Members of the ANL superfamily of enzymes are involved in primary and secondary metabolism throughout all domains of life and identify key pathways that contribute to essential physiological reactions as well as defense mechanisms to evade competition. Specifically, acetyl-CoA synthetases are directly involved in energy metabolism, while NonRibosoaml Peptide Synthetases and some Aryl-CoA Ligases produce secondary natural products that confer virulence for the producing organism. Due to the ANL superfamily's ubiquitous involvement in primary and secondary metabolism, gaining an understanding of how these enzymes work and identifying ways to regulate them could provide an alternative route for antibiotic targets. It is well documented that domain alternation is paramount for the ANL superfamily of enzymes including the adenylation and thioester-forming reactions of NRPS adenylation domains. This thesis utilizes structural and functional analysis in conjunction with computational methods to further our understanding of these unique enzymes.
In chapter 2 we present the structure of an adenylation:Peptidyl Carrier Protein di-omain NRPS from the cryptic PA1221 biosynthetic operon from Pseudomonas aeruginosa. The PA1221 structure is the second example of an adenylation:PCP in the PDB and validates the chimeric fusion interactions of EntE-B. The similar interacting regions are between the 2nd PCP helix and a helix in the N-terminal subdomain of the adenylation domain as well as the loop connecting the longest β-strands of the C-terminal subdomains interacting with loop 1 of the PCP.
Chapter 3 presents the structure of an acetoacetatyl-CoA Synthetase that is a confirmed substrate for a protein acetyltransferase, PatA, for inactivation through acetylation of the catalytic A10 lysine. This Streptomyces lividans acetoacetyl-CoA synthetase is the first structure to fully resolve the loop connecting C-terminal extension helix to the C-terminal subdomain. The C-terminal extension is only present in ACS proteins revealing an interaction where the C-terminal extension stabilizes the dynamic P-loop in the adenylate forming conformation.
In chapter 4 we further explore the PA1221 operon by functionally identifying the substrate preference of PA1215, the hypothetical fatty-acyl-CoA Ligase, that is proposed to acylate the charge PCP of PA1221. We computationally validate the substrate preference with a homology model and AutoDock to gain insight into the proteins slow kinetics. We also provide further insight into the biochemistry of a subset of ANL superfamily members, the phenylacetic acid CoA ligases, involved in the utilization of aryl-carboxylic acids as a carbon source as well as the derivatization of penicillin. We analyze their unique dimeric structures identifying structural motifs that are contributed through the dimeric interface, but are otherwise located to different sides of the enzyme in a monomeric form.
Finally, to help identify how the protein moves between the two productive conformations we subject members of the superfamily to computational dynamic simulations including Anisotropic Network Modeling, Interpolative Elastic Network Modeling, all-atom molecular dynamics, and analyze the output from these methods with Principal Component and Normal Mode Analysis. We developed a method to visualize a dynamic reaction coordinate through measuring the Conformation Determining Angle (defined by structural motifs that are present in superfamily members) and use this metric to interrogate all ANL superfamily member PDB entries for domain organization. Finally, we test our hypothesis that domain alternation proceeds through an extended, open conformation with structural comparisons and MD. Here we report functional and structural analysis of ANL superfamily members that are related through bacterial cell metabolism and natural product biosynthesis.
|Advisor:||Gulick, Andrew M.|
|Commitee:||Duax, William L., Gewirth, Daniel T., Malkowski, Michael G.|
|School:||State University of New York at Buffalo|
|School Location:||United States -- New York|
|Source:||DAI-B 75/02(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry, Bioinformatics|
|Keywords:||ANL enzymes, Adenylation-peptidyl carrier protein interaction, Domain alternation, Molecular dynamics, Nonribosomal peptide synthetases, X-ray crystallography|
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