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

https://doi.org/10.1074/jbc.RA120.013227Get rights and content
Under a Creative Commons license
open access

The lanthanide elements (Ln3+), those with atomic numbers 57–63 (excluding promethium, Pm3+), 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 Ca2+-PQQ cofactor. An important missing piece of the Ln3+ puzzle is defining what features distinguish enzymes that use Ln3+-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 La3+ bound in the active-site region and coordinated by Asp320. Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp320 is needed for in vivo catalytic function, in vitro activity, and La3+ coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La3+, coordinated Ca2+ 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 La3+. These results provide evidence that a Ln3+-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 Ln3+ metabolism.

one-carbon metabolism
metalloprotein
alcohol dehydrogenase (ADH)
crystallography
aspartate (aspartic acid)
cofactor coordination
ExaF
lanthanide
methanol dehydrogenase
XoxF
ethanol dehydrogenase
pyrroloquinoline quinone

Cited by (0)

This article contains supporting information.

Author contributions—N. M. G. and N. C. M.-G. conceptualization; N. M. G. and M. F. data curation; N. M. G., M. F., J. H., R. P. H., and N. C. M.-G. formal analysis; N. M. G., J. H., R. P. H., and N. C. M.-G. supervision; N. M. G. and M. F. validation; N. M. G., M. F., and K. D. investigation; N. M. G. and M. F. visualization; N. M. G. and M. F. methodology; N. M. G. writing-original draft; N. M. G., M. F., K. D., J. H., R. P. H., and N. C. M.-G. writing-review and editing; M. F., J. H., R. P. H., and N. C. M.-G. funding acquisition; J. H., R. P. H., and N. C. M.-G. project administration.

Funding and additional information—This material is based upon work supported by the National Science Foundation under Grants 1750003 (to N. C. M.-G. and N. M. G.) and CHE-1516126 (to R. P. H. and J. H.). M. F. was supported by University of Otago Health Sciences Postdoctoral Fellowship HSCDPD1703.

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

    PQQ

    pyrroloquinoline quinone

    DFT

    density functional theory

    MDH

    methanol dehydrogenase

    EDH

    ethanol dehydrogenase

    ADH

    alcohol dehydrogenase

    Ln-ADH

    Ln3+-dependent alcohol dehydrogenase

    IMAC

    immobilized metal affinity chromatography

    RMSD

    root mean square deviation

    PDB

    Protein Data Bank

    ANOVA

    analysis of variance

    PMS

    phenazine methosulfate

    DCPIP

    2,6-dichlorophenol indophenol

    ICP

    inductively coupled plasma

    OES

    optical emission spectroscopy

    OD

    optical density

    RMSE

    root mean square error

    TEV

    tobacco etch virus.

These authors contributed equally to this work.