Computational Redesign of Acyl-acp Thioesterases for Increased Substrate Specificity
Open Access
Author:
Gifford, Nathanael Paul
Graduate Program:
Chemical Engineering
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
July 02, 2014
Committee Members:
Michael John Janik, Thesis Advisor/Co-Advisor Costas D Maranas, Thesis Advisor/Co-Advisor
Keywords:
Thioesterase IPRO Computational Protein Engineering Chemical Engineering
Abstract:
Due to limited resources and the reality of global climate change, alternatives to petroleum resources will be necessary in the near future. Biologically produced fatty acids are precursors to biodiesel, an attractive alternative to naturally occurring fossil fuels. Due to the promiscuity of thioesterases, the enzymes responsible for terminating the growth cycle of fatty acids, it is difficult to produce fatty acids with the distribution of chain lengths desired for many applications. In this work, the iterative protein redesign and optimization (IPRO) procedure is used for the redesign of Thioesterase I/Protease I/Lysophospholipase L1 (TAP I) for increased substrate specificity for both lauric acid (12C fatty acid) and caprylic acid (8C fatty acid). IPRO uses molecular mechanics simulations alongside an MILP optimization problem formulation to generate recommended mutants for increased specificity. We also use ensemble refinement to assess the quality of mutants generated and to generate an energetic profile to approximate the specificities of the mutants for a range of fatty acid chain lengths. Mutants generated create a complex of charged residues which obstruct the binding crevice of TAP I, preventing fatty acids larger than the desired species from binding.