Electron transfer is an important phenomenon in various biological and synthetic protein systems. Nature frequently uses cofactors to carry electrons required for redox reactions. Some of these cofactors are mobile and freely diffuse (e.g. NAD(P)(H)) or they can be covalently bound to the enzyme (e.g. FADH). There are many applications where NAD(P)(H) dependent enzymes are desirable, but the cost and stability of the mobile cofactor can be limiting. We are working to develop coupled dehydrogenases with engineered electron transport pathways that will reduce and/or eliminate the need for the addition of soluble cofactors. In this talk, we explore the wiring of two redox proteins using designed collagen-like linker. For this purpose, we have first expressed synthetic genes encoding formate dehydrogenase (FDH) from S. cerevisiae and lactate dehydrogenase (LDH) from E. coli, and confirmed their specific activities. We have also designed and optimized sequences of collagen-like peptides (A, B, and C) that can self-assemble stably and specifically only when A, B, and C are mixed. Following this, collagen A has been genetically fused to the C-terminus of FDH and maleimide-functionalized collagen B has been covalently attached to LDH. We have characterized the ability of the collagen constructs to assemble using FRET spectroscopy. The most recent results, including the apparent kinetic constants and FRET efficiencies of collagen-enzyme constructs, will be presented. The approach presented here not only provides a new model for creating more efficient metabolic pathways, it also eliminates the need for an expensive, unstable cofactor.

Wiring of redox enzymes using a collagen heterotrimer protein

251st National Meeting (2016)

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