Enhancing Mn(II)-Binding and Manganese Peroxidase Activity in a Designed Cytochrome c Peroxidase through Fine-Tuning Secondary-Sphere Interactions

Abstract

Non-covalent second shell interactions are important in controlling metal-binding affinity and activity in metalloenzymes, but fine-tuning these interactions in designed metalloenzymes has not been fully explored. As a result, most designed metalloenzymes have low metal-binding affinity and activity. Here we identified three mutations in the second coordination shell of an engineered Mn(II)-binding site in cytochrome c peroxidase (called MnCcP.1, containing Glu45, Glu37, and Glu181 ligands) that mimics the native manganese peroxidase (MnP) and explored their effects on both Mn(II)-binding affinity and MnP activity. First, removing a hydrogen bond to Glu45 through Tyr36Phe mutation, enhanced Mn(II)-binding affinity, as evidenced by a decrease of KM of Mn(II) oxidation 2.8 fold. Second, introducing a salt bridge through Lys179Arg improved Glu35 and Glu181 coordination of Mn(II), decreasing KM 2.6 fold. Third, eliminating a steric clash that prevented Glu37 from orienting towards Mn(II) resulted in an 8.6 fold increase in kcat/KM, arising primarily from a 3.6 fold decrease in KM, with a KM value comparable to that of the native enzyme (0.28 mM vs. 0.2 mM for Pleurotus eryngii MnP PS3). We further demonstrated that while the effects of Tyr36Phe and Lys179Arg mutations are additive, other combinations of mutations were antagonistic. Finally, we showed that these MnCcP variants are functional models of MnP that mimic its activity both in Mn(II) oxidation and degradation of a phenolic lignin model compound and kraft lignin. Our results offer molecular insight into the role of non-covalent interactions around metal binding sites for improving metal binding and overall activity; such insight can be applied to rationally enhance these properties in other metalloenzymes and their models.

Design and engineering of artificial oxygen-activating metalloenzymes

“…15). 238 First, removing a hydrogen bond to Glu45 through Tyr36Phe mutation, enhanced Mn(II)-binding affinity, as evidenced by a decrease of K M of Mn(II) oxidation 2.8 fold. Second, introducing a salt bridge through Lys179Arg improved Glu35 and Glu181 coordination of Mn(II), decreasing K M 2.6 fold.…”

“…A main contributing factor to both issues is the non-covalent secondary sphere interactions around the primary coordination sphere that influence both the metal-binding affinity and enzymatic efficiency. While more recent reports have begun to address this issue, 113,162,165,170,238 more attention needs to be paid in designing non-covalent secondary sphere interactions.…”

Design of Heteronuclear Metalloenzymes

“…positive charge in a certain position – and in those cases the residue that satisfies this feature and fits the design scaffold best should be chosen. This method has been successfully used to enhance the manganese peroxidase activity in a designed mimic called MnC c P (Hosseinzadeh et al, 2016). Similarly; it has been used to increase the potential of Fe/Mn superoxide dismutase (Miller, 2008).…”

“…For example, by incorporating a Gly in the loop that contains primary Mn-binding ligands into MnC c P and giving the ligands more flexibility, this designed mimic of MnP could achieve binding affinities for Mn comparable to the native system (Hosseinzadeh et al, 2016). Removing a disfavored hydrogen bond to another Mn ligand in the same mimic increased the activity and binding affinity (Hosseinzadeh et al, 2016). …”

“…The inclusion body protocol is used extensively in models such as Cu B Mb and Fe B Mb and has been covered previously in those works (Springer & Sligar, 1987, Sigman et al, 2000, Sigman et al, 2000, Sigman et al, 2003, Pfister et al, 2005, Zhao et al, 2005, Zhao et al, 2006, Yeung et al, 2009, Lin et al, 2010, Lin et al, 2010, Miner et al, 2012, Chakraborty et al, 2014, Bhagi-Damodaran et al, 2014, Petrik et al, 2016). Additionally, cytosolic purification of C c P and its rationally designed mutants has also been described in great detail by our group (Yeung et al, 1997, Sigman et al, 1999, Feng et al, 2003, Pfister et al, 2007, Miner et al, 2014, Hosseinzadeh et al, 2016). After purification, the protein mass is determined by SDS-PAGE (Brunelle & Green, 2014) and ESI-MS (Strupat, 2005).…”