In-Silico Screening Compounds of Brassica rapa ssp. chinensis as Potential Inhibitor of Neutral Amino Acid Transporter B0AT1 as an Alternative Phenylketonuria Treatment

Phenylketonuria (PKU) is known as a severe autosomal recessive disease caused by mutations in the expression enzyme, namely the PAH (Phenylalanine Hydroxylase) enzyme that causes the build-up of phenylalanine in the body. Untreated PKU affected brain damage and developmental problems. One of the strategies to reduce phenylalanine in the body is inhibiting B0AT1 activity using carotenoid and terpenoids compounds from Bok choy (Brassica rapa ssp.chinensis). In this study, we evaluated the nine carotenoid and terpenoid compounds from Bok choy as B0AT1 inhibitors. Nine Bok choy compounds, including alpha-carotene, beta-carotene, dimethylallyl pyrophosphate, isopentenyl pyrophosphate, lutein, neoxanthin, violaxanthin, geranylgeranyl diphosphate, and zeaxanthin were downloaded from PubChem database, while the 3D structure of B0AT1 was retrieved from Protein Data Bank RCSB. The compounds and B0AT1 were prepared by PyRx 0.8 version and Discovery Studio ver 21.1.1, then docked with Hex 8.0.0 and analyzed using Discovery Studio ver 21.1.1. This screening implies that three terpenoid compounds dimethylallyl pyrophosphate, isopentenyl pyrophosphate, and geranylgeranyl diphosphate interacts in C domain of B0AT1 while six carotenoid compounds, alpha carotene, beta-carotene, lutein, neoxanthin, violaxanthin, and zeaxanthin interacts in A domain and have possibility to inhibit B0AT1, because it interact with same A domain and have a stronger binding energy than phenylalanine. Alpha carotene has a same residue with phenylalanine, Phe144, making it potentially greater than other compound as inhibitors. Brassica rapa ssp. chinensis is indeed good for consumption by people with phenylketonuria, but it is also necessary to do a further compound screening in other low-phenylalanine diet foods to know which one is better as alternative phenylketonuria treatment.

Structural insight on assembly-line catalysis in terpene biosynthesis

Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.

Geranylgeranyl diphosphate synthase deficiency hyperactivates macrophages and aggravates lipopolysaccharide-induced acute lung injury

Macrophage activation is a key contributing factor for excessive inflammatory responses of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Geranylgeranyl diphosphate synthase (GGPPS) plays a key role in the development of inflammatory diseases. Our group previously showed that GGPPS in alveolar epithelium have deleterious effects on acute lung injury induced by LPS or mechanical ventilation. Herein, we examined the role of GGPPS in modulating macrophage activation in ALI/ARDS. We found significant increased GGPPS expression in alveolar macrophages in ARDS patients compared to healthy volunteers and in ALI mice induced by LPS. GGPPS-floxed control (GGPPSfl/fl) and myeloid-selective knockout (GGPPSfl/flLysMcre) mice were then generated. Interestingly, using a LPS-induced ALI mouse model, we showed that myeloid-specific GGPPS knockout significantly increased mortality, aggravated lung injury, and increased the accumulation of inflammatory cells, total protein, and inflammatory cytokines in BALF. In vitro, GGPPS deficiency up-regulated the production of LPS-induced IL-6, IL-1β, and TNF-α in alveolar macrophages, bone marrow-derived macrophages (BMDMs), and THP-1 cells. Mechanistically, GGPPS knockout increased phosphorylation and nuclear translocation of NF-κB p65 induced by LPS. In addition, GGPPS deficiency increased the level of GTP-Rac1, which was responsible for NF-κB activation. In conclusion, decreased expression of GGPPS in macrophages aggravates lung injury and inflammation in ARDS, at least partly by regulating Rac1-dependent NF-κB signaling. GGPPS in macrophages may represent a novel therapeutic target in ARDS.

Synthesis of cembratriene-ol and cembratriene-diol in yeast via the MVA pathway

Background Cembranoids are one kind of diterpenoids with multiple biological activities. The tobacco cembratriene-ol (CBT-ol) and cembratriene-diol (CBT-diol) have high anti-insect and anti-fungal activities, which is attracting great attentions for their potential usage in sustainable agriculture. Cembranoids were supposed to be formed through the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, yet the involvement of mevalonate (MVA) pathway in their synthesis remains unclear. Exploring the roles of MVA pathway in cembranoid synthesis could contribute not only to the technical approach but also to the molecular mechanism for cembranoid biosynthesis. Results We constructed vectors to express cembratriene-ol synthase (CBTS1) and its fusion protein (AD-CBTS1) containing an N-terminal GAL4 AD domain as a translation leader in yeast. Eventually, the modified enzyme AD-CBTS1 was successfully expressed, which further resulted in the production of CBT-ol in the yeast strain BY-T20 with enhanced MVA pathway for geranylgeranyl diphosphate (GGPP) production but not in other yeast strains with low GGPP supply. Subsequently, CBT-diol was also synthesized by co-expression of the modified enzyme AD-CBTS1 and BD-CYP450 in the yeast strain BY-T20. Conclusions We demonstrated that yeast is insensitive to the tobacco anti-fungal compound CBT-ol or CBT-diol and could be applied to their biosynthesis. This study further established a feasibility for cembranoid production via the MVA pathway and provided an alternative bio-approach for cembranoid biosynthesis in microbes.