Structural Characterization of O‐ and C‐Glycosylating Variants of the Landomycin Glycosyltransferase LanGT2

Tam, Heng-Keat; Härle, Johannes; Gerhardt, S.; Rohr, Jürgen; Wang, Guojun; Thorson, Jon S.; Bigot, Aurélien; Lutterbeck, Monika; Seiche, Wolfgang; Breit, Bernhard; Bechthold, Andreas; Einsle, Oliver

doi:10.1002/anie.201409792

Cited by 26 publications

(27 citation statements)

References 42 publications

(38 reference statements)

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“…Therefore, we initially believed that the C-glycosylation activity of AbCGT was also dependent on the acyl group, similar to other known CGTs. However, to our surprise, AbCGT exhibited catalytic activity when phloroglucinol derivatives (20)(21)(22) without acyl groups attached to the aromatic rings were used as substrates ( Supplementary Figs. 34-36).…”

Section: Resultsmentioning

confidence: 98%

“…Plant CGTs usually recognize only substrates with the basic core structure of acyl phloroglucinol, which possesses three hydroxyl groups and one acyl group 15,[23][24][25][26]29,31,32 . Thus, to explore the effects of the number and position of phenolic hydroxyl groups on the catalytic activity of AbCGT, an acceptor library containing acyl phenols (1-3), acyl resorcinols (4-9), acyl phloroglucinols (10-19 and 23), phloroglucinol derivatives (20)(21)(22) without basic acyl groups, and coumarins (24)(25)(26) was used for the reactions (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…During the past decades, microbial CGTs have attracted considerable interest, and great progress has been achieved in identification of CGTs from bacteria [17][18][19][20][21] . However, studies on plant CGTs were rather limited until the year of 2009 22,23 .…”

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confidence: 99%

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Exploring and applying the substrate promiscuity of a C-glycosyltransferase in the chemo-enzymatic synthesis of bioactive C-glycosides

et al. 2020

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Bioactive natural C-glycosides are rare and chemical C-glycosylation faces challenges while enzymatic C-glycosylation catalyzed by C-glycosyltransferases provides an alternative way. However, only a small number of C-glycosyltransferases have been found, and most of the discovered C-glycosyltransferases prefer to glycosylate phenols with an acyl side chain. Here, a promiscuous C-glycosyltransferase, AbCGT, which is capable of C-glycosylating scaffolds lacking acyl groups, is identified from Aloe barbadensis. Based on the substrate promiscuity of AbCGT, 16 C-glycosides with inhibitory activity against sodium-dependent glucose transporters 2 are chemo-enzymatically synthesized. The C-glycoside 46a shows hypoglycemic activity in diabetic mice and is biosynthesized with a cumulative yield on the 3.95 g L‒1 scale. In addition, the key residues involved in the catalytic selectivity of AbCGT are explored. These findings suggest that AbCGT is a powerful tool in the synthesis of lead compounds for drug discovery and an example for engineering the catalytic selectivity of C-glycosyltransferases.

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

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Exploring and applying the substrate promiscuity of a C-glycosyltransferase in the chemo-enzymatic synthesis of bioactive C-glycosides

et al. 2020

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“…The LanGT2 has been engineered to catalyze C‐glycosylation through exchange of just 12 residues in the α3‐helix from the UrdGT2 template . The modification led to reduction in the size of the aglycone binding pocket in LanGT2 particularly by I58 and I62 . Docking studies indicated that subtle repositioning of the aglycone relative to the sugar nucleotide, with the C9 atom moving toward the cosubstrate and the C8 hydroxyl group away from the cosubstrate, leading to a switch in regiochemistry and even the catalytic mechanism of LanGT2 .…”

Section: Direct Modulation Of Enzyme Function In Biosynthetic Gene CLmentioning

confidence: 99%

A pharmaceutical model for the molecular evolution of microbial natural products

Fewer

Metsä‐Ketelä

2019

The FEBS Journal

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Microbes are talented chemists with the ability to generate tremendously complex and diverse natural products which harbor potent biological activities. Natural products are produced using sets of specialized biosynthetic enzymes encoded by secondary metabolism pathways. Here, we present a two‐step evolutionary model to explain the diversification of biosynthetic pathways that account for the proliferation of these molecules. We argue that the appearance of natural product families has been a slow and infrequent process. The first step led to the original emergence of bioactive molecules and different classes of natural products. However, much of the chemical diversity observed today has resulted from the endless modification of the ancestral biosynthetic pathways. The second step rapidly modulates the pre‐existing biological activities to increase their potency and to adapt to changing environmental conditions. We highlight the importance of enzyme promiscuity in this process, as it facilitates both the incorporation of horizontally transferred genes into secondary metabolic pathways and the functional differentiation of proteins to catalyze novel chemistry. We provide examples where single point mutations or recombination events have been sufficient for new enzymatic activities to emerge. A unique feature in the evolution of microbial secondary metabolism is that gene duplication is not essential but offers opportunities to synthesize more complex metabolites. Microbial natural products are highly important for the pharmaceutical industry due to their unique bioactivities. Therefore, understanding the natural mechanisms leading to the formation of diverse metabolic pathways is vital for future attempts to utilize synthetic biology for the generation of novel molecules.

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“…Surprisingly, multiple sequence comparison also revealed high homology of SchS7 to a known O‐GT, LanGT2 from S. cyanogenus , which catalyzes the first glycosylation step by attaching activated d ‐olivose to 8‐OH of the aglycone . Interestingly, an engineered version of LanGT2 was able to act as a C‐GT, showing activity for both C‐ and O‐glycosylation similar to UrdGT2 . Therefore, we expected SchS7 to be responsible for the attachment of d ‐amicetose to C‐9 of the polyketide aglycone.…”

Section: Discussionmentioning

confidence: 99%

New Insights into the Glycosylation Steps in the Biosynthesis of Sch47554 and Sch47555

et al. 2018

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Sch47554 and Sch47555 are antifungal compounds from Streptomyces sp. SCC-2136. The availability of the biosynthetic gene cluster made it possible to track genes that encode biosynthetic enzymes responsible for the structural features of these two angucyclines. Sugar moieties play important roles in the biological activities of many natural products. An investigation into glycosyltransferases (GTs) might potentially help to diversify pharmaceutically significant drugs through combinatorial biosynthesis. Sequence analysis indicates that SchS7 is a putative C-GT, whereas SchS9 and SchS10 are proposed to be O-GTs. In this study, the roles of these three GTs in the biosynthesis of Sch47554 and Sch47555 are characterized. Coexpression of the aglycone and sugar biosynthetic genes with schS7 in Streptomyces lividans K4 resulted in the production of C-glycosylated rabelomycin, which revealed that SchS7 attached a d-amicetose moiety to the aglycone core structure at the C-9 position. Gene inactivation studies revealed that subsequent glycosylation steps took place in a sequential manner, in which SchS9 first attached either an l-aculose or l-amicetose moiety to 4'-OH of the C-glycosylated aglycone, then SchS10 transferred an l-aculose moiety to 3-OH of the angucycline core.

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Structural Characterization of O‐ and C‐Glycosylating Variants of the Landomycin Glycosyltransferase LanGT2

Cited by 26 publications

References 42 publications

Exploring and applying the substrate promiscuity of a C-glycosyltransferase in the chemo-enzymatic synthesis of bioactive C-glycosides

Exploring and applying the substrate promiscuity of a C-glycosyltransferase in the chemo-enzymatic synthesis of bioactive C-glycosides

A pharmaceutical model for the molecular evolution of microbial natural products

New Insights into the Glycosylation Steps in the Biosynthesis of Sch47554 and Sch47555

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