Date of Award:


Document Type:


Degree Name:

Doctor of Philosophy (PhD)


Chemistry and Biochemistry


Sean J. Johnson


This work is focused on understanding protein function by describing how paralogous proteins with overlapping and distinct functions interact with their substrates and with other proteins. Two model systems are the subject of this research: (1) the stereospecific dehydrogenases R- and S-HPCDH, and (2) the zinc knuckle proteins Air1 and Air2.

R- and S-HPCDH are homologous enzymes that are central to the metabolism of propylene and epoxide in the soil bacterium Xanthobacter autotrophicus. The bacterium produces R- and S-HPCDH simultaneously to facilitate transformation of R- and S-enantiomers of epoxypropane to a common achiral product 2-ketopropyl-CoM (2-KPC). Both R- and S-HPCDH are highly stereospecific for their respective substrates as each enzyme displays less than 0.5% activity with the opposite substrate isomer. Presented here are substrate-bound x-ray crystal structures of S-HPCDH. Comparisons to the previously reported product-bound structure of R-HPCDH reveal structural differences that provide each enzyme with a distinct substrate binding pocket. These structures demonstrate how chiral discrimination by R- and S-HPCDH results from alternative binding of the distal end of substrates within each substrate binding pocket, providing a structural basis for stereospecificity displayed by R- and S-HPCDH.

Air1 and Air2 are homologous eukaryotic proteins that individually function within a trimeric protein complex called TRAMP. In the nucleus, TRAMP participates in RNA surveillance, processing, and turnover by stimulating the 3’-5’ exonucleolytic degradation of targeted RNAs by the nuclear exosome. Previous studies have indicated that within TRAMP Air1 and Air2 provide crucial protein-protein interactions that link the individual subunits of the complex. However, the mechanistic details of these protein-protein interactions are poorly understood. The work in this dissertation has characterized a previously unknown binding interface between Air2 and another TRAMP component, the helicase Mtr4. This interaction may explain how helicase activity is modulated in TRAMP. In addition to TRAMP protein interactions, preliminary studies have identified a small region of Air1 that is required for modulating the activity of a protein that is not found in TRAMP, the methyltransferase Hmt1. Collectively, these studies provide important characterization of Air1 and Air2 protein-binding interactions, and establish a foundation for future research efforts aimed at exploring Air protein function.

Included in

Biochemistry Commons