Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and enzymatic function.

The lanthanide elements (Ln ³⁺ ), those with atomic numbers 57-63 (excluding promethium, Pm ³⁺ ), form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs), expanding the range of biological elements and opening novel areas of metabolism and ecology. Other MDHs, known as MxaFIs, are related in sequence and structure to these proteins, yet they instead possess a Ca ²⁺ -PQQ cofactor. An important missing piece of the Ln ³⁺ puzzle is defining what features distinguish enzymes that use Ln ³⁺ -PQQ cofactors from those that do not. Here, using XoxF1 MDH from the model methylotrophic bacterium Methylorubrum extorquens AM1, we investigated the functional importance of a proposed lanthanide-coordinating aspartate residue. We report two crystal structures of XoxF1, one with and another without PQQ, both with La ³⁺ bound in the active-site region and coordinated by Asp ³²⁰ Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp ³²⁰ is needed for in vivo catalytic function, in vitro activity, and La ³⁺ coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La ³⁺ , coordinated Ca ²⁺ but exhibited little or no catalytic activity. We also generated the parallel substitution in ExaF to produce ExaF D319S and found that this variant loses the capacity for efficient ethanol oxidation with La ³⁺ These results provide evidence that a Ln ³⁺ -coordinating aspartate is essential for the enzymatic functions of XoxF MDHs and ExaF EDHs, supporting the notion that sequences of these enzymes, and the genes that encode them, are markers for Ln ³⁺ metabolism.
Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.
(© 2020 Good et al.)