Hydroxylase Activity of Met471Cys Tyramine β-Monooxygenase

Abstract

A series of mutations was targeted at the methionine residue, Met471, coordinating the Cu(M) site of tyramine beta-monooxygenase (TbetaM). The methionine ligand at Cu(M) is believed to be key to dioxygen activation and the hydroxylation chemistry of the copper monooxygenases. The reactivity and copper binding properties of three TbetaM mutants, Met471Asp, Met471Cys, and Met471His, were examined. All three mutants show similar metal binding affinities to wild type TbetaM in the oxidized enzyme forms. EPR spectroscopy suggests that the Cu(II) coordination geometry is identical to that of the WT enzyme. However, substrate hydroxylation was observed for the reaction of tyramine solely with Met471Cys TbetaM. Met471Cys TbetaM provides the first example of an active mutant directed at the Cu(M) site of this class of hydroxylases. The reactivity and altered kinetics of the Met471Cys mutant further highlight the central role of the methionine residue in the enzyme mechanism. The sole ability of the cysteine residue to support activity among the series of alternate amino acids investigated is relevant to theoretical and biomimetic investigations of dioxygen activation at mononuclear copper centers.

Interdomain Long-Range Electron Transfer Becomes Rate-Limiting in the Y216A Variant of Tyramine β-Monooxygenase

“…Product standard curves were generated as previously described using solutions that contained varying amounts of octopamine (50 μM–2 mM) and are in good agreement with published results (Figure S2 in Supporting Information) (29). The fit of the octopamine standard curve generated from the integrated peak area (224 nm) was used to quantify the amount of product generated in assay mixtures.…”

“…Details for the removal of the His-tag and for the expression and purification of TβM lacking the His-tag are identical to that reported previously (14). The details for copper reconstitution for oxidized and reduced Y216 TβM variant samples are also identical to previously published protocols (29). …”

Dioxygen Activation by Mononuclear Copper Enzymes: Insights from a Tripodal Ligand Mimicking Their CuM Coordination Sphere

“…11 This novel tripodal ligand will also be used in future computational studies, in particular to address more deeply the role of the thioether group. 12 Such investigations are in progress and will be reported in due course.…”

Effects of copper occupancy on the conformational landscape of peptidylglycine α-hydroxylating monooxygenase

“…The structures of the PHM and PAL domains of rat PAM, alone and in complexes with substrates, inhibitors, and other ligands, have been determined 11–15 . Other techniques used to study PHM have ranged from kinetics and kinetic isotope effect measurements 16–20 , to inhibitor design 21–24 , spectroscopy 25–29 including X-ray absorption spectroscopy (XAS) 17,30–33 , and computational studies 11,34–36 . These studies have provided significant insights into the mechanism of both domains, especially of PHM.…”