Part:BBa_K4348032
AKR1D1_V121toM_his
This is the gene for the human AKR1D1 enzyme with a mutation on the V121 residue, turning it into M. The mutation was done so it may have more activity on 4-cholesten-3-one, which is not its wild-type ligand.
Introduction
The McGill iGEM team set out to develop a cholesterol lowering probiotic as a preventative for cardiovascular disease. Both endogenously synthesized cholesterol and dietary cholesterol end up in the gut, where they are absorbed and sent around the body. McGill iGEM’s project consists of developing a novel metabolic pathway to convert cholesterol, which is absorbed in the gut, into coprostanol, a molecule that cannot be absorbed and is thus excreted from the gut. The metabolic pathway consists of a three step pathway with four metabolites: cholesterol, which is converted to cholestenone, then coprostanone and finally coprostanol. We repurposed existing enzymes to engineer a metabolic pathway to do this conversion, then packaged it in a probiotic bacterium. By converting intestinal cholesterol into coprostanol, our probiotic bacterium can prevent cholesterol absorption as a preventative for high cholesterol-induced cardiovascular disease.
Mutant Identification
Our goal was to identify residues in AKR1D1 that could be mutagenized to increase the catalytic efficiency of the enzyme, to increase the yield of our pathway. To do this, we aligned the human AKR1D1 enzyme that we used in our project with the AKR1D1 homologs of other related species, from monkeys to birds, to fish to pandas. Based on the known crystal structure, active site location and site of substrate binding in AKR1D1, we identified all the residues within a 4 angstrom radius of where we modeled our substrate to be in the active site of AKR1D1. These active site-proximal residues were likely the most important contributors to enzyme catalytic efficiency, so we reasoned that we would focus our mutagenesis efforts on these residues. We identified the residues in the active site of AKR1D1 that differed from species to species, meaning that they were not conserved between species and therefore not crucial for enzymatic function. Each mutant consists of one such amino acid switch from the human residue to the respective amino acid of another species.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 753
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 753
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 753
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 753
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 753
- 1000COMPATIBLE WITH RFC[1000]
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