Hormaomycin B

Structurally different and functionally diverse natural compounds - antitumour agents pyrrolo[1,4]benzodiazepines, bacterial hormone hormaomycin, and lincosamide antibiotic lincomycin - share a common building unit, 4-alkyl-L-proline derivative (APD). APDs arise from L-tyrosine through a special biosynthetic pathway. Its generally accepted scheme, however, did not comply with current state of knowledge. Based on gene inactivation experiments and in vitro functional tests with recombinant enzymes, we designed a new APD biosynthetic scheme for the model of lincomycin biosynthesis. In the new scheme at least one characteristic in each of five final biosynthetic steps has been changed: the order of reactions, assignment of enzymes and/or reaction mechanisms. First, we demonstrate that LmbW methylates a different substrate than previously assumed. Second, we propose a unique reaction mechanism for the next step, in which a putative γ-glutamyltransferase LmbA indirectly cleaves off the oxalyl residue by transient attachment of glutamate to LmbW product. This unprecedented mechanism would represent the first example of the C-C bond cleavage catalyzed by a γ-glutamyltransferase, i.e., an enzyme that appears unsuitable for such activity. Finally, the inactivation experiments show that LmbX is an isomerase indicating that it transforms its substrate into a compound suitable for reduction by LmbY, thereby facilitating its subsequent complete conversion to APD 4-propyl-L-proline. Elucidation of the APD biosynthesis has long time resisted mainly due to the apparent absence of relevant C-C bond cleaving enzymatic activity. Our proposal aims to unblock this situation not only for lincomycin biosynthesis, but generally for all above mentioned groups of bioactive natural products with biotechnological potential.

Covering: up to 2017This review covers the biosynthetic and evolutionary aspects of lincosamide antibiotics, antitumour pyrrolobenzodiazepines (PBDs) and the quorum-sensing molecule hormaomycin. These structurally and functionally diverse groups of complex natural products all incorporate rarely occurring 4-alkyl-l-proline derivatives (APDs) biosynthesized from l-tyrosine through an unusual specialized pathway catalysed by a common set of six proteins named Apd1-Apd6. We give an overview of APD formation, which involves unusual enzyme activities, and its incorporation, which is based either on nonribosomal peptide synthetase (PBDs, hormaomycin) or a unique hybrid ergothioneine-dependent condensation system followed by mycothiol-dependent sulphur atom incorporation (lincosamides). Furthermore, within the public databases, we identified 36 novel unannotated biosynthetic gene clusters that putatively encode the biosynthesis of APD compounds. Their products presumably include novel PBDs, but also novel classes of APD compounds, indicating an unprecedented potential for the diversity enhancement of these functionally versatile complex metabolites. In addition, phylogenetic analysis of known and novel gene clusters for the biosynthesis of APD compounds allowed us to infer novel evolutionary hypotheses: Apd3 methyltransferase originates from a duplication event in a hormaomycin biosynthetic gene cluster ancestor, while putative Apd5 isomerase is evolutionarily linked to PhzF protein from the biosynthesis of phenazines. Lastly, we summarize the achievements in preparing hybrid APD compounds by directing their biosynthesis, and we propose that the number of nature-like APD compounds could by multiplied by replacing l-proline residues in various groups of complex metabolites with APD, i.e. by imitating the natural process that occurs with lincosamides and PBDs, in which the replacement of l-proline for APD has proved to be an evolutionary successful concept.

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum-sensing 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 F420H2-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 4-substituted 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 F420H2-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.