Pyridoxal-5′-phosphate–dependent bifunctional enzyme catalyzed biosynthesis of indolizidine alkaloids in fungi

Abstract

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5′-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C–C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A−I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature’s arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.

Emergence of oxygen‐ and pyridoxal phosphate‐dependent reactions

“…First, CuaB catalyzes carbon‐carbon bond formation via Claisen condensation, similar to PLP‐dependent 8‐amino‐7‐oxononanoate synthase. Then, unlike 8‐amino‐7‐oxononanoate synthase, CuaB catalyzes an oxygen‐dependent hydroxylation, demonstrated through 18 O 2 labeling of the reaction products [117].…”

Oxygen reactivity with pyridoxal 5′-phosphate enzymes: biochemical implications and functional relevance

“…Recently, a new PLP enzyme, CuaB, has been identified in the gene cluster for the synthesis of an indolizidine alkaloid, curvulamine, in fungi (Dai et al 2020 ), underlining the fact that the reactivity of PLP enzyme with oxygen is a widespread feature. CuaB is a bifunctional enzyme: it catalyzes the formation of C–C bonds through Claisen condensation, as other PLP enzymes are able to do, such as 8-amino-7-oxononanoate synthase and, unexpectedly, a hydroxylation using dioxygen (Fig.…”

“…10 ). A detailed enzyme mechanism has been proposed on the basis of the collected products (Dai et al 2020 ). The external aldimine with the substrate is deprotonated to generate a quinonoid that follows the pathway of synthases (Fig.…”

Identification and Analysis of the Biosynthetic Gene Cluster for the Indolizidine Alkaloid Iminimycin in Streptomyces griseus

“…Curvulamine (Figure 1b) is produced by a marine‐derived fungus, Curvularia sp. IFB‐Z10, and exhibits antibacterial activity [8] . In curvulamine biosynthesis, a tetraketide diene synthesized by a highly reducing PKS, CuaA, is condensed with alanine, cyclized, and oxidized into a compound containing the 2,3‐dihydropyrrole core by a PLP‐dependent bifunctional aminotransferase, CuaB.…”