Investigation of Acid-Base Catalysis in Halimadienyl Diphosphate Synthase Involved in Mycobacterium tuberculosis Virulence.

The devastating human pathogenMycobacterium tuberculosis (Mtb) is able to parasitize phagosomal compartments within alveolar macrophage cells due, in part, to the activity of its cell-surface lipids. Prominent among these is 1-tuberculosinyl-adenosine (1-TbAd), a derivative of the diterpenoid tuberculosinyl (halima-5,13-dienyl) diphosphate produced by the class II diterpene cyclase encoded by Rv3377c, termed here MtHPS. Given the demonstrated ability of 1-TbAd to act as a virulence factor for Mtb and the necessity for Rv3377c for its production, there is significant interest in MtHPS activity. Class II diterpene cyclases catalyze a general acid-base-mediated carbocation cascade reaction initiated by protonation of the terminal alkene in the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate and terminated by deprotonation of the final cyclized (and sometimes also rearranged) intermediate. Here, structure-guided mutagenesis was applied to characterize the various residues contributing to activation of the enzymatic acid, as well as identify the enzymatic base in MtHPS. Particularly given the ability of conservative substitution for the enzymatic base (Y479F) to generate an alternative product (labda-7,13-dienyl diphosphate) via deprotonation of an earlier unrearranged intermediate, further mutational analysis was carried out to introduce potential alternative catalytic bases. The results were combined with mechanistic molecular modeling to elucidate how these mutations affect the catalytic activity of this important enzyme. This not only provided detailed structure-function insight into MtHPS but also further emphasized the inert nature of the active site of MtHPS and class II diterpene cyclases more generally.

Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis.

Triptolide is a trace natural product of Tripterygium wilfordii. It has antitumor activities, particularly against pancreatic cancer cells. Identification of genes and elucidation of the biosynthetic pathway leading to triptolide are the prerequisite for heterologous bioproduction. Here, we report a reference-grade genome of T. wilfordii with a contig N50 of 4.36 Mb. We show that copy numbers of triptolide biosynthetic pathway genes are impacted by a recent whole-genome triplication event. We further integrate genomic, transcriptomic, and metabolomic data to map a gene-to-metabolite network. This leads to the identification of a cytochrome P450 (CYP728B70) that can catalyze oxidation of a methyl to the acid moiety of dehydroabietic acid in triptolide biosynthesis. We think the genomic resource and the candidate genes reported here set the foundation to fully reveal triptolide biosynthetic pathway and consequently the heterologous bioproduction.

Identification of RoCYP01 (CYP716A155) enables construction of engineered yeast for high-yield production of betulinic acid.

Betulinic acid (BA) and its derivatives possess potent pharmacological activity against cancer and HIV. As with many phytochemicals, access to BA is limited by the requirement for laborious extraction from plant biomass where it is found in low amounts. This might be alleviated by metabolically engineering production of BA into an industrially relevant microbe such as Saccharomyces cerevisiae (yeast), which requires complete elucidation of the corresponding biosynthetic pathway. However, while cytochrome P450 enzymes (CYPs) that can oxidize lupeol into BA have been previously identified from the CYP716A subfamily, these generally do not seem to be specific to such biosynthesis and, in any case, have not been shown to enable high-yielding metabolic engineering. Here RoCYP01 (CYP716A155) was identified from the BA-producing plant Rosmarinus officinalis (rosemary) and demonstrated to effectively convert lupeol into BA, with strong correlation of its expression and BA accumulation. This was further utilized to construct a yeast strain that yields > 1 g/L of BA, providing a viable route for biotechnological production of this valuable triterpenoid.

Identification and functional characterization of diterpene synthases for triptolide biosynthesis from Tripterygium wilfordii.

Tripterygium wilfordii, which has long been used as a medicinal plant, exhibits impressive and effective anti-inflammatory, immunosuppressive and anti-tumor activities. The main active ingredients are diterpenoids and triterpenoids, such as triptolide and celastrol, respectively. A major challenge to harnessing these natural products is that they are found in very low amounts in planta. Access has been further limited by the lack of knowledge regarding their underlying biosynthetic pathways, particularly for the abeo-abietane tri-epoxide lactone triptolide. Here suspension cell cultures of T. wilfordii were found to produce triptolide in an inducible fashion, with feeding studies indicating that miltiradiene is the relevant abietane olefin precursor. Subsequently, transcriptome data were used to identify eight putative (di)terpene synthases that were then characterized for their potential involvement in triptolide biosynthesis. This included not only biochemical studies which revealed the expected presence of class II diterpene cyclases that produce the intermediate copalyl diphosphate (CPP), along with the more surprising finding of an atypical class I (di)terpene synthase that acts on CPP to produce the abietane olefin miltiradiene, but also their subcellular localization and, critically, genetic analysis. In particular, RNA interference targeting either both of the CPP synthases, TwTPS7v2 and TwTPS9v2, or the subsequently acting miltiradiene synthase, TwTPS27v2, led to decreased production of triptolide. Importantly, these results then both confirm that miltiradiene is the relevant precursor and the relevance of the identified diterpene synthases, enabling future studies of the biosynthesis of this important bioactive natural product.

Bifunctional abietadiene synthase: mutual structural dependence of the active sites for protonation-initiated and ionization-initiated cyclizations.

Abietadiene synthase from grand fir catalyzes two sequential, mechanistically distinct cyclizations, of geranylgeranyl diphosphate and of copalyl diphosphate, in the formation of a mixture of abietadiene isomers as the committed step of diterpenoid resin acid biosynthesis. Each reaction is independently conducted at a separate active site residing in what were considered to be structurally distinct domains typical of terpene cyclases. Despite the presence of an unusual 250-residue N-terminal insertional element, a tandem pair of charged residues distal to the insertion was shown to form a functional part of the C-terminal active site. Because abietadiene synthase resembles the ancestral plant terpene cyclase, this observation suggests an early evolutionary origin of catalytically important positively charged residues at the N-terminus of enzymes of this general class. A series of N- and C-terminal truncations of this enzyme were constructed and characterized, both alone and as mixtures of adjacent polypeptide pairs, to assess the proposed domain architecture, the function of the insertional element, and the role of presumptive interdomain contacts. These studies indicated a requirement for the insertional element in functional folding and allowed definition of the minimum primary structure of N- and C-terminal active site peptides. Most importantly, the results showed that, although the two active sites of abietadiene synthase are catalytically independent, substantial contact between the two regions is essential for the functional competence of this enzyme. Thus, the two cyclization sites of abietadiene synthase cannot be dissected into catalytically distinct domains, and, therefore, abietadiene synthase is unlikely to have arisen by fusion of two previously independent genes.