Kinetics Study Of Arom Complex Cascade Reaction In A Simulated Cellular Environment

The shikimate pathway is a seven-step biosynthetic pathway used by microorganisms and plants to catalyze the production of precursors for aromatic compounds.The AROM complex is a five-functional enzyme complex involved in catalyzing steps two through six of the shikimate pathway in brewing yeast.Multifunctional enzyme complexes typically have substrate channeling effects,but initial results indicate that individual enzyme domains of the AROM complex can function independently of substrate channeling in vitro.However,the real cellular environment in which the AROM complex is located is more complex than in vitro.The intracellular environment is filled with high concentrations of various large proteins and other substances（nucleic acids,polysaccharides,etc.）,which can result in crowding effects and chemical interactions with the AROM complex.Experimental conclusions drawn from in vitro studies cannot be fully applied to the intracellular environment.The activity of AROM complex in different environments was determined by constructing and expressing AROM,AROC,TrpEG and two AROM mutants(AROM^M1 and AROM^M2).The crowded environment in cells produces crowding effects and chemical effects on proteins,which are simulated by adding PEG-8k,Ficoll-70k,BSA,casein,albumin,Escherichia coli lysate and PMAA to the buffer.Two polymers,PEG-8k and Ficoll-70k,were used to simulate depletion interaction in cells,and no effect was found on the activity of AROM complex.When proteins such as BSA were used to studied the crowded environment in cells,the activity of AROM complex was significantly improved.The buffer containing PMAA（negatively charged polymer）also significantly increased the activity of AROM complex,demonstrating that negatively charged molecules in crowded environments increased the activity of AROM complex.It was verified that PMAA also promoted the catalytic activity of the DHQD,SD,SK and EPSPS functional enzyme active of AROM complex by two mutants,AROM^M1 and AROM^M2.By immobilizing the AROM complex onto nanoscale gold nanoparticles and submicron polystyrene microspheres,the changes in catalytic performance between the AROM complex in a free state and when fixed onto different scale carriers were studied.Preliminary results show that after immobilizing the AROM complex onto submicron-nanoscale carriers,the activity of AROM complex cascade catalysis was improved.