JOURNAL OF BACTERIOLOGY, July 1997, p. 4446–4450 0021-9193/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 179, No. 13
Cloning and Characterization of the Streptomyces peucetius dnmZUV Genes Encoding Three Enzymes Required for Biosynthesis of the Daunorubicin Precursor Thymidine Diphospho-L-Daunosamine SHAREE L. OTTEN,1 MARK A. GALLO,1† KRISHNAMURTHY MADDURI,1‡ XIAOCHUN LIU,1 AND C. RICHARD HUTCHINSON1,2* School of Pharmacy1 and Department of Bacteriology,2 University of Wisconsin, Madison, Wisconsin 53706 Received 6 December 1996/Accepted 23 April 1997
Characterization of the dnmZ, dnmU, and dnmV genes from the daunorubicin-producer Streptomyces peucetius by DNA sequence analysis indicated that these genes encode a protein of unknown function plus a putative thymidine diphospho-4-keto-6-deoxyglucose-3(5)-epimerase and thymidine diphospho-4-ketodeoxyhexulose reductase, respectively. Inactivation of each of the three genes by gene disruption and replacement in the wild-type strain demonstrated that all of them are required for daunosamine biosynthesis. more, disruptions of the dnmT (24) and dnmQ (formerly dnrQ) (21) genes indicate that they too are required for daunosamine biosynthesis although their enzymatic functions have not been established. In this report we extend our investigation of TDP- daunosamine biosynthesis through DNA sequencing and characterization of the dnmZ, dnmU, and dnmV genes that encode a protein of unknown function plus the putative TDP-4-keto-6-deoxyglucose-3(5)-epimerase and TDP-4-ketodeoxyhexulose reductase, respectively, of the daunosamine biosynthetic pathway. Sequence analysis of the dnmZ, dnmU, and dnmV genes. DNA sequencing of both strands of a 2,750-nucleotide (nt) AlwI-BglII fragment (Fig. 2), located between dnrY and dnmJ in the DXR biosynthesis gene cluster (Fig. 3), was carried out with single-stranded DNA templates subcloned in M13 vectors (33) as previously described (20). The CODONPREFERENCE program (6) was used to identify three complete open reading frames (ORFs), dnmZ, dnmU, and dnmV, and the C-terminal end of dnmJ (25) on the basis of the characteristic third-position G1C bias and codon usage typical of Streptomyces genes. The dnmZ gene has a likely start codon (ATG) at nt 25 preceded by a probable ribosome binding site (RBS), GAGG, at nt 17. A stop codon (TGA) located at nt 1053 suggests that this ORF would encode a polypeptide having 341 amino acid residues (ignoring the formylmethionyl) and an Mr of 36,751. Similarly, the dnmU gene has a likely start codon (ATG) at nt 1100 that is preceded by a probable RBS, GGAGG, at nt 1088. A stop codon (TGA) located at nt 1726 suggests that this ORF would encode a polypeptide having 208 amino acid residues and an Mr of 22,501. A likely start codon (ATG) for the dnmV gene is located at nt 1734 and is preceded by a probable RBS, GAGG, at nt 1725. A stop codon (TAG) at nt 2657 suggests that dnmV encodes a polypeptide having 307 amino acid residues and an Mr of 32,492. The adjacent dnmJ gene (25) is transcribed convergently to dnmV; however, the stop codons of dnmV and dnmJ are not separated by a transcriptional terminator; rather, they are separated by a single base pair. This gene arrangement is consistent with the transcriptional mapping of the convergent dnmZUV and dnrIdnmJ transcripts which showed that the 39 ends of the transcripts overlap extensively to produce complementary mRNA in the dnrIdnmJ/dnmZUV region (17) (Fig. 3).
Daunorubicin (DNR) and its C-14-hydroxylated derivative doxorubicin (DXR) (Fig. 1) are clinically important antitumor agents, and like many microbial secondary metabolites, they require a deoxyhexose component for their biological activity (7). These deoxyhexose constituents are commonly 6-deoxyhexoses including 2,6- and 4,6- dideoxy or trideoxy amino hexoses, as is the case for DNR and DXR, which contain the 2,3,6-trideoxy-3-aminohexose daunosamine. The biosynthesis of these biologically important deoxy sugars is not well understood, but progress is being made, fueled in part by the availability of DNA sequence data. These data facilitate the construction of mutant strains that are disrupted in potential sugar genes and permit the assignment of reasonable functions to deduced gene products by sequence comparisons to proteins of known function, such as those for the biosynthesis of rhamnose (16) and the 3,6-dideoxyhexoses (14) that are found in the lipopolysaccharides of gram-negative bacteria. Thus a putative glucose-1-phosphate thymidylyltransferase gene, dnmL (formerly dnrL) (8), which presumably governs the first step of daunosamine biosynthesis (Fig. 1), has been identified in the DNR gene cluster of the wild-type Streptomyces peucetius ATCC 29050 strain, and a thymidine-diphospho (TDP)-glucose-4,6-dehydratase for the formation of the 4-keto-6-deoxyhexulose nucleotide that is a key precursor in deoxyhexose biosynthesis has been purified (28). The gene encoding the putative TDP-glucose-4,6-dehydratase has been localized outside of the DNR gene cluster (8), while a TDP-glucose-4,6dehydratase homolog, dnmM (formerly dnrM) (8), found adjacent to dnmL, is apparently nonfunctional due to a frameshift mutation. Inactivation of the dnmJ (formerly dnrJ) gene (17) indicates that it is required for daunosamine biosynthesis, and the similarity between DnmJ and AscC (29) suggests that DnmJ is likely to be involved in the addition of the C-3 amino group to a daunosamine precursor (30). Further-
* Corresponding author. Mailing address: School of Pharmacy, University of Wisconsin, 425 N. Charter St., Madison, WI 53706. Phone: (608) 262-7582. Fax: (608) 262-3134. E-mail:
[email protected] .edu. † Present address: Biology Department, Niagara University, Niagara, NY. ‡ Present address: Biotechnology Department, DowElanco, Inc., Indianapolis, IN. 4446
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FIG. 1. Abbreviated pathway for the biosynthesis of DNR and DXR from propionyl-CoA, malonyl-CoA, and glucose-1-phosphate. Open arrows indicate multiple steps between precursor and product. The structures of substrates and the sequences of enzymatic reactions following the formation of TDP-4-keto-6-deoxy-D-glucose in the daunosamine pathway are hypothetical. 10-Carbomethoxy-13-deoxycarminomycin, rhodomycin D.
Characterization of the deduced product of dnmZ. Database comparisons of the deduced product of dnmZ with the BLASTP and TBLASTN programs (1) showed that DnmZ shares significant similarity (22 to 26% identity) with acyl coenzyme A (acyl-CoA) dehydrogenases (27), particularly shortchain fatty acid and isovaleryl-CoA dehydrogenases as found in rat (18) and human (19) cells. It also has a 26% identity with AidB, an adaptive response protein of Escherichia coli (13), and 74% identity with the Orf3 protein from the lkmB region of the DNR biosynthetic genes of Streptomyces griseus JA3933 (12). However, these relationships might mean that DnmZ is only a flavoprotein and not an acyl-CoA dehydrogenase. Gene disruption and replacement of dnmZ. To test the role of dnmZ in DNR biosynthesis, a 1.2-kb SmaI fragment from pUC4-KIXX (Pharmacia) containing the neomycin-kanamycin resistance gene was inserted into the MscI site (Fig. 2) in dnmZ. This disrupted copy of dnmZ was moved into the temperature-sensitive pKC1139 vector (3) as a 4.6-kb BamHI segment to give pWHM215. S. peucetius 29050 (pWHM215) transformants were selected on solid R2YE medium (11) at 30°C in the presence of kanamycin and apramycin. The cells were then grown in liquid R2YE medium containing no antibiotic at 39°C for 2 days, and samples of the culture were plated on solid R2YE medium containing kanamycin and incubated at 30°C to allow screening for apramycin-sensitive, kanamycin-resistant clones indicative of pWHM215 integration by homologous recombination at the dnmZ locus and loss
of the vector sequence. Southern hybridization of BamHIdigested genomic DNA from a representative apramycin-sensitive, kanamycin-resistant clone, WMH1629, established that the 3.4-kb BamHI fragment in the 29050 strain had shifted its mobility to 4.6 kb, as expected for replacement of the dnmZ gene with its disrupted form (data not shown). The WMH1629 dnmZ::aphII strain accumulated ε-rhodomycinone (RHO) when grown in a DNR production medium and analyzed for anthracycline metabolite production as previously described (20). Introduction of the dnmZ (a MluIBamHI segment [the MluI site is about 180-bp upstream of the BspHI site in dnmZ shown in Fig. 2] cloned in pWHM3 as pWHM216) or dnmUV (as pWHM218; see below) genes into this mutant by transformation did not restore DNR production, but this antibiotic was produced upon introduction of the dnmZUV genes (a MluI-BglII segment cloned in pWHM3 as pWHM219). Thus, dnmZ is essential for synthesis or attachment of daunosamine to RHO and the dnmZ::aphII mutation appears to be polar on dnmUV. (Since the actual transcription events for these genes have not been characterized beyond the information in reference 17, alternative explanations of the latter observation are possible.) If the DnmZ protein acts as an acyl-CoA dehydrogenase, it is not clear on the basis of current information (14) how this activity would form part of daunosamine biosynthesis. To explore this idea further, we tested the ability of the 29050 and WMH1629 strains to utilize fatty acids as the sole carbon and
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FIG. 2. The DNA sequence of the region containing dnmZUV. The proposed translational start and stop sites of the dnmZ, dnmU, and dnmV genes and the putative RBSs are doubly and singly underlined, respectively. Amino acid translations are shown for the dnmZ, dnmU, and dnmV gene products and the C-terminal end of the dnmJ (17, 25) gene product; stop codons are indicated with an asterisk. The underlined amino acid sequence in the dnmV gene product is a dinucleotide-binding domain. Only the restriction sites discussed in the text are shown above their recognition sites.
energy sources when grown in a minimal medium (11) containing butyrate, decanoate, or oleate along with Brij 58. There was only a very slight increase in the growth rate on butyrate and oleate of wild-type cells compared to dnmZ mutants. This slight variation is not the pattern expected if DnmZ is an acylCoA dehydrogenase required solely for this function. Moreover, when a 1.2-kb BspHI-BamHI fragment containing dnmZ (Fig. 2) was cloned into the NcoI-BamHI sites of pTRC99A (2) as pWHM211, a regulated, high-level expression of soluble DnmZ was observed in E. coli DH5aMCR as the appearance of a 43-kDa band after a 1-h induction with 10 mM isopropylb-D-thiogalactopyranoside. Introduction of pWHM211 into E. coli fad mutants defective in particular acyl-CoA dehydrogenases required for the metabolism of exogenous fatty acids (4) (LS6146 fadG106 fadR atoC(Con), deficient in short-chain de-
J. BACTERIOL.
hydrogenase; LS6149 fadF58, deficient in medium-chain dehydrogenase; and LS6150 fadE19, deficient in the acyl-CoA dehydrogenase electron transport flavoprotein) did not confer the ability to metabolize butyrate, decanoate, or oleate to any of these mutants, as reflected by the lack of any difference between the mutant and transformed fad strains in growth on minimal medium containing these fatty acids as the carbon sources. Consequently, DnmZ is likely to be a flavoprotein acting at some step in daunosamine biosynthesis instead of an acyl-CoA dehydrogenase. Characterization of the deduced product of dnmU. Database comparisons of the deduced product of dnmU with the BLASTP and TBLASTN programs (1) revealed similarities between DnmU and the NDP-4-keto-deoxyhexulose-3(5)-epimerases from sugar biosynthetic pathways that invert the configuration at C-3 and/or C-5 of the 4-keto-6-deoxy-hexulose derivatives which are common intermediates in the biosynthesis of many deoxysugars. The deduced product of dnmU is most similar to the product of ORF4 (84% identity by GAP analysis [6]) of S. griseus JA3933 (12) and StrM (45% identity), the putative TDP-4-keto-6-deoxyglucose-3(5)-epimerase from the streptomycin gene cluster of S. griseus (22). In addition, DnmU is 34% identical to RfbD, the TDP-4-keto-6-deoxyglucose-3(5)-epimerase, for rhamnose biosynthesis (16) and 31% identical to AscE, the CDP-4-keto-3,6-dideoxyhexulose-5-epimerase from the ascarylose biosynthetic pathway of Yersinia pseudotuberculosis (29, 31). Gene disruption and replacement of dnmU. The dnmU gene contained within a 4.4-kb BglII fragment was disrupted by replacing a 498-bp BspEI fragment containing most of dnmU with the kanamycin resistance gene from pUC4-KIXX. The disrupted dnmU gene, subcloned in the single-stranded DNA vector pDH5 (10), was introduced by transformation into the S. peucetius 29050 strain. Transformants containing the integrated plasmid were screened to identify colonies with the kanamycin-resistant, thiostrepton-sensitive phenotype resulting from gene replacement by a double crossover as previously described (20). A putative dnmU::aphII mutant strain (WMH1673) was obtained, and the NotI-digested chromosomal DNA from this strain was subjected to Southern analysis using a 1.1-kb NotI fragment containing dnmU as the probe. The probe DNA hybridized to the predicted 1.8-kb NotI fragment (composed of the 0.6-kb inactivated dnmU gene and the 1.2-kb aphII insert), verifying the disruption of dnmU. The WHM1673 strain was analyzed for antibiotic production (21) and was found to produce no DNR but instead accumulated RHO (69.6 mg/ml 6 17.42, n 5 3), indicating that DnmU is required for daunosamine biosynthesis. The dnmU::aphII mutant could be complemented by the dnmUV genes cloned as pWHM552 as a 1.7-kb BamHI-BglII fragment carried on the low-copy-number vector pWHM601 (9) or by dnmU cloned as pWHM553 as a 0.8-kb BamHI-NarI fragment in pWHM601. The complementation by dnmU alone was surprising since the mutation in dnmU was expected to have a polar effect on dnmV. However, analysis of plasmid DNAs by restriction endonuclease digestions and repetition of the complementation experiment con-
FIG. 3. Restriction map of the DNR gene cluster of S. peucetius 29050 showing the relative locations of all the genes believed to be involved in daunosamine (dnm) biosynthesis. The dnm gene designations are represented by letters. Arrows indicate direction of transcription. Restriction enzyme abbreviations: B, BamHI; Bg, BglII.
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firmed that the dnmU::aphII mutant strain was complemented by dnmU alone. Although this result raises the possibility that dnmZU and dnmV are in separate transcription units, we did not investigate this matter. The phenotype and complementation of the WHM1673 mutant strain indicate that DnmU is required for DNR biosynthesis. Together with the sequence similarities between the deduced product of dnmU and the NDP-4-keto-6-deoxyhexulose-3(5)-epimerases from other deoxysugar biosynthetic pathways, the results suggest that DnmU functions as the TDP-4keto-6-deoxyglucose-3(5)-epimerase for the formation of TDP-daunosamine, although we do not know if TDP-4-keto6-deoxyglucose is the actual substrate for this enzyme. Characterization of dnmV. Database comparisons of DnmV revealed strong similarity (64% identity) to the deduced product of ORF5, a gene of unknown function from the lkmB region of S. griseus (12) that has been suggested to be involved in the biosynthesis of daunosamine. Surprisingly, the deduced ORF5 gene product lacks the C-terminal 122 amino acids of DnmV due to an apparent frameshift at the position corresponding to nt 2291 of the dnmUV sequence (Fig. 2). The significance of this difference is not known; however, the length of the deduced DnmV peptide is similar to that of related proteins discussed below, and careful examination of the sequence data in this region did not reveal the presence of a sequencing error. DnmV is also distantly related to a group of proteins that are involved in the metabolism of nucleotide-activated hexoses. These proteins include the UDP-glucose 4-epimerases such as ExoB from Rhizobium meliloti (5), the NDP-glucose 4,6-dehydratases that catalyze 6-deoxygenation of nucleotide-activated hexoses such as StrE from S. griseus (22), and RfbC (16), the TDP-4-ketorhamnose ketoreductase that catalyzes the final step of rhamnose biosynthesis. The unifying properties of these proteins (which have different functions) are that they target the C-4 position of a nucleotide-activated hexose and possess a dinucleotide-binding domain (16, 32). This dinucleotide-binding domain is located near the N terminus of the protein and is characterized by a GXXGXXG motif which is present in DnmV as well (underlined in Fig. 2). Furthermore, dnmV resembles genes for putative NDP-4-ketodeoxyhexulose ketoreductases from Streptomyces spp. that are involved in the biosynthesis of the deoxysugar components of antibiotic molecules. These include avr-ORF4 (30% identity) and eryBIV (27% identity), which encode the putative TDP-4-ketodeoxyhexulose ketoreductases for oleandrose (15) and mycarose (26) biosynthesis, respectively. These data suggest that dnmV also functions in this manner and is likely to encode the TDP4-ketodeoxyhexulose ketoreductase of the daunosamine biosynthetic pathway. The role of dnmV was then established by gene disruption. A 1.67-kb BamHI-BglII fragment containing the dnmV and dnmU genes was cloned into the corresponding sites of pUC19, and then a 1.2-kb SmaI fragment containing the kanamycin resistance gene (see above) was cloned into the unique NotI site of dnmV (Fig. 2) after converting the sticky NotI ends to blunt ends according to standard procedures (23). The disrupted dnmV::aphII gene was cloned into pDH5 and used to replace the genomic copy of dnmV as described for dnmU above. Two dnmV::aphII mutants (WMH1621 and WMH1622) were obtained by this strategy, and both of them accumulated RHO but did not produce DNR when tested as described above. Since introduction of the dnmUV and dnmV genes into the WMH1621 strain restored DNR production, the dnmV gene is essential for daunosamine biosynthesis.
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TABLE 1. Functions of the TDP-L-daunosamine biosynthesis genes Gene
dnmL dnmM dnmZ dnmU dnmV dnmJ dnmT dnmQ dnmS
Presumed role of the gene product
Synthesis of TDP-D-glucose Natural frameshift mutant; functional equivalent would make TDP-4-keto-6-deoxyglucose Unknown Synthesis of the L stereoisomer of TDP-4-keto6-deoxyglucose or a later intermediate Reduction of the 4-ketone group Introduction of the C-3 amino group Unknown but speculated to be involved in C-2 deoxygenation Putative C-2 deoxygenation Glycosyltransferase for TDP-L-daunosamine
Reference(s) or source
8 8 This work This work This work 17, 30 24 21 21, 24
Concluding remarks. The important and diverse biological functions of the dideoxyhexoses (14), has lead to a growing interest in the genetics of deoxyhexose biosynthesis, and much information has been gained from comparisons of the deduced amino acid sequences of genes required for the synthesis of structurally related sugars. The amino acid sequences of the NDP-hexose 4,6-dehydratases from various sources are found to be substantially conserved, reflecting the expected conserved biochemical mechanism of 6-deoxygenation. The product of this reaction, an NDP-4-keto-6-deoxyhexulose, is thought to be the precursor of many other dideoxy and trideoxy sugars (14). In the synthesis of the 6-deoxyhexose rhamnose, the next step is a 3(5)-epimerization catalyzed by RfbD (16). The sequence similarity between RfbD and the products of dnmU and other putative 3(5)-epimerase genes suggests that they too may act on an NDP-4-keto-6-deoxyhexulose. Relative to the TDP-hexose-4,6-dehydratases and -3(5)-epimerases, DnmV and other putative 4-ketoreductases that have been identified so far share less overall homology. This observation may be a consequence of the different stereochemical functions of 4-ketoreductases or may indicate that 4-keto reduction occurs at a later step in the pathway when substrates have become structurally diverse. Homologs of several of the dnm genes are present in the cluster of erythromycin biosynthesis genes in Saccharopolyspora erythraea (26). There is a close similarity between the steps in the daunosamine pathway and the formation of 2,6dideoxy-L-mycarose, whereas the formation of 3,4,6-trideoxy3-amino-D-desosamine, the second deoxysugar present in erythromycin, has fewer features in common with daunosamine biosynthesis. Two enigmatic differences stand out as follows: (i) The homologous dnmQ and eryCII genes both are associated with deoxyamino sugar biosynthesis but are believed to involve C-2 deoxygenation and C-4 deoxygenation, respectively, and (ii) A dnmZ homolog is not involved in erythromycin biosynthesis (or in oleandrose formation [15]). Thus, the mechanisms of C-2, C-3, and C-4 deoxygenation seem to be different in the two pathways even though all of the other steps of deoxysugar formation are similar. Nucleotide sequence accession number. The DNA sequence data described in this paper have been deposited at EMBL and GenBank with accession number AF006633. We thank Doug MacNeil and Leonard Katz for unpublished sequence data and helpful discussions about deoxysugar biosynthesis. This work was supported by grants from the American Cancer Society (CH-524) and Farmitalia Carlo Erba (Pharmacia S.p.a.).
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