Structural and Functional Analysis of Angucycline C-6 Ketoreductase LanV Involved in Landomycin Biosynthesis

Abstract

Angucyclines are biologically active natural products constructed around a common benz[a]anthraquinone carbon frame. One key branching point in the biosynthesis of angucyclines is the ketoreduction at C-6, which results in the opposite stereochemistry of landomycins and urdamycins/gaudimycins. Here we present the 1.65 Å resolution crystal structure of LanV from Streptomyces cyanogenus S136 that is responsible for the 6R stereochemistry of landomycins. The enzyme displays the common architectural fold of short-chain alcohol dehydrogenases/reductases and contains bound nicotinamide adenine dinucleotide phosphate. Determination of the structure of LanV in complex with 11-deoxylandomycinone at 2.0 Å resolution indicated that substrate binding does not induce large conformational changes and that substrate recognition occurs mainly through hydrophobic interactions. Analysis of the electron density map of the ternary complex revealed that the catalytic reaction had most likely proceeded backward in the crystal, because the data could be best fit with a compound harboring a carbonyl group at C-6. A coordinated water molecule was atypically identified between the ligand and the conserved Tyr160 residue, which was confirmed to be critical for the catalytic activity by site-directed mutagenesis. A catalytic triad of Ser147, Tyr160, and Lys164 could be recognized on the basis of the crystal structure, and stereoselective labeling studies demonstrated that the transfer of hydride from reduced nicotinamide adenine dinucleotide phosphate to the substrate occurs from the 4-pro-S side of the cosubstrate. Importantly, Ser192 was identified as being involved in controlling the stereochemistry of the reaction, as assays with single mutant Ser192Ile led to accumulation of gaudimycin C with 6S stereochemistry as a minor product.

A pharmaceutical model for the molecular evolution of microbial natural products

“…B) by a short‐chain alcohol dehydrogenase/ reductase (SDR) UrdMred . LanV from the related landomycin pathway has diverged from these canonical 6‐ketoreductases (KR) to prefer the earlier 12‐hydroxylated pathway intermediate as a substrate and catalyze the ketoreduction with opposite stereochemistry . A single point mutation S192 to I192 in LanV, where the longer side‐chain pushes the substrate into a different angle above NADPH, was sufficient to convert the activity toward the use of C12,C12b‐hydroxylated substrates .…”

“…LanV from the related landomycin pathway has diverged from these canonical 6‐ketoreductases (KR) to prefer the earlier 12‐hydroxylated pathway intermediate as a substrate and catalyze the ketoreduction with opposite stereochemistry . A single point mutation S192 to I192 in LanV, where the longer side‐chain pushes the substrate into a different angle above NADPH, was sufficient to convert the activity toward the use of C12,C12b‐hydroxylated substrates . The importance of enzyme promiscuity in these redox cascades was highlighted under artificial conditions where native JadH and LanV were used to catalyze ancestral non‐native reactions leading to the formation of the noncognate urdamycin pathway product .…”

SimC7 Is a Novel NAD(P)H-Dependent Ketoreductase Essential for the Antibiotic Activity of the DNA Gyrase Inhibitor Simocyclinone

“…Although none is closely related to SimC7, several SDR ketoreductases have been identified that act on polyketides. These include SDR proteins that reduce the carbonyl group at position C-9 in the biosynthetic pathways for jadomycin (JadE), actinorhodin (ActKR), and hedamycin (HedKR) [32–34] and the SDR protein that reduces the carbonyl group at position C-6 in the landomycin pathway (LanV) [35] . Close homologs of these enzymes are encoded in the sim gene cluster, raising the possibility that SimA6 and SimA9 function to reduce the angucyclinone C-10 and C-6 carbonyl groups, respectively, during the biosynthesis of simocyclinone.…”

The Combinatorial Biosynthesis of “Unnatural” Products with Polyketides

“…Many researchers have studied the KR domain structure and its stereochemical mechanisms [36,38,39]. A mutation (Ser192Ile) in LanV, which is responsible for the 6R stereochemistry of landomycins in Streptomyces cyanogenus S136, led to the accumulation of gaudimycin C with 6S stereochemistry as a minor product [40].…”