Abstract
Cyclic mono- and di-nucleotides are essential signaling molecules involved in many cellular processes. Cellular levels of cyclic nucleotides are fine-tuned by metal-dependent phosphodiesterases (PDEs), such as the histidine-aspartate (HD)-domain proteins. The large histidine-aspartate (HD)-domain protein superfamily contain metalloproteins that share common structural features but catalyze vastly different reactions. This thesis focuses on the characterization of three HD-domain PDEs: i) the tandem domain HD-[HD-GYP] PDE, VCA0681 (V-cGAP1), ii) the prototypical single domain HD-GYP PDE, VCA0931 (V-cGAP3), both from the human pathogen Vibrio cholerae (Vc), and iii) the HD-domain Cas3 nuclease, which degrades foreign DNA in Type I clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems (Cas) system.Cellular levels of cyclic dinucleotides (CDNs) are regulated by EAL and HD-GYP proteins. Although both protein families were discovered almost at the same time, HD-GYPs are less well structurally and functionally characterized. There is an increasing number of annotated HD-GYP proteins, highlighting their importance in the regulation of CDNs. VCA0681 was previously identified as an HD-GYP PDE that showed significant hydrolysis of c-di-GMP and 3’3’c-GAMP in Vc. In chapter 2, we identified SO3491 as a homolog of VCA0681 and characterized both proteins with respect to their catalytic properties, range of metal ions and substrates. We also performed phylogenetic analyses so as to examine the distribution and functional relationships of the HD-[HD-GYP]s to the rest of HD-GYPs.
VCA0931 (V-cGAP3), a prototypical single domain HD-GYP from Vc, colocalizes phylogeneticaly with VCA0681 (V-cGAP1). However, the extant chemical makeup and steady-state parameters of the VCA0931 cofactor remain little explored. The simple architecture also makes it an ideal model for examining chemical roles of HD-GYP conserved residues. In chapter 3, we carry out an in-depth characterization of VCA0931 on its metal and substrate selectivity, as well as roles of conserved protein motifs.
Chapter 4 describes the characterization of Type I Cas3 nucleases. The nuclease Cas3 is also an HD-domain PDE, but little is known about the metallocofactor requirements for optimum activity. We map a promiscuous metal ion cofactor profile for the Cas3 from Thermobifida fusca (Tf) and show that Fe is not inhibitory as initially thought. We also explore the ability of Cas3 to hydrolyze cyclic mononucleotides and compare that activity to the homologous Conserved virulence factor A (CvfA), an HD-domain protein hydrolyzing 2’3’-cylic phosphodiester bonds at RNA 3’-termini.