Final Report Summary - MEDENZYMEDESIGN (Enzyme Design of Medical Interest)
Enzymes are the most efficient, specific and selective catalysts known up to date. Despite the enzyme advantages, not all synthetic processes present a natural enzyme to catalyze and accelerate the reactions. Hence, the design of new stable enzymes for those processes where no biocatalyst is known represents a major challenge for protein engineering and a stringent test to understand how natural enzymes work. In addition to that, the ability of designing specific active enzymes is of great interest due to the potential applications in biotechnology, biomedicine and industrial processes. In this proposal, the design of three enzymes of biological/medical interest was proposed. First, an enzyme to reverse the formation of Advanced Glycation End-Products (AGEs), mainly associated to diabetes-related disorders, but also to Alzheimer’s disease was suggested. Second, the design of an enzyme presenting Glucose-6-Phosphate Dehydrogenase (G6PD) activity was pursued. The latter might avoid the oxidative stress induced by many drugs in G6PD-deficient persons. Finally, the third objective of this proposal was the design of an enzyme with superoxide dismutase activity to avoid the oxidative stress produced in most of neurodegenerative diseases (i.e. Alzheimer’s, Parkinson’s, Huntington’s disorders). All designs were performed following the research methodology developed by the Prof. Houk group, which was already successfully applied for the design of active Kemp elimination, retro-aldolase and Diels-Alder enzymes. However, new QM/MM-MD strategies and DFT functionals developed at the outgoing host organization (IQCC) were introduced to improve some parts of the design process. The fellow researcher has had the chance to work on this pioneer project in a world leading research group and to transfer this knowledge to one of the best EU research institutes.
The initial design of an enzyme for reversing the formation of AGEs led to the development of an enzyme used in the production of the type 2 anti-diabetes drug known as Sitagliptin (Januvia®). The actual method of production is far from ideal because of its inadequate stereoselectivity and product contamination with the rhodium catalyst. We applied our inside-out computational protocol for designing a new transaminase for the production of sitagliptin. We established a collaboration with the biotechnology company Codexis for the experimental validation of the theoretical predictions. The application of the inside-out protocol in a case where an engineered enzyme was created with directed evolution (DE) was highly appealing and could provide important clues about the modest acceleration that computer created enzymes usually provide. The application of the computational protocol to this target was very challenging as the reaction proceeds through many transition states and intermediates, and unfortunately the computational designs did not improve upon the first mutant generated using the experimental technique called directed evolution. The low efficiency of the inside-out protocol in designing more challenging targets is not unexpected, as even in simple transformations like designing a mimic of chorismate mutase the protocol is unsuccessful.
In this direction, we started another collaboration with leader biochemistry (Prof. Y. Tang group) and crystallography groups at UCLA (Prof. T. Yeates group) to design new variants for the manufacture of the cholesterol-lowering drug Zocor® (Lovastatin acid is the active ingredient) and also to computationally evaluate the different generated mutants created by DE by the Codexis company. We used the Anton supercomputer and demonstrated the paramount importance of long MD simulations in the microsecond time scale to unveil the basis of improved catalysis achieved via DE. In this collaboration we aimed to understand the reason for the low efficiency of the computationally designed enzymes (kcat/kuncat values that range from 10^2 to 10^5 in contrast with average kcat/kuncat of ~10^11 for natural enzymes) to make the computational approach comparable to the expensive directed evolution technique. Our Anton simulations indicated that the improved catalysis obtained via directed evolution is achieved by a better pre-organization of the polar environment of the enzyme (i.e. the catalytic triad).
For the design of a small monomeric Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme and the SuperOxide Dismutase (SOD) enzyme, the initial steps of the computational protocol were accomplished during the first and last year of the fellowship, respectively. However, all our efforts to incorporate either a cofactor or a metal binding site in our designed enzymes were unsuccessful. Instead, we proposed some corrective actions which involve the re-design of natural NADP-dependent enzymes for presenting G6P dehydrogenase activity. We also performed some MD simulations to give some insight into the natural mechanism of the reactions and explore the role of certain mutations on the catalytic activity of the enzymes.
One of the main objectives of Horizon 2020 is to contribute to funding research related to the sustainable development for an efficient use of resources. This proposal was based on applying the current inside-out computational protocol for designing new biocatalysts of medical relevance. Apart from all the synthetic advantages of enzymes, biocatalysts are biodegradable, non-toxic, and their high selectivities and efficiencies reduce the number of work-up steps. They usually operate at ambient temperature, atmospheric pressure and neutral pH. Biocatalytic processes are therefore an attractive alternative due to their environmental advantages for chemical manufacturing. The successful development of a new and more potent computational protocol for designing new biocatalysts will in the near future contribute to enhance the attractiveness of the biocatalysts use in industry. This proposal was therefore also in line with the strategic research agenda of the European Technology Platform for Sustainable Chemistry.
The initial design of an enzyme for reversing the formation of AGEs led to the development of an enzyme used in the production of the type 2 anti-diabetes drug known as Sitagliptin (Januvia®). The actual method of production is far from ideal because of its inadequate stereoselectivity and product contamination with the rhodium catalyst. We applied our inside-out computational protocol for designing a new transaminase for the production of sitagliptin. We established a collaboration with the biotechnology company Codexis for the experimental validation of the theoretical predictions. The application of the inside-out protocol in a case where an engineered enzyme was created with directed evolution (DE) was highly appealing and could provide important clues about the modest acceleration that computer created enzymes usually provide. The application of the computational protocol to this target was very challenging as the reaction proceeds through many transition states and intermediates, and unfortunately the computational designs did not improve upon the first mutant generated using the experimental technique called directed evolution. The low efficiency of the inside-out protocol in designing more challenging targets is not unexpected, as even in simple transformations like designing a mimic of chorismate mutase the protocol is unsuccessful.
In this direction, we started another collaboration with leader biochemistry (Prof. Y. Tang group) and crystallography groups at UCLA (Prof. T. Yeates group) to design new variants for the manufacture of the cholesterol-lowering drug Zocor® (Lovastatin acid is the active ingredient) and also to computationally evaluate the different generated mutants created by DE by the Codexis company. We used the Anton supercomputer and demonstrated the paramount importance of long MD simulations in the microsecond time scale to unveil the basis of improved catalysis achieved via DE. In this collaboration we aimed to understand the reason for the low efficiency of the computationally designed enzymes (kcat/kuncat values that range from 10^2 to 10^5 in contrast with average kcat/kuncat of ~10^11 for natural enzymes) to make the computational approach comparable to the expensive directed evolution technique. Our Anton simulations indicated that the improved catalysis obtained via directed evolution is achieved by a better pre-organization of the polar environment of the enzyme (i.e. the catalytic triad).
For the design of a small monomeric Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme and the SuperOxide Dismutase (SOD) enzyme, the initial steps of the computational protocol were accomplished during the first and last year of the fellowship, respectively. However, all our efforts to incorporate either a cofactor or a metal binding site in our designed enzymes were unsuccessful. Instead, we proposed some corrective actions which involve the re-design of natural NADP-dependent enzymes for presenting G6P dehydrogenase activity. We also performed some MD simulations to give some insight into the natural mechanism of the reactions and explore the role of certain mutations on the catalytic activity of the enzymes.
One of the main objectives of Horizon 2020 is to contribute to funding research related to the sustainable development for an efficient use of resources. This proposal was based on applying the current inside-out computational protocol for designing new biocatalysts of medical relevance. Apart from all the synthetic advantages of enzymes, biocatalysts are biodegradable, non-toxic, and their high selectivities and efficiencies reduce the number of work-up steps. They usually operate at ambient temperature, atmospheric pressure and neutral pH. Biocatalytic processes are therefore an attractive alternative due to their environmental advantages for chemical manufacturing. The successful development of a new and more potent computational protocol for designing new biocatalysts will in the near future contribute to enhance the attractiveness of the biocatalysts use in industry. This proposal was therefore also in line with the strategic research agenda of the European Technology Platform for Sustainable Chemistry.