Substrate Specificity of the Macrolide‐Glycosylating Enzyme Pair DesVII/DesVIII: Opportunities, Limitations, and Mechanistic Hypotheses

Borisova, Svetlana A.; Zhang, Changsheng; Takahashi, Haruko; Zhang, Hua; Wong, Amy; Thorson, Jon S.; Liu, Hung‐wen

doi:10.1002/anie.200503195

Cited by 69 publications

(65 citation statements)

References 72 publications

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“…Previously, it was shown that the activation of some glycosyltransferases depends upon their interactions with auxiliary proteins. One glycosyltransferase, DesVII, which is involved in the biosynthesis of the macrolide antibiotics methymycin and pikromycin in Streptomyces venezuelae, was only activated if an auxiliary protein, DesVIII, was present (36)(37)(38). Similarly, other glycosyltransferases have been found to require another auxiliary protein for their activity (39)(40)(41).…”

Section: Discussionmentioning

confidence: 99%

“…Activity of the glycosyltransferase EryCIII, which is required for biosynthesis of the antibiotic erythromycin D, is dependent upon an auxiliary protein, EryCII (43). Although the roles of these auxiliary proteins are not clear, it has been proposed that these auxiliary proteins are required to induce conformational changes of the glycosyltransferases, which consequently activate these enzymes (36,42).…”

Section: Discussionmentioning

confidence: 99%

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Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Ghafoor

Jordens

Rehm

2013

Appl Environ Microbiol

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Pseudomonas aeruginosa produces three exopolysaccharides, Psl, Pel, and alginate, that play vital roles in biofilm formation. Pel is a glucose-rich, cellulose-like exopolysaccharide. The essential Pel biosynthesis proteins are encoded by seven genes, pelA to pelG. Bioinformatics analysis suggests that PelF is a cytosolic glycosyltransferase. Here, experimental evidence was provided to support this PelF function. A UDP-glucose dehydrogenase-based assay was developed to quantify UDP-glucose. UDP-glucose was proposed as the substrate for PelF. The isogenic pelF deletion mutant accumulated 1.8 times more UDP-glucose in its cytosol than the wild type. This suggested that PelF, which was found localized in the cystosol, uses UDP-glucose as substrate. Additionally, in vitro experiments confirmed that PelF uses UDP-glucose as substrate. To analyze the functional roles of conserved residues in PelF, site-directed mutagenesis was performed. The presence of the EX 7 E motif is characteristic for various glycosyltransferase families, and in PelF, E405/E413 are the conserved residues in this motif. Replacement of E405 with A resulted in a reduction of PelF activity to 30.35% ؎ 3.15% (mean ؎ standard deviation) of the wild-type level, whereas replacement of the second E, E413, with A did not produce a significant change in the activity of PelF. Moreover, replacement of both E residues did not result in a loss of PelF function, but replacement of the conserved R325 or K330 with A resulted in a complete loss of PelF activity. Overall, our data show that PelF is a soluble glycosyltransferase that uses UDP-glucose as the substrate for Pel synthesis and that conserved residues R325 and K330 are important for the activity of PelF.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Ghafoor

Jordens

Rehm

2013

Appl Environ Microbiol

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“…This was determined during the study of DesVII, a glycosyltransferase that catalyzes the attachment of desosamine onto 12-and 14-membered macrolactone rings during the biosynthesis of the macrolide antibiotics methymycin and pikromycin in Streptomyces venezuelae. For glycosylation to proceed efficiently, the presence of an auxiliary protein, DesVIII, was found to be necessary in vivo as well as in vitro (2,3,12,13). Since the discovery of the role of DesVIII in glycosylation, other pairs of glycosyltransferases and auxiliary proteins have been identified and characterized, and most of these are involved in macrolide biosynthesis (14,24,25).…”

mentioning

confidence: 99%

“…Indeed, it has been observed that the auxiliary protein is required for the initial activation of the glycosyltransferase, but then the activated glycosyltransferase can catalyze the glycosyltransfer alone. It was thus proposed that auxiliary proteins induce a one-time conformational change of the glycosyltransferase to its active form (2).…”

mentioning

confidence: 99%

Glycosylation Steps during Spiramycin Biosynthesis in Streptomyces ambofaciens : Involvement of Three Glycosyltransferases and Their Interplay with Two Auxiliary Proteins

Nguyen

Karray

Lautru

et al. 2010

Antimicrob Agents Chemother

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Streptomyces ambofaciens synthesizes spiramycin, a 16-membered macrolide antibiotic used in human medicine. The spiramycin molecule consists of a polyketide lactone ring (platenolide) synthesized by a type I polyketide synthase, to which three deoxyhexoses (mycaminose, forosamine, and mycarose) are attached successively in this order. These sugars are essential to the antibacterial activity of spiramycin. We previously identified four genes in the spiramycin biosynthetic gene cluster predicted to encode glycosyltransferases. We individually deleted each of these four genes and showed that three of them were required for spiramycin biosynthesis. The role of each of the three glycosyltransferases in spiramycin biosynthesis was determined by identifying the biosynthetic intermediates accumulated by the corresponding mutant strains. This led to the identification of the glycosyltransferase responsible for the attachment of each of the three sugars. Moreover, two genes encoding putative glycosyltransferase auxiliary proteins were also identified in the spiramycin biosynthetic gene cluster. When these two genes were deleted, one of them was found to be dispensable for spiramycin biosynthesis. However, analysis of the biosynthetic intermediates accumulated by mutant strains devoid of each of the auxiliary proteins (or of both of them), together with complementation experiments, revealed the interplay of glycosyltransferases with the auxiliary proteins. One of the auxiliary proteins interacted efficiently with the two glycosyltransferases transferring mycaminose and forosamine while the other auxiliary protein interacted only with the mycaminosyltransferase.Macrolide antibiotics consist of a polyketide macrolactone ring to which one or more deoxysugars are attached. They inhibit protein synthesis by binding to the large subunit of the ribosome and can also, in some cases, interfere with the assembly of the large subunit (reviewed in reference19). As with many other natural products, glycosylation of macrolides is essential for their biological activity. The determination of the three-dimensional structure of the 50S ribosomal unit in complex with various macrolides showed that the sugar moieties are involved in specific interactions with the 23S rRNA (11, 38). All macrolides block the ribosomal peptide exit tunnel, thus preventing peptide chain elongation. In addition to their role in binding, the sugars are also important for steric hindrance in the tunnel. For instance, it was shown that the C-5 disaccharide moieties of some 16-membered macrolides, such as spiramycin or tylosin, extend in the tunnel toward the peptidyl transferase center, and a correlation could be established between the length of the macrolide saccharide side chain and the number of amino acids which could be incorporated before peptide chain elongation was blocked (11).One of the most common resistance mechanisms to macrolide antibiotics encountered in pathogenic bacteria involves target modification. These modifications could include mutation or m...

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“…Among several well-known actinomycete heterologous hosts, a pikromycin (Pik)/methymycin-producing strain, Streptomyces venezuelae ATCC 15439, has recently been developed as a promising host in that it is amenable to genetic manipulation and requires a short culture period (3 to 4 days) for metabolite production compared to other Streptomyces species (7,8,19,23,24). In addition, the presence of two post-polyketide synthase (PKS) tailoring enzymes that have unusual substrate flexibility, DesVII glycosyltransferase (2,3,6,25) and PikC P450 monooxygenase (11,13,18), offers it another advantage as a heterologous host for combinatorial biosynthesis of novel compounds. However, one of the key challenges in developing S. venezuelae as a competent heterologous host is the relatively low production of heterologous polyketides (8).…”

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confidence: 99%

Enhanced Heterologous Production of Desosaminyl Macrolides and Their Hydroxylated Derivatives by Overexpression of the pikD Regulatory Gene in Streptomyces venezuelae

Jung

Jeong

Park

et al. 2008

Appl Environ Microbiol

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To elevate the production level of heterologous polyketide in Streptomyces venezuelae, an additional copy of the positive regulatory gene pikD was introduced into the pikromycin (Pik) polyketide synthase (PKS) deletion mutant of S. venezuelae ATCC 15439 expressing tylosin PKS genes. The resulting mutant strain showed enhanced production of both tylactone (TL) and desosaminyl tylactone (DesTL) of 2.7-and 17.1-fold, respectively. The notable increase in DesTL production strongly suggested that PikD upregulates the expression of the desosamine (des) biosynthetic gene cluster. In addition, two hydroxylated forms of DesTL were newly detected from the extract of this mutant. These hydroxylated forms presumably resulted from a PikDdependent increase in expression of the pikC gene that encodes P450 hydroxylase. Gene expression analysis by reverse transcriptase PCR and bioconversion experiments of 10-deoxymethynolide, narbonolide, and TL into the corresponding desosaminyl macrolides indicated that PikD is a positive regulator of the des and pikC genes, as well as the Pik PKS genes. These results demonstrate the role of PikD as a pathway-specific positive regulator of the entire Pik biosynthetic pathway and its usefulness in the development of a host-vector system for efficient heterologous production of desosaminyl macrolides and novel hydroxylated compounds.

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Substrate Specificity of the Macrolide‐Glycosylating Enzyme Pair DesVII/DesVIII: Opportunities, Limitations, and Mechanistic Hypotheses

Abstract: Two's Company: DesVII, a glycosyltransferase involved in the biosynthesis of macrolide antibiotics, is unusual in that it requires an additional protein partner, DesVIII, for its full activity. The level of substrate tolerance of the DesVII/DesVIII pair was explored.

Cited by 69 publications

References 72 publications

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Glycosylation Steps during Spiramycin Biosynthesis in Streptomyces ambofaciens : Involvement of Three Glycosyltransferases and Their Interplay with Two Auxiliary Proteins

Enhanced Heterologous Production of Desosaminyl Macrolides and Their Hydroxylated Derivatives by Overexpression of the pikD Regulatory Gene in Streptomyces venezuelae

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