Using Engineered Escherichia coli to Synthesize Squalene with Optimized Manipulation of Squalene Synthase and Mevalonate Pathway

Squalene is the metabolic precursor of sterols and naturally synthesized in the deep-dea shark liver and human sebum. The utilization of squalene is wide such as food, cosmetical, and pharmaceutical industries. This experiment used engineered Escherichia coli to construct the gene circuit for the biosynthesis of squalene. Human squalene synthase (hSQS) efficiently catalyzes the synthesis of squalene. Also, mevalonate (MVA) pathway would increase the yield of the precursor of squalene, farnesyl diphosphate, which then increased the yield of squalene. Meanwhile, the regulation of MVA pathway via different inducer IPTG concentrations and creation of selection pressure by antibiotics were investigated.

DESIGNING OF COUMARIN DERIVATIVES AS SQUALENE SYNTHASE INHIBITORS

Objective: The importance of this research work is to design a library of novel coumarin derivatives by docking evaluation of the designed coumarin derivatives as squalene synthase inhibitor.Methods: The three-dimensional structure of designed molecules of squalene synthase inhibitors was collected from Protein Data Bank. The designed molecules were docked onto the enzymes that are squalene synthase inhibitor - 3WCM, 3WCJ, and 3Q2Z protein using SYBYL-X 2.1. Using a standard protocol, the protein was subjected to minimization and protomol generation.Results: By this method, we visualized the possible binding and also estimated the protein interactions with our intended coumarin library, using SYBYL-X 2.1 software. Into the active site of the selected enzymes, all the 20 coumarins were docked and then the docking scores revealed that the compounds possess high affinity toward the selected enzymes.Conclusion: With the help of virtual evaluation, we have elaborated a fast synthetically accessible coumarin-based compounds, and it is an advanced and original scaffold in the area of probable human squalene synthase inhibitors. Some of the developed compounds show better binding property than ligand, and in 3q2Z, the compound 5d shows better binding property than the protein. Furthermore, 6g and 6c have good binding property. In 3 WCM, the compound 6f has better property. In 3 WCJ, the compounds 6g and 6f show better binding property than the protein.

Structural insights into the catalytic mechanism of human squalene synthase

Squalene synthase (SQS) is a divalent metal-ion-dependent enzyme that catalyzes the two-step reductive `head-to-head' condensation of two molecules of farnesyl pyrophosphate to form squalene using presqualene diphosphate (PSPP) as an intermediate. In this paper, the structures of human SQS and its mutants in complex with several substrate analogues and intermediates coordinated with Mg2+or Mn2+are presented, which stepwise delineate the biosynthetic pathway. Extensive study of the SQS active site has identified several critical residues that are involved in binding reduced nicotinamide dinucleotide phosphate (NADPH). Based on mutagenesis data and a locally closed (JK loop-in) structure observed in thehSQS-(F288L)–PSPP complex, an NADPH-binding model is proposed for SQS. The results identified four major steps (substrate binding, condensation, intermediate formation and translocation) of the ordered sequential mechanisms involved in the `1′–1' isoprenoid biosynthetic pathway. These new findings clarify previous hypotheses based on site-directed mutagenesis and biochemical analysis.