Abstract
Dehydrogenases are enzymes that consist of a flavin cofactor in their catalytic unit and a heme or an Fe4S4 cluster in their electron transfer subunit, thereby having the ability to undergo direct electron transfer processes. A significant advantage of using dehydrogenases over oxidases for enzyme-based biosensing applications is the ability to eliminate oxygen dependence. The goal of this dissertation was to investigate one such flavin dependent dehydrogenase (FDD) enzyme, the histamine dehydrogenase from Rhizobium sp. 4-9 (HaDHR) and determine the protein engineering criteria required of a dehydrogenase enzyme for use in in vivo biosensing applications. HaDHR belongs to a small family of dehydrogenases that contains a covalently attached FMN and an Fe4S4 as the redox cofactors. HaDHR can convert histamine to imidazole acetaldehyde and ammonia with a release of two electrons and is the only member of this family that does not show substrate inhibition, making HaDHR the best candidate for use in in vivo biosensing applications. The crystal structure of HaDHR at a resolution of 2.1 Å was determined, which facilitated elucidation of the internal electron transfer pathway from the active site to the protein’s surface. An artificial electron mediator Fc+ was covalently attached proximal to the exit point of the electron transfer pathway. We demonstrated that even in the absence of any other mediator, an Fc-modified HaDHR could deliver electrons from the enzyme to an electrode surface in the presence of histamine in a dose-dependent manner. We engineered HaDHR with noble metal affinity peptides to orient the enzyme on an electrode surface. Additionally, structural comparisons between HaDHR and other family members was undertaken to probe the origins of substrate inhibition. Together, these results provided a proof of concept that HaDHR is suitable for use in the fabrication of Gen 2.5 and Gen 3 biosensors, thereby contributing significantly to this rapidly evolving field.