Acceleration and control of spin-restricted oxygenation by cofactor-independent dioxygeanses

Lead Research Organisation: King's College London
Department Name: Randall Div of Cell and Molecular Biophy

Abstract

Humans like all other highly evolved organisms strictly depend on atmospheric oxygen for survival. Oxygen obtained via the respiration process is essential for the production of energy required to carry out our physiological functions as well as for the defence against various kinds of infections. Oxygen is also used for the degradation of various organic compounds and some bacteria use it to help breakdown molecules that are environmental pollutants. This is thanks to the action of particular enzymes, called oxygenases, which are able to promote reactions in which oxygen atoms are incorporated into molecules otherwise difficult to dispose of. The task of oxygenases is a difficult one because oxygen in its normal 'resting' state (the form present in the air) does not want to react with the vast majority of molecules for reasons related to its electronic structure. Oxygen needs activation to react. A major problem, however, is that once 'activated' oxygen can react indiscriminately with many biological molecules with detrimental consequences. For example, reactive oxygen species (ROS) are damaging forms of 'active oxygen' that play an important role in aging. Therefore, besides the generation of 'active oxygen', another challenge in oxygen biochemistry, is its control. ACTIVATION and CONTROL are critical keywords in oxygen-dependent biological processes. In this work we will investigate two bacterial oxygenases called with the acronyms of HOD and QDO which constitute a separate family from other oxygenases. Interestingly, they can bring oxygen into reactions with their organic substrates (ACTIVATION) and steer the reaction towards the desired products (CONTROL) with limited tools at their disposals. In fact, as oxygen activation is not an easy task, the vast majority of oxygenases rely on special additional components like metal and/or organic co-factors to form 'active oxygen'. HOD and QDO don't possess these additional features and therefore understanding how they work is particularly intriguing. Using a technique called X-ray crystallography which allows us to visualise at very high resolution the 3D structure of molecules as small as HOD and QDO (they are about ten thousand times smaller that the thickness of a human's hair) we now know in detail the shape of these enzymes. They do not look like other known dioxygenases; rather they have an architecture of another enzyme family which typically catalyses reactions not involving oxygen. Using the same X-ray technique we have also seen where the substrate binds to HOD when oxygen is not around and what specific interactions it makes with the enzyme. Similarly, we have seen how the reaction product is bound before leaving the enzyme for a new reaction cycle. These snapshots led us to formulate some hypotheses on how HOD/QDO work. We are now in an excellent position to study the most interesting aspects of how HOD/QDO work. These are on one hand the steps in which oxygen gets ACTIVATED and CONTROLLED to convert the substrate into products and on the other hand the reasons which allow a protein scaffold used typically for different reactions to be used here to host oxygen biochemistry. We will again use X-ray crystallography to visualise oxygen bound to the these enzymes, modern spectroscopic techniques to study important electronic properties at different stages of the reaction cycle, and advanced quantum mechanical theoretical methods to probe states that are not experimentally accessible. This multi-angle approach will allow novel insights into the biology of oxygen, an essential component of life on Earth.

Technical Summary

The classical conundrum in oxygen chemistry relates to the mechanism by which singlet-state organic molecules are made to react with the triplet-state molecular oxygen to produce singlet-state products circumventing the quantum chemical spin-restriction rule. The large majority of oxygenases use transition metal or organic co-factors for this purpose. These helpers allow electron shuttling engendering radical species that can react with dioxygen or directly activate it. A number of oxygenases and oxidases operate, however, in a cofactor-independent manner relying on very limited chemical tools to activate O2 for the oxygenolysis of their organic substrates. To gain an understanding of how dioxygen chemistry takes place in a cofactor-less manner we will use Arthrobacter nitroguajacolicus Rü61a 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) and Pseudomonas putida 33/1 1-H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO), for which we have recently obtained structural information in various catalytically relevant states. Interestingly, HOD and QDO adopt an alpha/beta-hydrolase fold. Albeit functionally heterogeneous, alpha/beta-hydrolase fold enzymes do not typically catalyze oxygenation processes. Employing an integrated approach combining X-ray crystallography, molecular dynamics, fast-reaction kinetics, electron spin resonance, and density functional theory calculations we aim at establishing how molecular oxygen is activated and its reactivity controlled in the absence of metal centres within a protein architecture generally employed to catalyze hydrolytic reactions rather than oxygenolytic ones.

Planned Impact

Catalysis and enzymes are central to life systems. Our understanding of catalysis underpins the exploitation of enzymes in biotechnology, through rational structure-based redesign and for therapeutic targeting of enzymes to maintain healthy physiological function in humans. Current research indicates that besides three-dimensional structure, dynamical properties and quantum effects also play a critical role in modulating enzymes' function, particularly in the case of redox processes. These higher-level mechanisms of control transcend a simplistic view of biological catalysis based purely on enzymes' architecture and their functional importance warrants that they should be explored in depth because, if we do not do so, we are not only left with a limited understanding of the natural world but also with the potential inability to exploit them for practical applications. Our multi-disciplinary approach to the study HOD/QDO cofactor-independent dioxygenases addresses the complex nature of biological catalysis in full, by looking not only at architectural properties, clearly of paramount importance, but also at the interplay between these and protein dynamics, preferential gas diffusion pathways and quantum chemistry. This in-depth mechanistic exploration coupled with the advantageous physicochemical properties of these enzymes might result in novel ideas for the design of robust oxidation catalysis. From the perspective of the PDRAs employed on this project, the training they will gain from this multi-disciplinary research, which combines X-ray structural studies, molecular dynamics, fast-reaction kinetics, EPR spectroscopy and quantum chemical calculations, will equip them with a rare and sought-after skill-set as well as a comprehensive overview of an inter-disciplinary study. This will enable them to make a valuable and practical contribution to the continued growth of molecular enzymology activities in the UK. Beyond these specific scientific skills, the coordination of this multi-disciplinary research project will give the individuals invaluable experience in a number of areas applicable to much of the employment sector. These include: people and time management; budgeting; responsible and thorough communication of results and ideas; coordination of personnel with a wide variety of expertise and interests in achieving a common aim. King's and Manchester will take advantage of various opportunities to disseminate our work together. For example, King's has an active role in the Aspire (Aimhigher South East London) Programme of Widening Participation events. Manchester will use the 'Discover days' hosted by the Faculty of Life Sciences to introduce school children to the science underpinning biological catalysis. This will be in addition to planned lectures at regional schools. King's College London has been awarded the title of 'Sunday Times University of the Year 2010/2011' in recognition of all-round excellence, encompassing a range of aspects of research, teaching, and the student experience. At King's we strive at maximising impact in our activities and a number of schemes have been introduced to facilitate this goal. Steiner has been nominated Health Innovation Fellow for the Randall Division. As Innovation Fellow, Steiner works with King's Business to support the development of contacts with industry and is involved in the School's bid in the 'BBSRC Excellence with Impact' scheme. Scrutton is currently Dean of Research in the Faculty of Life Sciences (FLS) at the University of Manchester and Director of the MIB, and is intimately involved in Faculty policy regarding the communication of research including the Faculty's bid in the 'BBSRC Excellence with Impact' scheme. All applicants have experience in communicating (in both written and oral presentations) complex, physical concepts to the general public.

Publications

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Description Humans like all other highly evolved organisms strictly depend on atmospheric oxygen for survival. Oxygen obtained via the respiration process is essential for the production of energy required to carry out our physiological functions as well as for the defence against various kinds of infections. Oxygen is also used for the degradation of various organic compounds and some bacteria use it to help breakdown molecules that are environmental pollutants. This is thanks to the action of particular enzymes, called oxygenases, which are able to promote reactions in which oxygen atoms are incorporated into molecules otherwise difficult to dispose of. The task of oxygenases is a difficult one because oxygen in its normal
"resting" state (the form present in the air) does not want to react with the vast majority of molecules for reasons related to its electronic structure. Oxygen needs activation to react. A major problem, however, is that once "activated" oxygen can react indiscriminately with many biological molecules with detrimental consequences. For example, reactive oxygen species (ROS) are damaging forms of "active oxygen" that play an important role in aging. Therefore, besides the generation of "active oxygen", another challenge in oxygen biochemistry, is its control. ACTIVATION and CONTROL are critical keywords in oxygen-dependent biological processes.

In this work we have investigated the bacterial oxygenase called with the acronym of HOD, a member of a separate family from other oxygenases. Interestingly, HOD can bring oxygen into reacting with its organic substrates (ACTIVATION) and steer the reaction towards the desired products (CONTROL) with limited tools at its disposals. In fact, as oxygen activation is not an easy task, the vast majority of oxygenases rely on special additional components like metal and/or organic co-factors to form "active oxygen". HOD does not possess these additional features and therefore understanding how it works is particularly intriguing. In this work we have addressed fundamental and contemporary questions in oxygen-dependent enzymology that afforded a profound understanding of cofactor-independent oxygenation.
Exploitation Route Our work has afforded important information on how dioxygenation can happen in the absence of complex co-factors. This in-depth mechanistic exploration coupled with the advantageous physicochemical properties of these enzymes might result in novel ideas for the design of robust oxidation catalysis.
Sectors Manufacturing, including Industrial Biotechology

 
Title 4CFS 
Description PDB model 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Researchers can freely download this protein stucture model and use it for their work 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4CFS
 
Title 4CW0 
Description PDB model 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Researchers can freely download this protein stucture model and use it for their work 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4CW0
 
Title 4CW2 
Description PDB file 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Researchers can freely download this protein stucture model and use it for their work 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4CW2
 
Title 4CW3 
Description PDB file 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Researchers can freely download this protein stucture model and use it for their work 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4CW3
 
Title 4CW6 
Description PDB file 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Researchers can freely download this protein stucture model and use it for their work 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4CW6
 
Title 4D12 
Description PDB file 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Researchers can freely download this protein stucture model and use it for their work 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4D12
 
Title 4D13 
Description PDB file 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Researchers can freely download this protein stucture model and use it for their work 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4D13
 
Title 4D17 
Description PDB model 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Researchers can freely download this protein stucture model and use it for their work 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4D17
 
Title 4D19 
Description PDB file 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Researchers can freely download this protein stucture model and use it for their work 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4D19