Crystal Structure of Aclacinomycin Methylesterase with Bound Product Analogues

Jansson, Anna; Niemi, Jarmo; Mäntsälä, Pekka; Schneider, Günter

doi:10.1074/jbc.m304008200

Cited by 34 publications

(27 citation statements)

References 41 publications

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“…4), serving as a unique substrate anchor that was not reported in other esterases. [23][24][25][26][27] The involvement of the Arg residues in the catalysis was also observed at the active site of tricorn protease 22 whose two Arg residues act as a C-terminal carboxylic group sinker of peptide substrates. Next to the catalytic Ser89, a thin hydrophobic pocket is formed by the conserved residues (see right pocket in Fig.…”

Section: Discussionmentioning

confidence: 82%

“…The complex crystal structures of esterases that degrade long hydrocarbon chain derivatives 26,27 also show a peculiar helical arrangement to construct the substrate-binding groove at the domain interface. However, their grooves differ from the observed L-shaped sack of the Ec_ybfF structure, since they are formed along the domain interface for a straight hydrocarbon chain of palmitate 27 or for a puckered hydrocarbon chain of the bound substrate 26 (Supplementary Fig. 1).…”

Section: Discussionmentioning

confidence: 92%

“…Instead, the helical cap directly contributes to the substrate binding, a property that was not observed in other esterase families. 23,[25][26][27] The conserved Arg123 that acts as a carboxylic group sinker in the malonate complex ( Figs. 4 and 7) is located on the β5-α4 loop, which is stabilized by polar interactions with the α/β hydrolase domain and the helical cap domain simultaneously.…”

Section: Discussionmentioning

confidence: 99%

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High-resolution Structure of ybfF from Escherichia coli K12: A Unique Substrate-binding Crevice Generated by Domain Arrangement

Park

Lee

et al. 2008

Journal of Molecular Biology

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Section: Discussionmentioning

confidence: 82%

Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

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High-resolution Structure of ybfF from Escherichia coli K12: A Unique Substrate-binding Crevice Generated by Domain Arrangement

Park

Lee

et al. 2008

Journal of Molecular Biology

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“…1) have been described as unstable and undergo spontaneous 10-decarboxylation on the rhodomycin and daunorubicin pathways, respectively (21,29). However, indirect evidence has suggested that the decarboxylation might also be enzymatically catalyzed by DnrP (21).…”

Section: Resultsmentioning

confidence: 99%

Divergent evolution of an atypical S -adenosyl- l -methionine–dependent monooxygenase involved in anthracycline biosynthesis

Grocholski

Dinis

Niiranen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial secondary metabolic pathways are responsible for the biosynthesis of thousands of bioactive natural products. Many enzymes residing in these pathways have evolved to catalyze unusual chemical transformations, which is facilitated by an evolutionary pressure promoting chemical diversity. Such divergent enzyme evolution has been observed in S-adenosyl-l-methionine (SAM)-dependent methyltransferases involved in the biosynthesis of anthracycline anticancer antibiotics; whereas DnrK from the daunorubicin pathway is a canonical 4-O-methyltransferase, the closely related RdmB (52% sequence identity) from the rhodomycin pathways is an atypical 10-hydroxylase that requires SAM, a thiol reducing agent, and molecular oxygen for activity. Here, we have used extensive chimeragenesis to gain insight into the functional differentiation of RdmB and show that insertion of a single serine residue to DnrK is sufficient for introduction of the monooxygenation activity. The crystal structure of DnrK-Ser in complex with aclacinomycin T and S-adenosyl-l-homocysteine refined to 1.9-Å resolution revealed that the inserted serine S297 resides in an α-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. Further experimental work indicated that the shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The channel is also closed in RdmB and may be important for monooxygenation in a solvent-free environment. Finally, we postulate that the hydroxylation ability of RdmB originates from a previously undetected 10-decarboxylation activity of DnrK.

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“…6). There is no space available within this pocket for more than one carbohydrate, and there is also no possibility for additional carbohy- drates to extend from the binding site into the solution as seen in other tailoring enzymes in anthracycline (38) (Fig. 6).…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure of a Ternary Complex of DnrK, a Methyltransferase in Daunorubicin Biosynthesis, with Bound Products

Jansson

Koskiniemi

Mäntsälä

et al. 2004

Journal of Biological Chemistry

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One of the final steps in the biosynthesis of the widely used anti-tumor drug daunorubicin in Streptomyces peucetius is the methylation of the 4-hydroxyl group of the tetracyclic ring system. This reaction is catalyzed by the S-adenosyl-L-methionine-dependent carminomycin 4-Omethyltransferase DnrK. The crystal structure of the ternary complex of this enzyme with the bound products S-adenosyl-L-homocysteine and 4-methoxy-⑀-rhodomycin T has been determined to a 2.35-Å resolution. DnrK is a homodimer, and the subunit displays the typical fold of small molecule O-methyltransferases. The structure provides insights into the recognition of the anthracycline substrate and also suggests conformational changes as part of the catalytic cycle of the enzyme. The position and orientation of the bound ligands are consistent with an S N 2 mechanism of methyl transfer. Mutagenesis experiments on a putative catalytic base confirm that DnrK most likely acts as an entropic enzyme in that rate enhancement is mainly due to orientational and proximity effects. This contrasts the mechanism of DnrK with that of other O-methyltransferases where acid/base catalysis has been demonstrated to be an essential contribution to rate enhancement.Daunorubicin and doxorubicin are aromatic polyketide antibiotics that exhibit high cytotoxicity and are widely applied in the chemotherapy of a variety of cancers (1, 2). These and related anthracyclines consist of a cyclic polyketide backbone, 7,8,9,10-tetrahydrotetracene-5,12-quinone, glycosylated at position C7 or C10 (Fig. 1). Diversity is generated by variations in the modification of the aglycone moiety and the composition of the attached carbohydrate. Biosynthesis of daunorubicin/doxorubicin starts with the formation of the polyketide backbone catalyzed by a class II polyketide synthase with subsequent cyclization of the polyketide chain (3). These steps lead to the formation of aklavinone, a common intermediate in the synthesis of most anthracyclines. This aglycone is then further modified through a series of steps, i.e. hydroxylation, glycosylation, methylester hydrolysis, decarboxylation, methylation, and, in

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Crystal Structure of Aclacinomycin Methylesterase with Bound Product Analogues

Cited by 34 publications

References 41 publications

High-resolution Structure of ybfF from Escherichia coli K12: A Unique Substrate-binding Crevice Generated by Domain Arrangement

High-resolution Structure of ybfF from Escherichia coli K12: A Unique Substrate-binding Crevice Generated by Domain Arrangement

Divergent evolution of an atypical S -adenosyl- l -methionine–dependent monooxygenase involved in anthracycline biosynthesis

Crystal Structure of a Ternary Complex of DnrK, a Methyltransferase in Daunorubicin Biosynthesis, with Bound Products

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