New Concept of the Biosynthesis of 4-Alkyl-L-Proline Precursors of Lincomycin, Hormaomycin, and Pyrrolobenzodiazepines: Could a γ-Glutamyltransferase Cleave the C–C Bond?

Abstract

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.

The Novel Transcriptional Regulator LmbU Promotes Lincomycin Biosynthesis through Regulating Expression of Its Target Genes in Streptomyces lincolnensis

“…In contrast, the lmbA, lmbB1, lmbB2, lmbW, lmbX, and lmbY genes are involved in PPL biosynthesis (13). Among these, lmbA, lmbB1, and lmbB2 share the same operon (2) (Fig.…”

“…In the recent report, it was proposed that product 1 undergoes methylation by LmbW to generate product 2 (13,14). The function of the putative ␥-glutamyltransferase LmbA appears to indirectly remove the oxalyl residue of the product 2 to produce intermediate 3 (13).…”

An Activator of an Adenylation Domain Revealed by Activity but Not Sequence Homology

“…However, this would not be consistent with its ability to adenylate l ‐Thr that is not involved in the dihydropyrrole synthesis (Figure S8). Many of the purported intermediates in sibiromycin biosynthesis remain speculative and might require revisions similar to those recently published on the formation of a dihydropyrrole intermediate in lincomycin A . Results from gene‐deletion studies on this related pathway indicate that methylation appears to target 1 rather than 2 a .…”

“…Results from gene‐deletion studies on this related pathway indicate that methylation appears to target 1 rather than 2 a . In addition, the SibY and SibS homologues in lincomycin A biosynthesis have been reassigned as a hydrolase and isomerase, respectively; this challenges the previous suggestion of possible endocyclic and exocyclic unsaturation in 3 and 4 .…”

Recent advances in the biosynthesis of lincosamide antibiotics

“…PPL单元后续的生物合成途径目前仍不明确, 从 化学结构来看, 还原中间体56/58或者57/59中的共轭 双键是必须的一步. lmbY编码一个F 420 依赖的还原酶, 推测该酶与上述双烯体的还原有关 [37,41] . 根据体内 基因敲除的结果, 推测LmbX与PPL中正丙基的构型 有关 [41] .…”