A Catalytic Role of XoxF1 as La3+-Dependent Methanol Dehydrogenase in Methylobacterium extorquens Strain AM1

Abstract

In the methylotrophic bacterium Methylobacterium extorquens strain AM1, MxaF, a Ca2+-dependent methanol dehydrogenase (MDH), is the main enzyme catalyzing methanol oxidation during growth on methanol. The genome of strain AM1 contains another MDH gene homologue, xoxF1, whose function in methanol metabolism has remained unclear. In this work, we show that XoxF1 also functions as an MDH and is La3+-dependent. Despite the absence of Ca2+ in the medium strain AM1 was able to grow on methanol in the presence of La3+. Addition of La3+ increased MDH activity but the addition had no effect on mxaF or xoxF1 expression level. We purified MDH from strain AM1 grown on methanol in the presence of La3+, and its N-terminal amino acid sequence corresponded to that of XoxF1. The enzyme contained La3+ as a cofactor. The ΔmxaF mutant strain could not grow on methanol in the presence of Ca2+, but was able to grow after supplementation with La3+. Taken together, these results show that XoxF1 participates in methanol metabolism as a La3+-dependent MDH in strain AM1.

Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth

“…Strains lacking mxaF were unable to grow because the Ca-dependent MeDH had been eliminated, while strains lacking both xoxF1 and xoxF2 were unable to grow because XoxF is required for expression of mxaF (22). As reported by Nakagawa et al (11), addition of La allowed wild-type growth of an mxaF mutant strain in methanol medium (Fig. 1B).…”

“…The studies addressing how methylotrophic bacteria sense lanthanides and differentially regulate expression of the mxa and xox genes are limited and conflicting. In M. extorquens AM1, it was initially reported that La does not affect expression of the mxa or xox1 operon genes (11), which conflicted with the subsequent findings described by Skovran and Martinez-Gomez (17). Following these reports, Farhan et al showed that in the methanotrophic bacterium Methylosinus trichosporium OB3b, exogenous Ce upregulates expression of both xoxF copies in its genome and represses expression of the mxaFI genes (28).…”

“…A key step in this process is the oxidation of methanol to formaldehyde, which is carried out by different enzymes, including methanol dehydrogenase (MeDH) and alcohol oxidase, depending on the specific methylotroph (6,7). Recently, it was discovered that some types of MeDHs require rareearth elements, specifically lanthanides, as cofactors (8)(9)(10)(11) [Yb], and lutetium [Lu]) and two chemically similar elements (scandium [Sc] and yttrium [Y]). The term "rare earth" is deceptive, as these lanthanide elements are relatively abundant in the Earth's crust, found at levels similar to those seen for copper and zinc (Ce, 66 ppm; La 39 ppm; Cu, 60 ppm, Zn, 70 ppm) (12).…”

“…However, in some environments, such as volcanic mud pots, concentrations of soluble lanthanides can reach the low micromolar range (8). Studies have shown that plants can effectively concentrate lanthanides like La from the soil, with measurements ranging from 0.18 to 3.1 g La/g dry leaf mass (11). These studies have had profound impacts on the study of methylotrophic organisms, as different methylotrophic bacteria have now been shown to use lanthanides directly for MeDH activity and addition of lanthanides to growth media has facilitated the growth of organisms that had been difficult or impossible to culture before (8,11,17).…”

“…It was formally possible that XoxF-MeDH required Ca for the oxidation of methanol but was upregulated in the presence of La. In 2012, this was resolved when Nakagawa et al demonstrated that MeDH, purified from M. extorquens AM1 cells grown in medium containing La, consisted of a XoxF dimer containing 1.24 atoms of La and lacked Ca (11). Since these findings, XoxF has been purified from a variety of methylotrophs, displaying differences in subunit composition, optimal enzyme assay parameters, and oxidation products depending on the organism studied (9,10,25).…”