Catalytic innovation underlies independent recruitment of polyketide synthases in cocaine and hyoscyamine biosynthesis

Abstract

Tropane alkaloids such as hyoscyamine and cocaine are of importance in medicinal uses. Only recently has the hyoscyamine biosynthetic machinery become complete. However, the cocaine biosynthesis pathway remains only partially elucidated. Here we characterize polyketide synthases required for generating 3-oxo-glutaric acid from malonyl-CoA in cocaine biosynthetic route. Structural analysis shows that these two polyketide synthases adopt distinctly different active site architecture to catalyze the same reaction as pyrrolidine ketide synthase in hyoscyamine biosynthesis, revealing an unusual parallel/convergent evolution of biochemical function in homologous enzymes. Further phylogenetic analysis suggests lineage-specific acquisition of polyketide synthases required for tropane alkaloid biosynthesis in Erythroxylaceae and Solanaceae species, respectively. Overall, our work elucidates not only a key unknown step in cocaine biosynthesis pathway but also, more importantly, structural and biochemical basis for independent recruitment of polyketide synthases in tropane alkaloid biosynthesis, thus broadening the understanding of conservation and innovation of biosynthetic catalysts.

Discovery and Engineering of the Cocaine Biosynthetic Pathway

“…Quite interestingly, two PKSs ( En PKS1/2) involved in the generation of 3 in E. novogranatense have recently been characterized, providing further evidence supporting a role of MPOA ( 3 ) as a physiological intermediate in the biosynthesis of cocaine. However, while En CYP81AN15 and En MT4 are strong candidates for catalyzing the tropane ring forming steps of cocaine biosynthesis, in the absence of in planta characterization, we cannot exclude the possibility that additional enzymes catalyze these reactions.…”

“…novogranatense by an HMM (hidden Markov model) search. Based on the tissue-specific expression of cocaine biosynthetic genes in buds and young leaves (Figure S1), − six candidate genes ( EnMT1 – 5 and EnMT9 ), exhibiting expression in these tissues [fragments per kilo base of transcript per million mapped fragments (FPKM) > 5.0], were cloned from E. novogranatense cDNA and transiently expressed in Nicotiana benthamiana leaves mediated by Agrobacterium tumefaciens LBA4404.…”

“…novogranatense was unknown, we designed to produce 10 in tobacco (N. benthamiana) using three known functional proteins ( En ODC, Ab PMT1 from Atropa belladonna , and Nt MPO1 from N. tabacum ) (Figure a) as the same precursor 10 is implicated in the biosynthesis of cocaine ( 1 ), hyoscyamine ( 2 ), and nicotine. , We also expressed five downstream cocaine biosynthesis-related proteins ( En PKS1, En CYP81AN15, En MT4, En MecgoR, and En CS), as well as a benzoyl-CoA biosynthesis-related protein ( Aa 4HBCL: benzoic acid CoA ligase from Anthoceros agrestis). This yielded 797.4 ± 19.5 ng cocaine ( 1 ) per milligram of dry plant weight with the supplement of benzoic acid (Figure b,c).…”

The Evolutionary Pattern of Cocaine and Hyoscyamine Biosynthesis Provides Strategies To Produce Tropane Alkaloids

“…Indeed, cocaine biosynthesis is initiated in leaves by the formation of the arginine derivative ornithine. In the primary steps, an ornithine decarboxylase (ODC) and two redundant polyketide synthases (PKS1 and 2) respectively govern the conversion of ornithine to putrescine and malonyl‐CoA to oxo‐glutaric acid [2,3] . Later down the pathway, methylecgonone reductase (MecgoR) and cocaine synthase (CS) perform the two last steps of methylecgonone to cocaine conversion through reduction and acylation [4,5] .…”

“… [10] Interestingly, previous phylogenetic studies performed on enzymatic actors involved in the earliest and last stages of cocaine and hyoscyamine routes have revealed a common‐distinct reaction mechanism. For example, the structural analysis of ketide synthase homologs from the early rate‐limiting step of hyoscyamine and cocaine biosynthesis has revealed a distinct architecture of their catalytic sites even if they conduct the same biochemical reaction [3] . Additionally, molecular actors required for the later stages of TA biosynthesis from Erythroxylaceae and Solanaceae are also distinct.…”