Structure-driven development of a biomimetic rare earth artificial metalloprotein.

Abstract

The 2011 discovery of the first rare earth-dependent enzyme in methylotrophic <i>Methylobacterium extorquens</i> AM1 prompted intensive research toward understanding the unique chemistry at play in these systems. This enzyme, an alcohol dehydrogenase (ADH), features a La³⁺ ion closely associated with redox-active coenzyme pyrroloquinoline quinone (PQQ) and is structurally homologous to the Ca²⁺-dependent ADH from the same organism. AM1 also produces a periplasmic PQQ-binding protein, PqqT, which we have now structurally characterized to 1.46-Å resolution by X-ray diffraction. This crystal structure reveals a Lys residue hydrogen-bonded to PQQ at the site analogously occupied by a Lewis acidic cation in ADH. Accordingly, we prepared K₁₄₂A- and K₁₄₂D-PqqT variants to assess the relevance of this site toward metal binding. Isothermal titration calorimetry experiments and titrations monitored by UV-Vis absorption and emission spectroscopies support that K₁₄₂D-PqqT binds tightly (<i>K</i>_d = 0.6 ± 0.2 μM) to La³⁺ in the presence of bound PQQ and produces spectral signatures consistent with those of ADH enzymes. These spectral signatures are not observed for WT- or K₁₄₂A-variants or upon addition of Ca²⁺ to PQQ ⸦ K₁₄₂D-PqqT. Addition of benzyl alcohol to La³⁺-bound PQQ ⸦ K₁₄₂D-PqqT (but not Ca²⁺-bound PQQ ⸦ K₁₄₂D-PqqT, or La³⁺-bound PQQ ⸦ WT-PqqT) produces spectroscopic changes associated with PQQ reduction, and chemical trapping experiments reveal the production of benzaldehyde, supporting ADH activity. By creating a metal binding site that mimics native ADH enzymes, we present a rare earth-dependent artificial metalloenzyme primed for future mechanistic, biocatalytic, and biosensing applications.