How is cofactor specificity determined in metalloenzymes?
Lead Research Organisation:
Newcastle University
Department Name: Inst for Cell and Molecular Biosciences
Abstract
Essential metal ions are required for the function of nearly half of all enzymes. Most metalloenzymes are highly specific for their cognate metal, exhibiting reduced or abrogated activity when loaded with alternative metal ions. This is especially true for the subset of metalloenzymes that utilise a redox-active metal cofactor such as manganese (Mn) or iron (Fe). This specificity has been proposed to be due to 'redox tuning', whereby the structural architecture of the active site within the folded protein precisely controls the redox potential of the metal ion to optimise reactivity. This redox tuning concept has a long history in the literature, but there are few structural clues from any metalloenzyme as to how it is achieved.
In a recent study, a BBSRC DTP-funded PhD student in the Waldron lab identified a pair of highly related (75% sequence identity) superoxide dismutase (SOD) enzymes that show disparate metal specificities. Staphylococcus aureus SodA is a classical Mn-dependent SOD, whereas its paralogue SodM is 'cambialistic', i.e. it's equally active with either Mn or Fe cofactors (Garcia et al, 2017, PLoS Pathog. 13:e1006125).
The close sequence homology but disparate specificities make this an ideal model system to investigate how redox tuning is achieved at atomic resolution. A limited mutagenesis study (manuscript in preparation for Nature Chemical Biology) in which evolutionarily-selected mutations located spatially close to the active site were swapped between SodA and SodM identified three residues that play a key role in determining cofactor specificity, but these mutations are insufficient to fully explain the enzymes' differing metal specificities. An electron paramagnetic resonance (EPR) spectroscopy study of the two wild type proteins, in collaboration with Sun Un (CEA Saclay, Paris) (Barwinska-Sendra et al, 2018, Phys Chem Chem Phys 20:2363) has identified other residues that play a role in modulating the reduction potential of the catalytic centre.
In this project we will use targeted mutagenesis, based on our existing crystal structures of SodA and SodM, to identify which residues control metal specificity. We will solve crystal structures for all resulting mutant variant enzymes with Dr Pohl, and will also study the electronic structure of their active sites by EPR and determine their reduction potentials via existing collaborations with Un (above) and Dr Alison Parkin (York). Collectively, these data will determine how subtle changes in the active site structure, through changes in the metal's secondary coordination sphere, control the metal's reactivity through redox tuning.
This project will combine core training in biochemistry, molecular biology and microbiology (Waldron) with cutting-edge biophysical studies with each of the collaborative team. This will include X-ray crystallographic protein structure determination (Pohl), electronic spectroscopy using cutting-edge EPR technology (Un), and advanced techniques for determination of metalloenzymes' reduction potentials (Parkin). The student will thus develop a multi-disciplinary skill set that can be applied to a wide spectrum of bioscience-relevant careers.
Given that approximately one-half of all enzymes require a metal ion, the results of this study will be far-reaching. This project will develop our understanding of how metalloenzymes are optimised for their cognate metal cofactor, which will in turn advance our capability to modify metalloenzymes rationally. Numerous attempts have been made to develop new synthetic metalloenzymes with novel, non-natural catalytic functions, an area where this project will be extremely relevant. In addition, attempts to utilise high-value metalloenzymes in synthetic biology applications will be dependent on the ability of those metalloenzymes to acquire their metal within the heterologous chassis. Thus this project is relevant to the World Class Underpinning Bioscience priority.
In a recent study, a BBSRC DTP-funded PhD student in the Waldron lab identified a pair of highly related (75% sequence identity) superoxide dismutase (SOD) enzymes that show disparate metal specificities. Staphylococcus aureus SodA is a classical Mn-dependent SOD, whereas its paralogue SodM is 'cambialistic', i.e. it's equally active with either Mn or Fe cofactors (Garcia et al, 2017, PLoS Pathog. 13:e1006125).
The close sequence homology but disparate specificities make this an ideal model system to investigate how redox tuning is achieved at atomic resolution. A limited mutagenesis study (manuscript in preparation for Nature Chemical Biology) in which evolutionarily-selected mutations located spatially close to the active site were swapped between SodA and SodM identified three residues that play a key role in determining cofactor specificity, but these mutations are insufficient to fully explain the enzymes' differing metal specificities. An electron paramagnetic resonance (EPR) spectroscopy study of the two wild type proteins, in collaboration with Sun Un (CEA Saclay, Paris) (Barwinska-Sendra et al, 2018, Phys Chem Chem Phys 20:2363) has identified other residues that play a role in modulating the reduction potential of the catalytic centre.
In this project we will use targeted mutagenesis, based on our existing crystal structures of SodA and SodM, to identify which residues control metal specificity. We will solve crystal structures for all resulting mutant variant enzymes with Dr Pohl, and will also study the electronic structure of their active sites by EPR and determine their reduction potentials via existing collaborations with Un (above) and Dr Alison Parkin (York). Collectively, these data will determine how subtle changes in the active site structure, through changes in the metal's secondary coordination sphere, control the metal's reactivity through redox tuning.
This project will combine core training in biochemistry, molecular biology and microbiology (Waldron) with cutting-edge biophysical studies with each of the collaborative team. This will include X-ray crystallographic protein structure determination (Pohl), electronic spectroscopy using cutting-edge EPR technology (Un), and advanced techniques for determination of metalloenzymes' reduction potentials (Parkin). The student will thus develop a multi-disciplinary skill set that can be applied to a wide spectrum of bioscience-relevant careers.
Given that approximately one-half of all enzymes require a metal ion, the results of this study will be far-reaching. This project will develop our understanding of how metalloenzymes are optimised for their cognate metal cofactor, which will in turn advance our capability to modify metalloenzymes rationally. Numerous attempts have been made to develop new synthetic metalloenzymes with novel, non-natural catalytic functions, an area where this project will be extremely relevant. In addition, attempts to utilise high-value metalloenzymes in synthetic biology applications will be dependent on the ability of those metalloenzymes to acquire their metal within the heterologous chassis. Thus this project is relevant to the World Class Underpinning Bioscience priority.
