Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Room: Exhibit Hall A
Stephen Gonzalez - California State University, Fullerton
Co-Author(s): Seth A. Wiley, University of Michigan, Ann Arbor, MI; Dr. Stephen W. Ragsdale, University of Michigan, Ann Arbor, MI
The quest for solving the global warming crisis and to produce renewable carbon based sources is of great global interest. The Wood-Ljungdahl pathway of CO and CO2 fixation offers a method to develop renewable energy supplies while reducing and utilizing waste CO2. In the pathway, two key anaerobic metalloenzymes in a heterotetrameric complex, Carbon Monoxide Dehydrogenase (CODH) and Acetyl-CoA Synthase (ACS), help produce acetate which is a fundamental growth product of acetogenic bacteria or incorporate acetyl-CoA into cell carbon or regenerate ATP. CODH reduces CO2 to CO in the first reaction by which CO travels through an interprotein channel to ACS. In the second reaction, ACS in conjunction with coenzyme A and a methyl group donated by corrinoid iron-sulfur protein (CFeSP) synthesizes acetyl-CoA. During each binding event, ACS shuffles between two different conformations: open and closed. In order to fully understand the underlying mechanism in substrate specificity to the A-cluster, we must test out some identified key residues which may play an interesting role in each mechanism. Recently, a residue mutated near the A-cluster displayed severe impact upon binding to CO in comparison to the wild-type. In order to understand the role the local environment plays on the ACS mechanism, we prepared three active site mutants for kinetic and spectroscopic experiments by carbonylation and methylation. One specific variant, ACS-F512A, was expressed, purified, and reconstituted in strictly anaerobic conditions before performing subsequent experiments. To understand the significance that F512 plays in substrate binding, we compared F512A to the ACS wildtype using Electron Paramagnetic Resonance (EPR) spectroscopy for ACS CO-binding and UV-Visible kinetics to monitor ACS methylation. EPR spectroscopy was used to assess the ACS Nip1+-CO signal upon exposure to CO in relation to wildtype ACS and revealed a lower binding affinity for CO than was anticipated in comparison to the wildtype. Analysis of methylation to ACS-F512A was assayed using methylcobinamide and data provides preliminary insight into how ACS gets methylated in the variant compared to the wildtype. Our current studies suggest that residue F512 may be important to the reactions ACS is involved in the carbonyl branch. Future plans for ACS-F512A are metal analysis and further studies by EPR and radiolabeled exchange assays. Following supporting spectroscopic and kinetic evidence, it will be sent out for X-ray Absorption Spectroscopy (XAS) to analyze its active site structure.
Funder Acknowledgement(s): This research was funded by NSF award number 1851985 through the Interdisciplinary REU Program in the Structure and Function of Proteins at the University of Michigan- College of Pharmacy and by NIH grant # R37-GM39451 on Enzymology of the Reductive Acetyl-CoA Pathway.
Faculty Advisor: Stephen W. Ragsdale, email@example.com
Role: I created three single point amino acid mutations on ACS (F512A, F512W, and I146A). My poster covers data over the summer on F512A which I personally gathered. All other data that will be presented contains my graduate mentor's data.