Mechanism of the 2-Deoxygenation Step in the Biosynthesis of the Deoxyhexose Moieties of the Antibiotics Granaticin and Oleandomycin

Draeger, Gerald; Park, Jin Young; Floss, Heinz G.

doi:10.1021/ja9837250

Cited by 83 publications

(107 citation statements)

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“…Two enzymes are required to catalyze this deoxygenation, a dehydratase and a reductase. Experimental biochemical evidence for the activity of these two enzymes has been reported for the biosynthesis of granaticin and oleandomycin in Streptomyces violaceoruber Tü22 and in S. antibioticus Tü99, respectively (8), and for the biosynthesis of tylosin in S. fradiae (5). 2,3-Dehydration is first catalyzed by a small family of Zn 2ϩ -dependent dehydratases (5) that share a high degree of similarity at the protein level.…”

Section: Discussionmentioning

confidence: 99%

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Functional Analysis of OleY l -Oleandrosyl 3- O -Methyltransferase of the Oleandomycin Biosynthetic Pathway in Streptomyces antibioticus

Rodrı́guez

Olano

et al. 2001

J Bacteriol

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Oleandomycin, a macrolide antibiotic produced by Streptomyces antibioticus, contains two sugars attached to the aglycon: L-oleandrose and D-desosamine. oleY codes for a methyltransferase involved in the biosynthesis of L-oleandrose. This gene was overexpressed in Escherichia coli to form inclusion bodies and in Streptomyces lividans, producing a soluble protein. S. lividans overexpressing oleY was used as a biotransformation host, and it converted the precursor L-olivosyl-erythronolide B into its 3-O-methylated derivative, L-oleandrosyl-erythronolide B. Two other monoglycosylated derivatives were also substrates for the OleY methyltransferase: L-rhamnosyl-and L-mycarosyl-erythronolide B. OleY methyltransferase was purified yielding a 43-kDa single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme showed a molecular mass of 87 kDa by gel filtration chromatography, indicating that the enzyme acts as a dimer. It showed a narrow pH range for optimal activity, and its activity was clearly stimulated by the presence of several divalent cations, being maximal with Co 2؉ . The S. antibioticus OleG2 glycosyltransferase is proposed to transfer L-olivose to the oleandolide aglycon, which is then converted into L-oleandrose by the OleY methyltransferase. This represents an alternative route for L-oleandrose biosynthesis from that in the avermectin producer Streptomyces avermitilis, in which L-oleandrose is transferred to the aglycon by a glycosyltransferase.Oleandomycin (Fig. 1A) is a clinically important 14-membered macrolide antibiotic produced by Streptomyces antibioticus. Structurally it contains a macrolactone ring (oleandolide) which is synthesized by the assembly of one starter acetylcoenzyme A (CoA) and six extender methylmalonyl CoA units. These reactions are catalyzed by a modular type I polyketide synthase comprising three large polypeptides, named OleA1, OleA2, and OleA3 (24,25). The oleandomycin biosynthetic gene cluster has been fully sequenced and characterized (1,15,16,18,19,24,25) (Fig. 1B). Two DNA regions flanking the genes encoding the three multifunctional polypeptides of the polyketide synthase contain a number of genes that code for other enzymatic functions required for the biosynthesis of the two sugars and their transfer to the aglycon (1, 16, 18), genes encoding an ABC transporter system responsible for secretion (15), genes encoding an inactivating-reactivating enzymatic system that confers self-resistance to the producer organism (18, 26), and a gene encoding a cytochrome P-450 oxygenase involved in epoxidation of the macrolactone ring at carbon 8 (19, 24). The oleandomycin macrolactone ring is glycosylated by two 6-deoxysugars (L-oleandrose and D-desosamine). Two glycosyltransferases (OleG1 and OleG2) are responsible for sugar transfer. Insertional inactivation of oleG1 produced a mutant that accumulates the macrolactone 8,8a-deoxyoleandolide, because both OleG1 and OleG2 glycosyltransferases were affected in this mutant by a polar effect (16). Complementatio...

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Section: Discussionmentioning

confidence: 99%

“…Two mechanisms exist for catalyzing the C-3 reduction step. In one way, exemplified by the Graorf26 or the Tü99-orf11, the reduction forms a sugar in which the C-3 hydroxy group is equatorial (8). In the other approach, exemplified by TylC2, the resulting hydroxy group is axial (5).…”

Section: Discussionmentioning

confidence: 99%

Functional Analysis of OleY l -Oleandrosyl 3- O -Methyltransferase of the Oleandomycin Biosynthetic Pathway in Streptomyces antibioticus

Rodrı́guez

Olano

et al. 2001

J Bacteriol

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“…After that, only a few functional studies have been carried out on this cluster. [2][3][4][5] The functions of several gra genes remain totally unknown. Knowledge about how the production is regulated is still limited.…”

mentioning

confidence: 99%

Streptomyces vietnamensis GIMV4.0001: a granaticin-producing strain that can be readily genetically manipulated

2011

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Granaticin belongs to a class of aromatic polyketides, benzoisochromanequinone antibiotics, among which actinorhodin is the most well-known member. The biosynthetic gene cluster of actinorhodin have been intensively studied and serves as a model for studying the type II polyketide synthatase (PKS) pathways. Granaticin has a similar basic skeleton but unique fine structure compared to actinorhodin (Figure 1). The opposite stereochemistry of the pyran ring and the unusual sugar attachment presented in granaticin structure has drawn much attention among chemical and biochemical researchers. The granaticin biosynthetic gene cluster (gra) was identified originally from Streptomyces violaceoruber Tü22, 1 and the functions of most biosynthetic genes were assigned based on sequence homology. After that, only a few functional studies have been carried out on this cluster. [2][3][4][5] The functions of several gra genes remain totally unknown. Knowledge about how the production is regulated is still limited. The fact that S. violaceoruber Tü22 is recalcitrant to plasmid transformation 1 has imposed a significant barrier to its study.S. vietnamensis GIMV4.0001 (CCTCC M 205143, hereafter referred as GIMV4.0001), the type strain of a newly designated streptomycete species by our laboratory, 6 was found to be a novel granaticin producer, and the gra cluster was sequenced by a sequential cloning strategy (unpublished result, GenBank accession number: GU233672). In this study, we report the establishment of an efficient and stable conjugation system between Escherichia coli and GIMV4.0001 and applying a modified PCR-targeted disruption method to GIMV4.0001.All strains and plasmids used in this study are listed in Supplementary Table S1. To screen putative exconjugants derived from GIMV4.0001, we first carried out an antibiotic sensitivity test using an agar diffusion method (Supplementary Information, Materials and methods). All the plates containing GIMV4.0001 showed clear zones of inhibition around the holes with apramycin, kanamycin, thiostrepton, streptomycin or spectinomycin at 10 mg ml À1 , suggesting that there were adequate choices of selectable marker for genetic manipulation of GIMV4.0001.Two kinds of plasmids, auto-replicating pHZ1358 and integrative pSET152, were used to test the feasibility to introduce foreign DNA into GIMV4.0001. The basic procedures of conjugation between E. coli and GIMV4.0001 were as described by Kieser et al., 7 but some modifications were made (Supplementary Information, Materials and methods). The optimal medium for conjugation was screened. Exconjugants were confirmed by PCR amplification with an aac3(IV)-specific primer pair (for pSET152; Supplementary Information, Table S2) or by plasmid isolation and restriction mapping (for pHZ1358). For pSET152, putative exconjugants were obtained from YD, SFM, YMS and Gauze's synthetic agar, but the efficiency differed. The conjugation frequency for YD, SFM, YMS and Gauze's synthetic agar were 8Â10 À4 , 3Â10 À4 , 5Â10 À6 and 2Â10 À5 , respectively. F...

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“…Alternatively, 2-deoxygenation might occur first (Summers et al, 1997), in which case the stereochemistry at C-3 following the enoylreductase reaction would dictate the mode of subsequent epimerization. Support for the latter route, at least in the biosynthesis of other 2,6-dideoxyhexoses, came from recent in vitro studies (Draeger et al, 1999) involving pairs of purified enzymes that catalyse 2-deoxygenation of dTDP-4-keto-6-deoxy--glucose (Fig. 3, compound 1) during the synthesis of -rhodinose in Streptomyces violaceoruber Tu$ 22, and of -oleandrose in Streptomyces antibioticus Tu$ 99.…”

Section: The Biosynthetic Route To Mycarosementioning

confidence: 99%

The mycarose-biosynthetic genes of Streptomyces fradiae, producer of tylosin The GenBank accession number for the sequence determined in this work is AF147704.

et al. 2000

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The tylCK region of the Streptomyces fradiae genome was sequenced, revealing an incomplete set of five tylC genes encoding all-but-one of the enzymes involved in the biosynthesis of mycarose. The latter is a 6-deoxyhexose sugar required during production of the macrolide antibiotic, tylosin. The missing mycarose-biosynthetic gene, tylCVI, was found about 50 kb distant from its functional partners, on the other side of the tylG (polyketide synthase) gene complex. Mutational analysis, involving targeted gene transplacement, was employed to confirm the functions of specific genes, including tylCVI. Particularly interesting was the similarity between the tylosin-biosynthetic mycarosyltransferase enzyme, TylCV, and proteins of the macrolide glycosyltransferase (MGT) family that inactivate macrolides via glycosylation of attached sugar residues and are involved in resistance and/or antibiotic efflux. The arrangement of genes within the ' mycarose cluster ' would allow their expression as two short operons with divergent, and perhaps co-regulated, promoters. Whether displacement of tylCVI relative to the other tylC genes provides additional regulatory opportunities remains to be established.

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Mechanism of the 2-Deoxygenation Step in the Biosynthesis of the Deoxyhexose Moieties of the Antibiotics Granaticin and Oleandomycin

Cited by 83 publications

References 30 publications

Functional Analysis of OleY l -Oleandrosyl 3- O -Methyltransferase of the Oleandomycin Biosynthetic Pathway in Streptomyces antibioticus

Functional Analysis of OleY l -Oleandrosyl 3- O -Methyltransferase of the Oleandomycin Biosynthetic Pathway in Streptomyces antibioticus

Streptomyces vietnamensis GIMV4.0001: a granaticin-producing strain that can be readily genetically manipulated

The mycarose-biosynthetic genes of Streptomyces fradiae, producer of tylosin The GenBank accession number for the sequence determined in this work is AF147704.

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