National Science Foundation     |     Directorate for Engineering  (ENG)
Division of Chemical, Bioengineering, Environmental, & Transport Systems  (CBET)
CBET Research Highlights 
Notable Accomplishments from CBET Awards
1401 - Part A - Nanostructured Enzyme Complexes Give More Efficient Catalysis
Neal Woodbury  -  Arizona State University

Outcome or Accomplishment:  Dr. Jinglin Fu, working with Professors Hao Yan and Neal Woodbury in the Biodesign Institute at Arizona State University, have used an extremely small nanostructure made from DNA to assemble a two-enzyme complex with the enzymes in precisely-controlled positions.  They then investigated the dependence of the overall reaction rate and efficiency on the distance between the enzymes.  The results reveal that at very close distances hydration shells (layer of water molecules) of the enzymes overlap, substrate transfer between enzymes occurs much more efficiently, thereby dramatically increasing the rate of the coupled reaction.

Neal Woodbury Image 1
    Figure 1.
(A) Two enzymes, Glucose oxidase (GOx, orange) and Horseradish peroxidase (HRP, purple) have been organized on a DNA nanoscaffold (brown) in precisely controlled positions.  The hydrogen peroxide intermediate (red and green molecule) that is made by GOx and used by HRP transfers along the hydrated protein surface (pink shell) much more efficiently than it would through solution.  Multi-enzyme complexes assembled as in (A) can exhibit more than a 15-fold higher activity than enzymes free in solution, as shown in (B).
Neal Woodbury Image 2
    Figure 2.
Placing a protein bridge (tan color) between the two enzymes connects their protein surfaces and provides a facilitated pathway for the hydrogen peroxide intermediate that is made by GOx and used by HRP to transfer along the hydrated protein surface (pink shell) rather than diffusing through solution.  The ability to build bridges between enzymes increases the design options for creating functional enzyme complexes.
Credit for All Images:  Jinglin Fu, Minghui Liu, Neal Woodbury and Hao Yan at Arizona State University, Tempe, AZ

Impact:  In this work, an approach for organizing multiple enzymes on self-assembled DNA nanoscaffolds (see Figure 1.A) is demonstrated and design principles for utilizing this approach to create efficient nanoscale catalytic complexes are elucidated.  These approaches and principles should find utility in the development of the catalysts for production of high-value products in industry and bioenergy as well as diagnostic applications in biomedicine.

Explanation/background: Cells use complex, multi-step metabolic pathways both to derive energy and to make the molecules that are important to life.  Many of the enzymes in these pathways are spatially organized to improve both the speed and specificity of the overall process.  In particular, this organization facilitates the transfer of specific pathway intermediates from one enzyme to another.  Very little is currently known about how the geometric arrangement of enzymes (e.g. position, orientation, enzyme ratio) affects the overall activity of systems involving multiple enzymes.
In research supported by the CBET division of the NSF, self-assembled DNA nanoscaffolds are being used to organize a multi-enzyme complex with precisely-controlled distances between enzymes.  This makes it possible to systematically explore the mechanisms of substrate transfer between enzymes.  The Arizona State University researchers have found that at very close distances, the transfer suddenly becomes extremely efficient because the substrate can move directly along the surface of one enzyme onto the other.  These studies have both increased the current understanding of the enzymology of biological metabolic pathways and have provided the tools and design principles for creating man-made catalytic complexes that are similarly efficient.

CBET Research Highlight - Part B - Engineering Technical Information

1401 - Spacial Organization of Enzyme Systems on Self-Assembled Scaffolds

Neal Woodbury  -  Arizona State University

Background:  Cellular metabolism takes place via complex, multi-enzyme synthetic pathways that exhibit extraordinary yield and specificity.  The enzymes in these multi-enzyme complexes are usually spatially organized to facilitate efficient transfer of intermediates between them.  Understanding the effect of spatial organization on enzymatic activity in multi-enzyme systems is not critical to our fundamental understanding of biochemistry, but is also important in the design and fabrication of man-made catalytic complexes that are both more active and more specific than individual catalysts alone.  Up until now, few methods have been available to systematically evaluate how the geometric arrangement (e.g. position, orientation, enzyme ratio) of the component enzymes influence the overall activity of multi-enzyme systems.
DNA nanotechnology has emerged as a reliable way to organize nanoscale systems due to the fact that DNA self-assembles; pieces of DNA can come together to form specific structures and furthermore can be "programmed" by properly selecting the set of DNA sequences involved.  In this way, almost any nanoscale structure can be made.  Further, such structures can be made with tags - specific addresses where other molecules like enzymes can be attached.  In research supported by the CBET division of NSF, Dr. Jinglin Fu working with Professors Hao Yan and Neal Woodbury in the Biodesign Institute at Arizona State University have employed self-assembled DNA nanoscaffolds to arrange enzymes that work together in a pathway, rather like the metabolic pathways of living cells.  These enzymes can be positioned with nanometer accuracy making it possible to study how the geometry of interaction between enzymes affects their coupled function.

Results:  The research team has taken two coupled enzymes, glucose oxidase which converts glucose and oxygen to hydrogen peroxide, and horse radish peroxidase which uses the hydrogen peroxide as a reactant, and attached them to a DNA nanostructure.  They then systematically explored how the distance between these two enzymes changes the ability of the enzymes to work efficiently together.  What they found is that at very short distances, distances so close that the layer of water molecules that surround the enzymes overlaps, a large increase in coupled enzyme activity occurred.  In other words, when two enzymes are right next to each other, they pass intermediates from one to the other much more efficiently than one would expect from simple diffusion.  Apparently, the intermediates stay within the layer of water that is right next to the protein surface, so it passes directly from one enzyme to the next.  This sort of "limited dimensional diffusion" is also found in other biological processes, where it is important to have molecules find specific sites of action quickly.  In fact, the researchers were even able to build protein bridges between the enzymes (Figure B.1), and create a sort of preferred pathway for efficient transfer of the substrate.  This opens the door for the design of much more complicated multi-enzyme systems that have practical applications in areas such as drug synthesis, bioenergy and waste management.

Scientific Uniqueness: By combining enzymology and DNA nanotechnology, a whole new area of precisely engineered catalytic complexes is opened for investigation and development.

Strategic Outcome Goals:
- 1Discovery:  The research is aimed at systematically exploring the spatial parameters of self-assembled multi-enzyme complexes based on DNA nanoscaffolds.  This provides a unique approach to understanding the enzymology under circumstances where the geometry of multi-enzyme complexes is precisely contolled.
- 2Research Infrastructure:  The nanoscale tools and design principles being developed in this work are of broad significance to the research community, enabling the design, fabrication and exploration of a wide range of different catalytic and structural complexes.
- 3Learning:  This project has provided training and research experience to one postdoctoral researcher, two PhD candidate graduate students and two undergraduate students in the Department of Chemistry and Biochemistry, and Biodesign Institute at Arizona State University.  In addition, the team participates in the Summer High School Internship Program (SIP) at Biodesign Institute at ASU, which provides a unique opportunity for high school students to work closely with researchers in a world-class research institute, gaining hands-on experience with current research projects.  The program encourages students to become better thinkers and problem solvers.

Transformative Research:  The ability to simply and precisely organize catalytic modules at the nanoscale literally opens up a whole new field of geometrically controlled complex chemical reactions that the scientific community is only beginning to explore.

Intellectual Merit:  The spatial organization of multi-enzyme pathways using self-assembled DNA nanostructures represents a unique protein immobilization technology that can significantly improve the activities and specificities of surface-immobilized enzyme pathways.  This could lead to advances in a variety of sensing and biocatalytic applications based on multi-step enzyme systems.  A major aspect of this work is the kinetic analysis and modeling of enzyme systems assembled on these rationally-designed surfaces.  The outcome is of particular interest for the understanding and design of multi-enzyme reaction pathways in which the ability of one enzyme to directly pass a product to the next is critically dependent on the relative positions of the enzymes involved. This approach not only enables a predictable framework for the fabrication of man-made enzymatic circuitry, it also is providing new insights into the mechanisms of biological multi-enzyme pathways.

The Broader Impacts of this research include:
- 1Benefits to society:  The potential applications of engineered, self-assembled multi-enzyme systems are many and include: carbon dioxide fixation (bioenergy), high value compound synthesis (biomedicine, industrial chemistry), diagnostic applications (biomedicine), an chemical control processes.
- 2Broadening participation of underrepresented groups:  Through our participation in high school summer research programs, we reach out to young students of all ethnicities.
- 3Advancing discovery and understanding while promoting teaching, training, and learning:  This research provides an avenue for systematically exploring the role of geometric arrangement in enzyme complexes, an area that previously has been difficult to address. The researchers are involved in student training and outreach at all levels: ASU graduate students and undergraduate students, as well as high school students and their teachers in summer research experience programs.  Research within the Biodesign Institute at ASU exposes students to a highly interdisciplinary environment which involves people from different backgrounds including chemistry and biochemistry, engineering, physics, and computer and life sciences.  We anticipate that the interdisciplinary training opportunity made possible by this project will encourage a spectrum of creative thinking and inspire a greater interest in science and technology.
- 4Enhancing the infrastructure for research and education:  One of the primary goals of this project is to create a new toolset for arranging functional biological molecules with nanoscale accuracy.  This toolset should have broad application both in fundamental research and the design and fabrication of new molecular devices.
- 5Results disseminated broadly to enhance scientific and technological understanding:  The findings from this research are being disseminated broadly in international scientific journals as well as through presentations made at international conferences.

Program Director:
George Antos
CBET Program Director - Catalysis and Biocatalysis
NSF Award Number:   1033222
Award Title:   Enzymology of Multi-enzyme Systems on Self-assembled Surfaces
Principal Investigator:   Neal Woodbury
Institution Name:   Arizona State University
Program Element Code:   1401
CBET Research Highlight:   Fiscal Year 2012
Approved by CBET on:   23 March 2012

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This CBET Research Highlight was Updated on 9 April 2012.