Insights into the Branched-Chain Formation of Mycarose: Methylation Catalyzed by an (S)-Adenosylmethionine-Dependent Methyltransferase

Chen, Huawei; Zhao, Zongbao K.; Hallis, Tina M.; Guo, Zhihong; Liu, Hung‐wen

doi:10.1002/1521-3757(20010202)113:3<627::aid-ange627>3.0.co;2-f

Cited by 13 publications

(5 citation statements)

References 21 publications

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“…EryBVI and EryBII, which catalyze C2-deoxygenation of TDP-4-keto-6-deoxy-D-glucose (B) to yield TDP-4-keto-2,6-dideoxy-D-glucose (H), are structurally and functionally distinct from the corresponding enzymes catalyzing the C-3 deoxygenation in the biosynthesis of 3,6-dideoxysugars [27]. TylCIII, a 46.4-kDa C-methyl transferase, catalyzes C-3 methylation of the 2,6-dideoxysugar (H) to produce TDP-4-keto-3-methyl-2,6-dideoxy-D-glucose (I), and uses S-AdoMet as a cosubstrate [28]. C5-epimerization is catalyzed by TylCVII [29], whereas EryBIV, a 4-ketoreductase, converts TDP-4-keto-3-methyl-2,6-dideoxy-L-glucose (J) to TDP-L-mycarose (K) [12].…”

Section: Resultsmentioning

confidence: 99%

Bioassay-Guided Evolution of Glycosylated Macrolide Antibiotics in Escherichia coli

Lee

Khosla

2007

PLoS Biol

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Macrolide antibiotics such as erythromycin are clinically important polyketide natural products. We have engineered a recombinant strain of Escherichia coli that produces small but measurable quantities of the bioactive macrolide 6-deoxyerythromycin D. Bioassay-guided evolution of this strain led to the identification of an antibiotic-overproducing mutation in the mycarose biosynthesis and transfer pathway that was detectable via a colony-based screening assay. This high-throughput assay was then used to evolve second-generation mutants capable of enhanced precursor-directed biosynthesis of macrolide antibiotics. The availability of a screen for macrolide biosynthesis in E. coli offers a fundamentally new approach in dissecting modular megasynthase mechanisms as well as engineering antibiotics with novel pharmacological properties.

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Section: Resultsmentioning

confidence: 99%

Bioassay-Guided Evolution of Glycosylated Macrolide Antibiotics in Escherichia coli

Lee

Khosla

2007

PLoS Biol

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“…2.1.1.-) using S-adenosylmethionine (adoMet) as the methyl donor. A role for this enzyme has been demonstrated in the biosynthesis of L-mycarose, erythromycin, and avilamycin by S. fradiae [45], S. erythraea [46], and S. viridochromogenes [47], respectively. In C. burnetii , the product of the CBU_0691 locus has 44% (E value 1e-127), 44% (E value 3e-121) and 42% (E value 7e-119) amino acid identity with TylCIII from S. fradiae (AAD41823), EryBIII from S. erythrae ( YP_001102998), and aviGI from S. viridochromogenes (AAK83176), respectively.…”

Section: Resultsmentioning

confidence: 99%

“…Although this enzyme has only been demonstrated to be active with TDP-sugars, there is no a priori reason that it might not be able to accommodate a range of NDP-sugars. The results from mechanistic studies indicate that the methylation reaction catalyzed by TylCIII proceeds with an inversion of hydroxyl groups at C-3’ [45]. Thus, the equatorial methyl group at C-3’ position and the axial C-3’ hydroxyl group that are structural features of D- (12) and L-virenose (13) [16,17] are reversed.…”

Section: Resultsmentioning

confidence: 99%

In silico biosynthesis of virenose, a methylated deoxy-sugar unique to Coxiella burnetii lipopolysaccharide

et al. 2012

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BackgroundCoxiella burnetii is Gram-negative bacterium responsible for the zoonosis Q-fever. While it has an obligate intracellular growth habit, it is able to persist for extended periods outside of a host cell and can resist environmental conditions that would be lethal to most prokaryotes. It is these extracellular bacteria that are the infectious stage encountered by eukaryotic hosts. The intracellular form has evolved to grow and replicate within acidified parasitophorous vacuoles. The outer coat of C. burnetii comprises a complex lipopolysaccharide (LPS) component that includes the unique methylated-6-deoxyhexose, virenose. Although potentially important as a biomarker for C. burnetii, the pathway for its biosynthesis remains obscure.ResultsThe 6-deoxyhexoses constitute a large family integral to the LPS of many eubacteria. It is believed that precursors of the methylated-deoxyhexoses traverse common early biosynthetic steps as nucleotide-monosaccharides. As a prelude to a full biosynthetic characterization, we present herein the results from bioinformatics-based, proteomics-supported predictions of the pathway for virenose synthesis. Alternative possibilities are considered which include both GDP-mannose and TDP-glucose as precursors.ConclusionWe propose that biosynthesis of the unique C. burnetii biomarker, virenose, involves an early pathway similar to that of other C-3’-methylated deoxysugars which then diverges depending upon the nucleotide-carrier involved. The alternatives yield either the D- or L-enantiomers of virenose. Both pathways require five enzymatic steps, beginning with either glucose-6-phosphate or mannose-6-phosphate. Our in silico results comprise a model for virenose biosynthesis that can be directly tested. Definition of this pathway should facilitate the development of therapeutic agents useful for treatment of Q fever, as well as allowing improvements in the methods for diagnosing this highly infectious disease.

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“…The functions of each of the enzymes in TDP-β-L-mycarose ( 13 ) biosynthesis in the tylosin producer, S. fradiae , have been verified in vitro and are shown in Scheme 4 (Chen et al, 1999; Chen et al, 2001; Takahashi et al, 2005). From TDP-4-keto-6-deoxy-α-D - glucose ( 6 ), TylX3 and TylC1 catalyze the formation of 9 .…”

Section: Introductionmentioning

confidence: 98%

“…The reaction is allowed to proceed for 1 h at room temperature to convert 6 to 13 . The cloning, expression, and purification of the mycarose biosynthetic enzymes have been previously described (Chen et al, 1999; Chen et al, 2001; Takahashi et al, 2005). TDP-β-L-mycarose ( 13 ) is purified from the reaction mixture by FPLC using a MonoQ 10/10 column that is eluted with H 2 O over 2-column volumes, followed by a linear gradient from 0–280 mM NH 4 HCO 3 buffer (pH 7.0) over 2-column volumes at a flow rate of 1 mL/min.…”

Section: Introductionmentioning

confidence: 99%

Chapter 21 Enzymatic Synthesis of TDP-Deoxysugars

White-Phillip

Thibodeaux

Liu

2009

Methods in Enzymology

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Many biologically active bacterial natural products contain highly modified deoxysugar residues that are often critical for the activity of the parent compounds. Most of these deoxysugars are secondary metabolites that are biosynthesized in the form of nucleotide diphosphate (NDP) sugars prior to their transfer to natural product aglycones by glycosyltransferases. Over the past decade, many biosynthetic pathways that lead to the formation of these unusual sugars have been unraveled, and the mechanisms of many key enzymatic transformations involved in these pathways have been elucidated. However, obtaining workable quantities of NDP-deoxysugars for in vitro studies is often a difficult task. This limitation has hindered an in-depth investigation of the substrate specificity of deoxysugar biosynthetic enzymes, many of which are promiscuous with respect to their NDP-sugar substrates and are, thus, potentially useful catalysts for natural product glycoengineering. Presented in this review are procedures for the enzymatic synthesis and purification of a variety of NDP-deoxysugars, including some early intermediates in NDP-deoxysugar biosynthetic pathways, and highly modified NDP-deoxysugars that are late intermediates in their respective biosynthetic pathways. The procedures described herein could be used as general guidelines for the development of specific protocols for the synthesis of other NDP-deoxysugars.

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Insights into the Branched-Chain Formation of Mycarose: Methylation Catalyzed by an (S)-Adenosylmethionine-Dependent Methyltransferase

Cited by 13 publications

References 21 publications

Bioassay-Guided Evolution of Glycosylated Macrolide Antibiotics in Escherichia coli

Bioassay-Guided Evolution of Glycosylated Macrolide Antibiotics in Escherichia coli

In silico biosynthesis of virenose, a methylated deoxy-sugar unique to Coxiella burnetii lipopolysaccharide

Chapter 21 Enzymatic Synthesis of TDP-Deoxysugars

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