Overview
During 2014, iGEM ITESM CEM Team worked on the development of a metabolic pathway for 7-ketocholesterol degradation by enzyme therapy of human macrophages, using two hypothetic enzymes of the microorganism Rhodococcus jostii, active when the microbe grows using 7-ketocholesterol as a sole source of carbon. These enzymes (oxoacyl reductase and 7-dehydratase) were first described as potentially being used to catalyse these reactions by Mathieu (1); and were cloned and purified by iGEM ITESM CEM Team using E. coli and a diverse array of expression vectors.
However, in order to properly asses and predict the behaviour of both proteins in the cytosol of human cells, it is first necessary to numerically model their interaction and catalysis over their substrates. In order to do so, the proposed pathway must firstly be analysed.
Figure 1.- Theoretical metabolic pathway for 7-ketocholesterol degradation in Rhodococcus jostii. Red arrows indicated the most intuitive order of reactions.
Figure 1 shows the general array of chemical reactions in the pathway, were red arrows indicate the direction proposed by iGEM ITESM CEM Team. Even though reactions can occur in almost any order, the most intuitive arrangement is that in which 7-ketocholesterol is converted to 7-βOH-cholesterol, which is finally transformed to regular cholesterol; these two reactions are supposed to be catalysed by oxoacyl reductase and 7-dehydratase respectively.
7-ketocholesterol is commonly present throughout the bloodstream in the form of esters (with a fatty acid chain attached to the 3rd position of the core rings) as a part of oxidized lipoproteins (usually oxLDL); 7-ketocholesterol is released from these particles by a transesterification reaction catalysed by cholesterol oxidase (the third enzyme cloned by the team). As for cholesterol, it is able to enter a wide variety of naturally occurring metabolic pathways such as hormone synthesis, bile salt production or, in a lesser extent, complete mineralization (CO2 is formed). On the other hand, cholesterol is at the same time synthesized endogenously by human cells, and a small fraction of it comes from an animal product-containing diet.
Inside human macrophages (cells that are responsible for 7-ketocholesterol degradation throughout the development of atherosclerosis), this metabolic pathway does not exist. This is mainly due to the lack of specific enzymes that act upon oxidized derivatives of sterols, particularly 7-ketocholesterol. This is the main reason why using proteins derived from microbial genomes (in this study, Rhodococcus jostii) seems a reasonable approach in order to enhance 7-ketocholesterol metabolism, and minimize its accumulation on the inner walls of human arteries.
Since all the previously described chemical reactions are properly modelled using single-substrate enzyme kinetics, Michaelis-Menten equations were used. In order to simplify the proposed mathematical model, cholesterol metabolism was not included among the equations, and only the two enzymes directly involved in 7-ketone removal were modelled. This permits us to analyse the impact of variations in kinetic constants (Km and Rmax) over sterol metabolism, which lets us in turn asses the efficiency of a microbial pathway when compared to the endogenous degradation of 7-ketocholesterol in human cells.
Model Description
The general Michalies-Menten model was used, which was stated in a differential form, as the following:
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