Payne, Christina M*

Selectivity and Stability of Cytochrome P450BM3 for Membrane-based Enzymatic Remediation

Contaminated wastewater is an unfortunate byproduct of many industrial processes and society's increasing reliance upon chemical products such as fertilizers, pesticides, and pharmaceuticals. Adapting membrane technology to facilitate safe and efficient deconstruction of toxic molecules offers a promising approach toward environmental remediation, either prior to large-scale environmental contamination or as part of associated cleanup efforts. Coupling enzymes to carbon nanotube membrane solid supports localizes biocatalytic conversion of a broad range of contaminates to lower toxicity products and allows high-throughput wastewater treatment without loss of activity, minimizing issues of low protein expression and/or catalytic efficiency. To maximize applicability of such a technology, the attached enzymes are ideally both promiscuous and tolerant of extreme conditions. The proposed objectives of this project develop a molecular-level understanding of protein dynamics governing stability and substrate selectivity in cytochrome P450BM3, which has been selected for its specificity toward common contaminates. These findings will form the foundation of a structure-function relationship toward rational design of P450BM3 for a variety of membrane applications.

Student

Inacrist M Geronimo (Postdoc)

Project 1:

Computational studies of recalcitrant polysaccharide degradation

This project examines chitin, a biopolymer closely related to cellulose, and the associated degrading enzymes, chitinases. Chitin is the linear polymer of β-1,4-N-acetyl-D-glucosamine occurring naturally as a well-ordered crystal. As a major component of the exoskeletons of crustaceans, insects, and mollusks as well as the cell wall material of fungi and algae, chitin is the second most abundant biological material on Earth after cellulose. To deconstruct these carbohydrates from the polymer crystal, it is hypothesized that processive enzymes acquire single chains from the surface and cleave linkages without detaching for several monomeric units. The nature of this processive action, particularly within the enzyme active site, is not well characterized at the molecular level. We apply molecular dynamics simulations in glycoside hydrolase Family 18 Serratia marcescens chitinases to understand how chemical composition defines behavior. We will use NSF XSEDE resources to perform free energy calculations related to this project.

Students

Suvamay Jana (graduate student)
Shawn Nigam (graduate student)
Christina M. Payne (PI)

Project 2:

Identifying the physiological ligand of the mammalian glycoprotein YKL-40

YKL-40, a mammalian glycoprotein, is a known biomarker associated with progression, severity, and prognosis of chronic inflammatory diseases and a multitude of cancers. Despite this well-documented association, conclusive identification of the lectin’s physiological ligand, and accordingly the biological function, has proven experimentally difficult. Clinical studies suggest YKL-40’s biological function may be related to collagen fibril formation or degeneration as a result of injury or disease, inferring drug resistance and increasing cell migration leading to progression of cancer, or protection from pathogens. Investigation of binding to potential physiological ligands has yielded consistently measureable affinity only in the case of chitin, a polysaccharide not naturally present in vivo. While clues to YKL-40’s physiological ligand and biological function exist, the mechanism and affinity by which YKL-40 recognizes and binds ligands remains elusive. As a potential therapeutic target, these fundamental insights are critical to the ability to rationally design YKL-40 antagonists. We apply molecular simulation and free energy calculations to define the molecular-level recognition mechanisms and binding affinity of YKL-40 to potential physiological ligands including both polysaccharides, collagen, and procollagen. Free energy calculations will be performed using NSF XSEDE resources given the significant computing requirements.

Students

Abhishek Kognole (graduate student)
Christina M. Payne (PI)

Project 3:

Understanding the Molecular Mechanism of Biological Desulfurization to Improve Sulfur Removal from Petroleum.

Biodesulfurization represents a unique complement to conventional petroleum upgrading processes. However, commercialization hinges upon successful biocatalytic activity improvements. To develop a fundamental understanding of the molecular-level mechanisms responsible for activity, we propose a computational investigation of the desulfinase enzyme that catalyzes the final, rate-limiting step in the biodesulfurization of dibenzothiophene. We will perform molecular simulations of the desulfinase bound to substrates and inhibitors in an oil-like solvent to characterize contributions to specificity and inhibition.

Students:

Anderson, Nolan
Gibson, Codell
Karnes, Tyler
Yu, Yue
Gado, Japheth

Collaborators:

Englert, Derek - OISTL

Project 4:

Carbohydrate Recognition in Type B CBMs

Type B CBMs are thought to selectively bind oligomeric chains. However, a subset of cellulose-specific CBM families has been shown to bind to non-crystalline cellulose more tightly than oligomers and in a manner that discriminates between surface topology. These CBMs also often appear in tandem, harnessing multivalent interactions to enhance affinity. Biochemical studies have painted a broad picture of Type B CBM carbohydrate recognition, yet the structural and dynamical features that contribute to substrate binding remain unknown. We address the hypothesis that Type B CBM carbohydrate recognition mechanisms are different for oligomeric and non-crystalline substrates, which may account for the evolutionary occurrence of tandem binding modules. Molecular dynamics simulations and enhanced sampling free energy methods will be used to evaluate the molecular-level origins of oligomeric carbohydrate recognition within and across three families of cellulose-specific, Type B CBMs. Non-crystalline cellulose-binding CBMs will be modeled bound to the insoluble substrate, and alchemical free energy pathways will be used to determine the free energy of binding to non-crystalline cellulose. The outcome of this study will provide an unprecedented level of insight into the complex solid and soluble carbohydrate substrate recognition mechanisms of CBMs, the findings of which hold considerable promise for enhancing biomass conversion technology.

Students

Abhishek Kognole (graduate student)::
Nolan Anderson (undergraduate student)


Methodology
Computational methodology is common to both described projects, and thus, is described once. We will apply classical MD simulations and free energy calculations including thermodynamic integration and free energy perturbation. Model systems are setup and equilibrated using CHARMM, a commercially available code. This is not currently available to every researcher at UK. I have purchased a license for my group and locally installed the source code for my group’s use. Production simulations (all methods)are performed using NAMD, which is currently available at UK and is freely distributed. When force field development is required, Gaussian09 is occasionally used by our group as part of these projects.

Funding:

1. “Understanding the Molecular Mechanism of Biological Desulfurization to Improve Sulfur Removal from Petroleum,” (PI), American Chemical Society – Doctoral New Investigator, Total: $100,000, 09/1/2014 - 08/31/2016 (time devoted to project 0.5 months), awarded (#23861-DNI4).
2. Inhibition of the mammalian glycoprotein YKL-40: Identification of the physiological ligand,” (PI), Kentucky Science and Engineering Foundation, Total: $30,000, 07/1/2013 - 06/30/2014 (time devoted to project 0 months), awarded (#KSEF-2815-RDE-016).

Publications:

Publications from DLX

1. G.T. Beckham, J. Ståhlberg, B.C. Knott, M.E. Himmel, M.F. Crowley, M. Sandgren, M. Sørlie, and C.M. Payne*, “Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases,” Curr. Opin. Biotechnol., 27, 96-106 (2014).
2. C.M. Payne*‡, W. Jiang‡, M.R. Shirts, M.E. Himmel, M.F. Crowley, G.T. Beckham, “Glycoside hydrolase processivity is directly related to oligosaccharide binding free energy,” J. Am. Chem. Soc., 135(50), 18831-18839 (2013).
3. C.M. Payne‡, M.G. Resch‡, L. Chen‡, M.F. Crowley, M.E. Himmel, L.E. Taylor, M. Sandgren, J. Ståhlberg, I. Stals, Z. Tan, and G.T. Beckham, “Glycosylated Linkers in Multi-Modular Lignocellulose Degrading Enzymes Dynamically Bind to Cellulose,” Proc. Natl. Acad. Sci. U.S.A., 110, 14646-14651 (2013).
4. M. Kern, J.E. McGeehan, S.D. Streeter, R.N.A. Martin, K. Besser, L. Elias, W. Eborral, G.P. Malyon, C.M. Payne, M.E. Himmel, K. Schnorr, G.T. Beckham, S.M. Cragg, N.C. Bruce, S.J. McQueen-Mason, “Structural Characterization of a Family 7 Cellobiohydrolase from a Marine Animal Reveals Potential Mechanisms of Cellulase Salt Tolerance,” Proc. Natl. Acad. Sci. U.S.A., 110, 10189-10194 (2013).
5. R. Kushwaha, and A.B. Downie, and C.M. Payne, “Uses of Phage Display in Agriculture: Sequence Analysis and Comparative Modeling of Late Embryogenesis Abundant Client Proteins Suggests Protein-Nucleic Acid Binding Functionality,” Comput. Math. Methods Med., 2013, 470390 (2013).
6. R. Kushwaha, C.M. Payne, and A.B. Downie, “Uses of Phage Display in Agriculture: A Review of Food-Related Protein-Protein Interactions Discovered by Biopanning over Diverse Baits,” Comput. Math. Methods Med., 2013, 653759 (2013).
7. M.H. Momeni‡, C.M. Payne‡, H. Hansson, N.E. Mikkelsen, J. Svedberg, J., Å, Engström, Å., M. Sandgren, G.T. Beckham, and J. Ståhlberg, “Structural, Biochemical, and Computational Characterization of the Glycoside Hydrolase Family 7 Cellobiohydrolase of the Tree-killing Fungus Heterobasion irregulare,” J. Biol. Chem., 288, 5861-5872 (2013).
8. D.W. Sammond, C.M. Payne, R. Brunecky, M.E. Himmel, M.F. Crowley, and G.T. Beckham, “Cellulase Linkers are Optimized Based on Domain Type and Function: Insights from Sequence Analysis, Biophysical Measurements, and Molecular Simulation,” PLoS ONE, 7, e48615 (2012).
9. C.M. Payne, J. Baban, S.J. Horn, P.H. Backe A.S. Arvai, B. Dalhus, M. Bjørås, V.G.H. Eijsink, M. Sørlie, G.T. Beckham, and G. Vaaje-Kolstad, “Hallmarks of processivity in glycoside hydrolases from crystallographic and computational studies of the Serratia marcescens chitinases,” J. Biol. Chem., 287, 36322-36330 (2012).
10. G.T. Beckham, Z. Dai, J.F. Matthews, M. Momany, C.M. Payne, W.S. Adney, S.E. Baker, and M.E. Himmel, “Harnessing Glycosylation to Improve Cellulase Activity,” Curr. Opin. Biotechnol., 23, 338-345 (2012).

Grants

1. Payne, Christina M SusChEM: Carbohydrate Recognition in Type B Carbohydrate Binding Modules (CHE-1404849) Role: PI National Science Foundation: Chemistry for Life Processes; 08/15/2014 – 07/31/2017; Total: $225,000
2. Payne, Christina M Oak Ridge Associated Universities Ralph E. Power Junior Faculty Enrichment Award (FY2014_419) Role: PI ORAU and UK Center for Computational Sciences; 06/01/2014 – 05/31/2015; Total: $10,000
3. Payne, Christina M PRF53861-DN14 Computational characterization of a desulfinase for enhanced crude oil desulfurization $100,000 American Chemical Society 1/1/2014 8/31/2016
4. Payne, Christina M KSEF-148-502-13-307 KSEF RDE: Inhibition of the mammalian glycoprotein YKL-40: Identification of the physiological ligand $30,000 KY Science and Technology Co Inc 7/1/2013 6/30/2014
5. Payne, Christina M Understanding the Molecular Mechanism of Biological Desulfurization to Improve Sulfur Removal from Petroleum (#23861-DNI4) Role: PI American Chemical Society Petroleum Research Fund; 09/14 – 08/16; Total: $100,000
6. Payne, Christina M Developing the Foundation of Sequence-based Performance Prediction in Fungal Glycoside Hydrolases Role: August T. Larsson Guest Researcher; Hosts: Henrik Hansson and Jerry Stahlberg Swedish University of Agricultural Sciences; 05/14 – 08/17; Total: $195,000
7. Payne, Christina M NSF EPSCoR: Powering the Kentucky Bioeconomy for a Sustainable Future Role: Faculty Participant; PIs: Rodney Andrews, Bruce Hinds, Seth DeBolt, Yang-Tse Cheng) National Science Foundation; 07/14 – 06/19; $20,000,000 award of Total: $25,859