Alcohol dehydrogenase D from Pyrococcus furiosus is a thermostable rare earth element binding enzyme: Characterization of metal-binding and chromatographic separation of critical materials.

Abstract

Metal ions are essential in biological systems, serving structural and signaling functions, and acting as enzyme cofactors. The d-block transition metals are most commonly found in metalloenzymes, however, recent discoveries have highlighted the biological relevance of the rare earth elements (REEs). Despite growing evidence of lanthanide utilization in biology, our understanding of REE handling and function, especially beyond pyrroloquinoline quinone (PQQ)-dependent enzymes in methanotrophic systems, remains limited. In this work, we investigated and biochemically characterized the REE-binding properties of alcohol dehydrogenase D (AdhD), a thermostable aldo-keto reductase (AKR) from Pyrococcus furiosus. We identified and characterized a high-affinity REE binding site with low micromolar affinity, consistent with bioavailable free REE concentrations in the Pyrococcus furiosus natural niche, near geothermal marine sediments. Taking advantage of the REE binding site, we developed an AdhD-based, citrate-assisted REE separation protocol. This purification strategy provides an efficient, single-stage purification platform with high resolution to separate REEs from common divalent contaminants, and it can resolve heavy REEs from each other as an alternative to traditional solvent extraction-based purifications. This strategy offers an alternative to conventional, environmentally detrimental separation methods. These findings expand the understanding of REE binding in noncanonical systems, and showcases AdhD as a robust, thermostable scaffold for REE separations.