A Benchmark for Homomeric Enzyme Active Site Structure Prediction Highlights the Importance of Accurate Modeling of Protein Symmetry.

Accurate prediction and modeling of an enzyme's active site are critical for engineering efforts as well as providing insight into an enzyme's naturally occurring function. Previous efforts demonstrated that the integration of constraints enforcing strict geometric orientations between catalytic residues significantly improved the modeling accuracy for the active sites of monomeric enzymes. In this study, a similar approach was explored to evaluate the effect on the active sites of homomeric enzymes. A benchmark of 17 homomeric enzymes with known structures and a bound ligand relevant to the established chemistry were identified from the protein data bank. The enzymes identified span multiple classes as well as symmetries. Unlike what was observed for the monomeric enzymes, upon the application of catalytic geometric constraints, there was no significant improvement observed in modeling accuracy for either the active site of the protein structure or the accuracy of the subsequently docked ligand. Upon further analysis, it is apparent that the symmetric interface being modeled is inaccurate and prevented the active sites from being modeled at atomic-level accuracy. This is consistent with the challenge others have identified in being able to predict de novo protein symmetry. To further improve the accuracy of active site modeling for homomeric proteins, new methodologies to accurately model the symmetric interfaces of these complexes are needed.

Combination of type II fatty acid biosynthesis enzymes and thiolases supports a functional ß-oxidation reversal.

An engineered reversal of the ß-oxidation cycle (r-BOX) and the fatty acid biosynthesis (FAB) pathway are promising biological platforms for advanced fuel and chemical production in part due to their iterative nature supporting the synthesis of various chain length products. While diverging in their carbon-carbon elongation reaction mechanism, iterative operation of each pathway relies on common chemical conversions (reduction, dehydration, and reduction) differing only in the attached moiety (acyl carrier protein (ACP) in FAB vs Coenzyme A in r-BOX). Given this similarity, we sought to determine whether FAB enzymes can be used in the context of r-BOX as a means of expanding available r-BOX components with a ubiquitous set of well characterized enzymes. Using enzymes from the type II FAB pathway (FabG, FabZ, and FabI) in conjunction with a thiolase catalyzing a non-decarboxylative condensation, we demonstrate that FAB enzymes support a functional r-BOX. Pathway operation with FAB enzymes was improved through computationally directed protein design to develop FabZ variants with amino acid substitutions designed to disrupt hydrogen bonding at the FabZ-ACP interface and introduce steric and electrostatic repulsion between the FabZ and ACP. FabZ with R126W and R121E substitutions resulted in improved carboxylic acid and alcohol production from one- and multiple-turn r-BOX compared to the wild-type enzyme. Furthermore, the ability for FAB enzymes to operate on functionalized intermediates was exploited to produce branched chain carboxylic acids through an r-BOX with functionalized priming. These results not only provide an expanded set of enzymes within the modular r-BOX pathway, but can also potentially expand the scope of products targeted through this pathway by operating with CoA intermediates containing various functional groups.