Why does Nature use modular enzyme architectures for biological catalysis?

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This proposal brings together a multi-disciplinary strength of Liverpool and Manchester to address a serious gap in our knowledge of some of the fundamental processes that underpin catalysis by redox enzymes involving proton-electron transfer. We will pursue an integrated programme drawing on the unique collaborative expertise formed by the applicants with expertise in biophysical, kinetic and enzymology studies (Manchester) and metalloenzyme crystallography, SFX and SEC-SAXS (Liverpool). Together we have published some significant papers on NiRs: JBC (2009), PNAS (2011), Biochemistry (2011) & Nature Comm (2014). Crucially, we provided recent evidence for long-range effects on enzyme mechanism resulting from mutations remote from the catalytic site, thus showing that efficient catalysis is controlled by basic design features of a number of elements that are inter-linked. Such knowledge is needed to design/redesign catalytic modules for synthetic biology in a predictable way.

We have invested significantly in obtaining important supporting data for this proposal. Key elements that make this proposal timely are (i) the development of new FRET-stopped flow approaches engineering to follow dynamics; (ii) discovery of 4-domain NiRs with additional cupredoxin and cytochrome domains fused to the core Cu1-Cu2 NiR, (iii) atomic structures of two members of cupredoxin-Cu1-Cu2NiR tethered complexes; (iv) supporting molecular biology/biochemical studies that enable expression and mutagenesis of most of the target proteins; (v) continued development of Liverpool's pioneering SEC-SAXS-DLS (size-exclusion chromatography-small angle X-ray scattering-dynamic light scattering) facility for use with proteins and their complexes. Our programme will provide deep understanding of how coordinated protein movements impact on (i) mechanisms of long-range ET, ii) localised chemical change (bond formation / breakage) and (iii) the utilization of coupled redox sites in a catalytic core.

Beneficiaries. The beneficiaries of the research program are academic and industrial scientists within the international scientific community, as a strong knowledge base for predictive design of redox components will inform on future exploitation of these parts in synthetic biology and biotechnology. There is also a strong enabling methods theme in the program, which will benefit the wider dynamic structural science communities and time resolved spectroscopy community. We already engage strongly with instrument manufacturers, assisting them in the authoring of specialised application notes and adapting instruments for new capabilities by hosting industrial colleagues in our groups to assist in technology development. This should facilitate commercial developments from basic science research. We will continue to operate in this way with the explicit aim of enhancing laser spectroscopy and structural biology applications, and in developing wider appreciation of our novel laboratory-based methods (e.g. SEC-SAXS) being developed with both academic and commercial users.

Internationalisation. There is a strong international aspect to our program. We have teamed up with expert scientists at international facilities and we are working with technical scientists at Bruker Karlsruhe (Germany). This will enhance links with expert researchers in these areas, enabling exchange of research scientists (Bruker has established strong connections within the national EPR centre at Manchester). Extended visits by the PDRA/applicants to these groups will facilitate import of the knowledge back to the UK.
We will establish a series of workshops/symposia that will enable us to develop further links with international groups in the area of redox enzymes, structural biology, XFEL and predictive design for synthetic biology thereby facilitating better integration of their expertise in the UK research programs. These will take place at Liverpool and Manchester and we will also contribute to workshops at national/international facilities and BBSRC SBRC workshops.

Outreach. We will take advantage of the 'Discover days' hosted by the Faculty of Life Sciences at Manchester to introduce school children to the science underpinning biological catalysis. This is addition to our planned lectures at regional schools and other outreach activities (podcasts and participation in the annual MIB schools open day). Hasnain and Scrutton have, and continue to lecture to schoolchildren at science focus meetings on a variety of scientific topics. We will exhibit at both regional and national science exhibitions.

Communication. We will communicate and develop our infrastructure and approaches through frequent networking events with external stakeholders at structured workshops, showcase events and industry focussed meetings within the MIB (Manchester) and Life Sciences (Liverpool). In particular, we are developing new biophysical capabilities of broad interest to the academic and commercial communities. We will host a major symposium focused on the power of integrating biophysical and structural methods in the field of dynamic structural science. An important aspect here will be training and scientific development of younger workers. Our PDRAs will engage in science exhibitions and public lectures such as Science and Society Lectures at Liverpool and Manchester Science Festival, and we hope (with the applicants) at a Royal Society summer science exhibition.