Different Reaction Specificities of F₄₂₀H₂-Dependent Reductases Facilitate Pyrrolobenzodiazepines and Lincomycin To Fit Their Biological Targets

Abstract

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorumsensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of L-tyrosine-or L-leucine-derived 4-alkyl-L-proline derivatives (APDs) in their structures. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F 420 H 2 -dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4substituted Δ1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F 420 H 2 -dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, our bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated L-proline-like precursors and likely in the microbial central metabolism.

On the diversity of F420‐dependent oxidoreductases: A sequence‐ and structure‐based classification

“… 38 Hitherto, biochemical details of these other members are scarce. Recently, Steiningerova et al 32 demonstrated that the actinobacterial enzymes LmbY, 39 HrmD, 40 among others, are F 420 H 2 ‐dependent reductases displaying the TIM barrel fold. These enzymes are involved in the last step of 4‐alkyl‐ l ‐proline derivatives (APDs) biosynthesis.…”

Mycoplanecins, potent anti-tuberculosis antibiotics: Elucidation of unusual biosynthesis and DnaN-targeting mode of action

“…Previously, 4-EtPro and 4-PrPro were characterized to be derived from L-tyrosine in the biosynthesis of lincomycins (Supplementary Figs. 11 and 12a), 22 however, no homologous genes were found in the MP pathway. As reported in GM biosynthesis, (2S,4R)-4-MePro was the product of a series of enzymatic modifications of leucine (Fig.…”

Lincosamide Antibiotics: Structure, Activity, and Biosynthesis

“…In 2020, Kamenik and coworkers reported the functional analysis of six Apd6 reductases: LmbY (lincosamycin), Por15 (porothramycin), SibT (sibiromycin), HrmD (hormaomycin), Lim12 (limazepine), and GriH (griselimycin). [36] In vitro analyses revealed that these enzymes strictly recognize F 420 H 2 as a cofactor to produce 44-49 (Figure 7B and C). Moreover, Por15, SibT, Lim12, HrmD reduced only the endocyclic imine bond of substrates, while LmbY and GriH also catalyzed the reduction of the exocyclic double bond in addition to the endocyclic imine bond.…”