Biochemical and Structural Characterization of XoxG and XoxJ and Their Roles in Lanthanide‐Dependent Methanol Dehydrogenase Activity

Abstract

Lanthanide (Ln)‐dependent methanol dehydrogenases (MDHs) have recently been shown to be widespread in methylotrophic bacteria. Along with the core MDH protein, XoxF, these systems contain two other proteins, XoxG (a c‐type cytochrome) and XoxJ (a periplasmic binding protein of unknown function), about which little is known. In this work, we have biochemically and structurally characterized these proteins from the methyltroph Methylobacterium extorquens AM1. In contrast to results obtained in an artificial assay system, assays of XoxFs metallated with LaIII, CeIII, and NdIII using their physiological electron acceptor, XoxG, display Ln‐independent activities, but the Km for XoxG markedly increases from La to Nd. This result suggests that XoxG′s redox properties are tuned specifically for lighter Lns in XoxF, an interpretation supported by the unusually low reduction potential of XoxG (+172 mV). The X‐ray crystal structure of XoxG provides a structural basis for this reduction potential and insight into the XoxG–XoxF interaction. Finally, the X‐ray crystal structure of XoxJ reveals a large hydrophobic cleft and suggests a role in the activation of XoxF. These studies enrich our understanding of the underlying chemical principles that enable the activity of XoxF with multiple lanthanides in vitro and in vivo.

Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster’s Blue in methanol dehydrogenase assays

“…Here, methanol conversion is coupled to electron acceptors that are, in turn, linked to oxygen in an oxygensensitive electrode [16,17]. Studies with the natural electron acceptor cytochrome c L and a bovine or equine heart cytochrome as terminal electron acceptor and dye have also been reported [15,16,18]. Recently, the oxidation of methanol by Eu-MDH via cytochrome c GJ driven by electrocatalytic voltammetry was also demonstrated [14].…”

Biological Function of Lanthanide in Plant-Symbiotic Bacteria: Lanthanide-Dependent Methanol Oxidation System

“…As shown in Fig. 3, an Ln-uptake system has already been postulated in methylotrophic and methanotrophic bacteria (Cotruvo Jr, 2019;Featherston et al, 2019;Groom et al, 2019;Ochsner et al, 2019;Roszczenko-Jasińska et al, 2019;Cotruvo Jr et al, 2018). In this system, the cells are believed to uptake Ln species bonded by a lanthanophore, and the Ln species is transferred into the cell using a TonB-dependent system, which is suspected to be similar to siderophore-and chalcophore-mediated iron and copper uptake (Daumann, 2019).…”

“…Kawai et al, 2007Hibi et al, 2011Nakagawa et al, 2012Fitriyanto et al, 2011aWang et al, 2019Pastawan et al, 2020Fitriyanto et al, 2011bPol et al, 2014Wehrmann et al, 2020 In the case of Mr. extorquens AM1, MxaFI is encoded on the mxa cluster ( Fig. 2B) and the gene cluster includes several genes for methanol oxidation; mxaF and mxaI, encoding subunits α and β of MDH, respectively (Nunn and Lidstrom, 1986a;1986b); mxaG, encoding cytochrome cL, which is electron acceptor for MxaFI (Amaratunga et al, 1997); mxaJ, encoding a putative chaperone for MxaFI (Featherston et al, 2019); mxaA, encoding a putative chaperone for Ca 2+ insertion to MxaF (Morris et al, 1995); and many other genes of unknown function (Anderson et al, 1990;Toyama et al, 1998). On the other hand, XoxF is encoded on the xox1 cluster (Chistoserdova and Lidstrom 1997).…”

“…On the other hand, XoxF is encoded on the xox1 cluster (Chistoserdova and Lidstrom 1997). The gene composition of the xox1 cluster is very simple compared to that of the mxa cluster: xoxF, encoding Ln-dependent MDH (Nakagawa et al, 2012); xoxG, encoding cytochrome cL (Chistoserdova and Lidstrom 1997); and xoxJ, encoding a putative chaperone for XoxF (Featherston et al, 2019). In the genome of Mr. extorquens AM1, there is another gene cluster for PQQ-dependent MDHs, i.e., the xox2 cluster (Vuilleumier et al, 2009), but its function is still unknown.…”

“…In particular, XoxF is able to utilize only L-Ln species, i.e., La 3+ , Ce 3+ , Pr 3+ , and Nd 3+ , as a cofactor, and it chooses La 3+ preferentially over Nd 3+ (Wang et al, 2020). Moreover, it is known that Ln species as a cofactor affect several enzymatic properties of XoxF, such as the dye-linked MDH activity, thermal stability, and the interaction with XoxG (Featherston et al, 2019;Good et al, 2019;Wang et al, 2020). In short, the preference of XoxF for L-Ln species has a definitive effect on its methanol-oxidizing activity, and L-Ln species existing in growth environments affect the methylotrophic growth of strains in the Methylobacterium group (Vu et al, 2016;.…”