Abiological catalysis by myoglobin mutant with a genetically incorporated unnatural amino acid

Abstract

To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compound I in the catalytic oxidation process by participating in an additional hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The reduction potential values of WT (E° = - 260 mV) and mutant Mb (E° = - 300 mV), w.r.t. Ag/AgCl reference electrode, in the presence of hydrogen peroxide, indicated an additional H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We observed that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, respectively, as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochemical studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.

How to Turn an Electron Transfer Protein into a Redox Enzyme for Biosensing

“…The second strategy, which is applied in combination with the former, is based on the possibility of effectively modulating and controlling the reactivity of heme proteins by rationally designed mutations in key positions [ 32 , 33 , 34 , 35 , 36 ]. Of particular interest is the replacement of five-coordinated heme b -containing proteins (which feature an open heme coordination site for substrate binding) with engineered single and multi-heme cytochromes c as the core constituents of the hybrid sensing interfaces [ 8 , 9 , 15 , 37 , 38 , 39 , 40 ].…”

Noncanonical Amino Acids in Biocatalysis

“…NcAAs have also been introduced to the distal pocket of heme proteins to tune the properties of the heme cofactor and augment catalytic function. Mutation of the distal pocket His64 of Mb to DOPhe led to a 54- and 10-fold increase in catalytic rate toward benzaldehyde oxidation and thioanisole sulfoxidation, respectively . These improvements were proposed to arise from increased stabilization of the ferryl intermediate compound I from additional hydrogen bonding interactions in the His64DOPhe mutant, with the ncAA performing the hydrogen bonding role of the distal pocket His/Arg residues that are conserved in natural peroxidases.…”

Synthetic Biology: Bottom-Up Assembly of Molecular Systems

“…Through incorporation of non-native reactive groups, such as ketones, proteins could be selectively targeted even in vivo by suitable chemical probes, such as hydrazine derivatives of fluorescent dyes . A wide variety of chemistries has since been introduced into proteins, including ring-substituted aromatics, − halogenated derivatives, − β- and γ-amino acids, − photo- − or redox-reactive − groups, metal binding moieties, − fluorescent probes, − and many more . The introduction of p -aminophenylalanine into the Lactococcus lactis multidrug resistance regulator protein significantly increased the hydrazine and oxime formation and showcases how catalytic activities can be improved using UAAs .…”