm the existence of the transcription start points detected by low resolution nuclease-S1 assays. To locate the transcription initiation site 5` to napH, we used a probe (Fig. 6) covering its 5` coding region and the noncoding region between this gene and orfA. The nuclease assay showed a protected fragment of about 500 bp (Fig. 6), which indicated that napH transcription started around position 679, 41 bp upstream of the translational initiator GTG (Fig. 2). Possible -10 and -35 regions are indicated in Fig. 2. To identify a possible transcription initiation site 5` of pur3, the probe used (Fig. 6), covered the 5` end of this ORF and the region between pur5 and pur3. A protected fragment of approximately 290 bp (Fig. 6) suggested the presence of a transcription initiation signal around position 8210, 43-bp upstream of the pur3 initiator ATG (Fig. 2). No clear -10 and -35 regions were found. Transcription initiation upstream of pur8 was examined using a probe that covered the 5` end of pur8 and all the noncoding region up to orf1(9) . A protected fragment of 124 bp (Fig. 6) indicated the existence of transcription initiation 59-bp upstream of the pur8 initiator ATG(9) .
Figure 6:
Transcription analysis. Total RNA used was
isolated (14) from S. alboniger samples taken at 5.0 A(10) , when puromycin was actively
produced as shown by a Pac assay. RNA samples (50 µg) were
hybridized to the
P-labeled probes described below. High
resolution S1 mapping was performed as described(11) . Lanes G, A, T, and C contain standards from
sequencing reactions of known sequences from pur (B,
C, and D), and a fragment from M13 mp18 (A). For D,
29 digested with HindIII and labeled with
P was also used as size standard. Numbers on the left margins indicate the size (in nucleotides) of the
standards and those on the right the size (in nucleotides) of
the DNA fragments protected from the nuclease S1 digestion. Lines 1 and 2 represent S1 assays performed with S. alboniger RNA and yeast tRNA, respectively. Line 3 represents DNA
probes. A, transcription initiation of napH. The
2.07-kb BglII-XhoI fragment from pur (nucleotides 363-2429; Fig. 2) was cloned in Bluescript.
From the resulting construct (pPS1.8), an 855-bp SplI
(internal site to the napH coding sequence, Fig. 2)-SacI (from the polylinker of the vector)
fragment was isolated, labeled at the SplI end and then used
as a probe. B, transcription initiation of pur3. From
plasmid pPS5.2 (5.5-kb XhoI-EcoRI fragment from pur, cloned in pUC19; XhoI, nucleotide 7523, Fig. 2. EcoRI, right end, Fig. 1), a 984-bp SalI (internal site to the pur3 coding sequence; Fig. 2)-PstI (from the polylinker of the vector)
fragment was isolated, labeled at the SalI end and then used
as a probe. C, transcription initiation of pur8. The
2-kb SalI-EcoRI rightmost fragment of pur was cloned in Bluescript. From the resulting plasmid (pPS2.0; 9),
a 743-bp BamHI (internal site to the pur8 sequence)-PstI (from the polylinker of the vector)
fragment was isolated, labeled at the BamHI end, and then used
as a probe. D, transcription termination of pur5.
From plasmid pPS1.0 (984-bp SalI-PstI fragment
described above, cloned in Bluescript), a 1.08-kb StyI
(internal site to pur5 sequence; Fig. 2)-SspI
(from the vector polylinker) fragment, was isolated, labeled at StyI and used as a probe.
The stem-loop 3` to pur5 referred to above suggested that transcription termination could take place at this region. To examine this possibility, a high resolution S1 protection experiment was performed using a probe (Fig. 6) that comprised all the DNA between pur5 and pur3, including the stem-loop (Fig. 2), plus a Bluescript tail. The results indicated the presence of major protected fragments of approximately 840 and 512 bp, and minor protected ones of 535-545 and 1080 bp sizes (Fig. 6). The 1080-bp fragment corresponds to probe/probe reannealing. The 840-bp fragment corresponds to full protection of the probe minus the non-homologous plasmid sequence, thus confirming the existence of a transcript that extends through the stem-loop into pur3. The protected band of approximately 512 bp may correspond to transcription termination close to the 5` end of the stem-loop. The minor protected bands suggest that transcription termination occasionally occurs at the region close to or covering the loop located within the inverted repeat 3` to pur5. However, since these protected fragments have not been found 3` to the putative transcription terminator, it is questionable that these terminations really take place in vivo, and other possible roles for this stem-loop must be considered (see ``Discussion''). Therefore, these studies and those reported elsewhere (12) show that the pur cluster is transcribed into at least two mRNA species: one of them is a polycistronic messenger that covers nine ORFs, and the other one is a monocistronic messenger transcribed in the opposite direction that corresponds to pur8. In addition, there may be two other transcripts that span napH through pur5 and pur3 through dmpM, respectively (Fig. 1).
Figure 7: Proposed biosynthetic pathway of puromycin. Roman numerals indicate compounds referred to in the text.
It has been proposed that tyrosine would be attached
by its -COOH group to the 3` -NH group of
3`-amino-3`-deoxyadenosine to produce N
,N
,O-tridemethylpuromycin (5, 7) . According to our proposal, formation of this
bond should take place on the 5`-phosphoderivative (Fig. 7),
thus removing it from the nucleotide pool to prevent any additional
phosphorylation. This reaction would be equivalent to a peptide bond
formation step, which would previously require an activation of
tyrosine. Amino acid activation is performed by aminoacyl-tRNA
synthetases, antibiotic peptide synthetases, the bacterial
peptidoglycan precursor synthetases, and condensing enzymes of the
-lactams
antibiotics(79, 80, 81, 82) .
Assuming that the relevant gene(s) is(are) not on a separate
locus(loci), the only putative enzyme, by elimination, of the puromycin
biosynthetic pathway to which this function could be attributed is
Pur6. Although it presents low level similarities to these enzymes and
apparently lacks any AMP binding domain, this might not be an impeding
difficulty. Thus, LeuS, a leucyl-tRNA synthetase, has low similarities
to members of the peptide antibiotic synthetases and lacks an apparent
AMP binding site(83) . The only ORF from data banks which
presents a similarity to Pur6 is LmbC from the lincomycin biosynthetic
gene cluster. This ORF appears to belong to the peptide synthetase
family of proteins. It has been proposed to be an activating enzyme of
either tyrosine, before conversion to L-dihydroxyphenylalanine, or propylproline (a
tyrosine-derivative intermediate), prior to condensation at the
-NH
group of the sugar moiety of lincomycin(47) .
In our case, the activation of tyrosine could be performed by a
different enzyme (i.e. a tyrosinyl-tRNA synthetase) and Pur6
might only catalyze its linkage to 3`-amino-3`-deoxy-AMP to produce N
,N
,O-tridemethylpuromycin-5`-phosphate
(compound V; Fig. 7). Given the rarity of the linkage attributed
to Pur6, a sequence deviation from peptide bond forming enzymes should
not be surprising. Indeed, Pur6 could be a member of a variety of
enzymes which are implicated in the biosynthetic pathways of certain
nucleoside antibiotics of bacterial and fungal origin, like
chriscandin, A201A, and the agricultural fungicides polyoxins, where a
variety of polycarbon chains are attached to the amino-ribofuranosyl
moieties by forming a peptide bond-like linkage(1) .
It has
been proposed that N,N
,O-tridemethylpuromycin
is dimethylated at N
(5) . However, the
resulting intermediate inhibits protein synthesis(84) , and to
prevent it, a Pac-dependent inactivation by N-acetylation of
the -NH
group of the tyrosinyl moiety of
tridemethylpuromycin has been suggested(7) . According to our
proposal, this acetylation should take place on compound V (Fig. 7). The resulting intermediate (compound VI; Fig. 7) would be N
-dimethylated by Pur5,
which presents a typical motif of AdoMet-dependent N-methylases (Fig. 4). Curiously, AdoMet-dependent N
-methylation of either N
,N
,O-tridemethylpuromycin
or N
,O-didemethylpuromycin with cell
extracts from S. alboniger has not been detected. This result
was attributed to the possible requirement of a precursor derivative
such as a phosphate ester(5) . Indeed, our proposal suggests
that this modified precursor is compound VI (Fig. 7). The
resulting N-acetyl-O-demethylpuromycin-5`-phosphate
(compound VIII, via compound VII; Fig. 7) would be O-methylated by DmpM(5, 7) . However, the
affinity of DmpM for N-acetyl-O-demethylpuromycin
(compound IX; Fig. 7) is very high (K
= 2.3 µM; 7), which suggests that this,
instead of VIII, is the real substrate for this enzyme. Therefore, the
5`-phosphate group could be removed by Pur3 prior to O-methylation. If so, this removal could take place even at an
earlier step (i.e. from compound V; Fig. 7). This
sequence of reactions would produce N-acetylpuromycin
(compound X; Fig. 7), which would be secreted to be N-hydrolyzed by NapH, the puromycin (compound XI; Fig. 7) activating enzyme ((8) ; Fig. 7).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X92429[GenBank].