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. 2008 Aug 25;15(8):842-53.
doi: 10.1016/j.chembiol.2008.06.011.

Biochemical and Structural Insights of the Early Glycosylation Steps in Calicheamicin Biosynthesis

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Free PMC article

Biochemical and Structural Insights of the Early Glycosylation Steps in Calicheamicin Biosynthesis

Changsheng Zhang et al. Chem Biol. .
Free PMC article

Abstract

The enediyne antibiotic calicheamicin (CLM) gamma(1)(I) is a prominent antitumor agent that is targeted to DNA by a novel aryltetrasaccharide comprised of an aromatic unit and four unusual carbohydrates. Herein we report the heterologous expression and the biochemical characterization of the two "internal" glycosyltransferases CalG3 and CalG2 and the structural elucidation of an enediyne glycosyltransferase (CalG3). In conjunction with the previous characterization of the "external" CLM GTs CalG1 and CalG4, this study completes the functional assignment of all four CLM GTs, extends the utility of enediyne GT-catalyzed reaction reversibility, and presents conclusive evidence of a sequential glycosylation pathway in CLM biosynthesis. This work also reveals the common GT-B structural fold can now be extended to include enediyne GTs.

Figures

Fig. 1
Fig. 1. Representative naturally occurring enediynes
ncluding the 10-membered enediynes calicheamcin γ1I (1), esperamicin (2), dynemicin (3) and 9-membered chromoprotein enediynes C-1027 (4), neocarzinostatin (5), and maduropeptin (6). The CLM aryltetrasaccharide is highlighted in blue.
Fig. 2
Fig. 2. Preparation of CLM T0 (9) from CLM α3I (7)
(A) Schematic of the strategy – (a) refluxing in acetone, 65°C, 20 h; (b) incubated in 0.2% TFA, RT, 4 h. (B) HPLC analyses of the preparation - (i) the starting material 7; (ii) refluxed for 20 h; (iii) purified 8 from the reaction mixture of (ii); (iv) 8 incubated with 0.2% TFA at RT for 2 h; and (iv) 8 incubated with 0.2% TFA at RT for 4 h.
Fig. 3
Fig. 3. CalG3-catalyzed reverse reaction and ‘sugar exchange’ reaction
(A) Schematic of the CalG3-catalyzed formation of 10 from 9 via reverse catalysis and the production of 10 novel CLM variants 9a–j via ‘sugar exchange’. (B) HPLC analyses of CalG3-catalyzed reverse reactions. In these reactions, 50 µM 9 was incubated with 7.5 µM CalG3 for 2 h at 30 °C in the presence of (i) 2 mM ADP; (ii) 2 mM GDP; (iii) 2 mM UDP; (iv) 2 mM CDP; (v) 2 mM TDP and (vi) no NDP. Percent conversions were indicated in the parentheses. (C) The production of 9a–j via CalG3-catalyzed ‘sugar exchange’. 50 µM 9 was incubated with 7.5 µM CalG3 at 30°C overnight in the presence of various TDP-sugars (300 µM, Fig. S4). Percent conversions were indicated in the parentheses.
Fig. 4
Fig. 4. Structure of CalG3
(A) A ribbon diagram of the CalG3 dimer with monomers color-coded in red and cyan. (B) The CalG3 monomer is formed by closely opposed N-terminal- (cyan) and the C-terminal-domains (khaki). These distinct domains are connected by a linker (yellow) and their interaction is stabilized by the C-terminal helix (green). The blue arrow indicates the putative catalytic loop, the magenta arrow points to a pyrophosphate-binding tetraglycine loop spanning residues 285–288. An ordered portion of a polyethylene glycol molecule (brown) has been found in the cavity formed by the N-domain. The inset highlights Cot-trace of CalG3 (cyan, yellow, khaki) in the active site with putative catalytic diad residues H11 and E115 highlighted. The gray Cα-trace is that of a docked model, which incorporates experimentally-observed conformational changes in the pyrophosphate binding loop (magenta) and modeled changes of the "catalytic loop" (blue, black arrow). (C) Manually docked model of the CalG3 with CLM T0 (carbon-cyan, oxygen-red, sulfur-yellow, nitrogen-blue) and a dinucleotide TDP (carbon-yellow, oxygen-red, nitrogen-blue, phosphorus-orange) in the active site.
Fig. 5
Fig. 5. Differential reactions with 9 and 12 in the presence of CalG2 and CalG3
(i) 50 µM 9, 300 µM 12 in the presence of 7.5 µM CalG2 at 30 °C overnight; (ii) 50 µM 9, 300 µM 12 in the absence of enzymes at 30 °C overnight; (iii) 50 µM 9, 300 µM 12 in the presence of 7.5 µM CalG3 at 30 °C overnight.
Fig. 6
Fig. 6. The proposed CLM glycosylation pathway

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