Lanthanides: New life metals?

Lanthanides (Ln³⁺) that are Rare Earth Elements, until recently thought to be biologically inert, have recently emerged as essential metals for activity and expression of a special type of methanol dehydrogenase, XoxF. As XoxF enzyme homologs are encoded in a wide variety of microbes, including microbes active in important environmental processes such as methane and methanol metabolism, Ln³⁺ may represent some of the key biogeochemical drivers in cycling of carbon and other elements. However, significant gaps in understanding the role of Ln³⁺ in biological systems remain as the functions of most of the proteins potentially dependent of Ln³⁺ and their roles in specific metabolic networks/respective biogeochemical cycles remain unknown. Moreover, enzymes dependent on Ln³⁺ but not related to XoxF enzymes may exist, and these so far have not been recognized. Through connecting the recently uncovered genetic divergence and phylogenetic distribution of XoxF-like enzymes and through elucidation of their activities, metal and substrate specificities, along with the biological contexts of respective biochemical pathways, most parsimonious scenarios for their evolution could be uncovered. Generation of such data will firmly establish the role of Ln³⁺ in the biochemistry of Life inhabiting this planet.     

Isolation and genomic characterization of Novimethylophilus kurashikiensis gen. nov. sp. nov., a new lanthanide‐dependent methylotrophic species of Methylophilaceae

Recently, it has been found that two types of methanol dehydrogenases (MDHs) exist in Gram‐negative bacterial methylotrophs, calcium‐dependent MxaFI‐MDH and lanthanide‐dependent XoxF‐MDH and the latter is more widespread in bacterial genomes. We aimed to isolate and characterize lanthanide‐dependent methylotrophs. The growth of strain La2‐4^T on methanol, which was isolated from rice rhizosphere soil, was strictly lanthanide dependent. Its 16S rRNA gene sequence showed only 93.4% identity to that of Methylophilus luteus Mim^T, and the name Novimethylophilus kurashikiensis gen. nov. sp. nov. is proposed. Its draft genome (ca. 3.69 Mbp, G + C content 56.1 mol%) encodes 3579 putative CDSs and 84 tRNAs. The genome harbors five xoxFs but no mxaFI. XoxF4 was the major MDH in the cells grown on methanol and methylamine, evidenced by protein identification and quantitative PCR analysis. Methylamine dehydrogenase gene was absent in the La2‐4^T genome, while genes for the glutamate‐mediated methylamine utilization pathway were detected. The genome also harbors those for the tetrahydromethanopterin and ribulose monophosphate pathways. Additionally, as known species, isolates of Burkholderia ambifaria, Cupriavidus necator and Dyadobacter endophyticus exhibited lanthanide dependent growth on methanol. Thus, lanthanide can be used as an essential growth factor for methylotrophic bacteria that do not harbor MxaFI‐MDH.     

Use of rare‐earth elements in the phyllosphere colonizer Methylobacterium extorquens PA1

Until recently, rare‐earth elements (REEs) had been thought to be biologically inactive. This view changed with the discovery of the methanol dehydrogenase XoxF that strictly relies on REEs for its activity. Some methylotrophs only contain xoxF, while others, including the model phyllosphere colonizer Methylobacterium extorquens PA1, harbor this gene in addition to mxaFI encoding a Ca²⁺‐dependent enzyme. Here we found that REEs induce the expression of xoxF in M. extorquens PA1, while repressing mxaFI, suggesting that XoxF is the preferred methanol dehydrogenase in the presence of sufficient amounts of REE. Using reporter assays and a suppressor screen, we found that lanthanum (La³⁺) is sensed both in a XoxF‐dependent and independent manner. Furthermore, we investigated the role of REEs during Arabidopsis thaliana colonization. Element analysis of the phyllosphere revealed the presence of several REEs at concentrations up to 10 μg per g dry weight. Complementary proteome analyses of M. extorquens PA1 identified XoxF as a top induced protein in planta and a core set of La³⁺‐regulated proteins under defined artificial media conditions. Among these was a REE‐binding protein that is encoded next to a gene for a TonB‐dependent transporter. The latter was essential for REE‐dependent growth on methanol indicating chelator‐assisted uptake of REEs.     

XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

“Candidatus Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “Ca. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “Ca. Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “Ca. Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity (V_max of 10 μmol min⁻¹ mg⁻¹ protein, K_m of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “Ca. Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described.     

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

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.     

Crystal structure of quinone‐dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates

The quinone‐dependent alcohol dehydrogenase (PQQ‐ADH, E.C. 1.1.5.2) from the Gram‐negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three‐dimensional (3D) structures of the native form, with PQQ and a Ca²⁺ ion, and of the enzyme in complex with a Zn²⁺ ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ‐ADH displays an eight‐bladed β‐propeller fold, characteristic of Type I quinone‐dependent methanol dehydrogenases. However, three of the four ligands of the Ca²⁺ ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ‐ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ‐dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.     

Multiphyletic origins of methylotrophy in Alphaproteobacteria,exemplified by comparative genomics of Lake Washington isolates

We sequenced the genomes of 19 methylotrophic isolates from Lake Washington, which belong to nine genera within eight families of the Alphaproteobacteria, two of the families being the newly proposed families. Comparative genomic analysis with a focus on methylotrophy metabolism classifies these strains into heterotrophic and obligately or facultatively autotrophic methylotrophs. The most persistent metabolic modules enabling methylotrophy within this group are the N‐methylglutamate pathway, the two types of methanol dehydrogenase (MxaFI and XoxF), the tetrahydromethanopterin pathway for formaldehyde oxidation, the serine cycle and the ethylmalonyl‐CoA pathway. At the same time, a great potential for metabolic flexibility within this group is uncovered, with different combinations of these modules present. Phylogenetic analysis of key methylotrophy functions reveals that the serine cycle must have evolved independently in at least four lineages of Alphaproteobacteria and that all methylotrophy modules seem to be prone to lateral transfers as well as deletions.     

Influence of rare earth elements (Y,Gd and Lu) on the luminescent properties of green phosphor ZnMoO4:Tb3+

(Zn,Ln_x)MoO₄:Tb³⁺ (Ln = Y³⁺, Gd³⁺ and Lu³⁺) were prepared using the co‐precipitation method. Phase impurity, morphology and composition were investigated by power X‐ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). The experimental results show that crystal structure is not destroyed after doping an appropriate amount of Y³⁺, Gd³⁺ and Lu³⁺. EDS analysis reveals that Y, Gd and Lu have been successfully doped into ZnMoO₄. In addition, the morphology of the phosphors is notably improved, exhibiting homogeneous dispersion morphology and irregular shapes of particle size ~ 0.5–1 µm. The luminescent intensity of (Zn,Ln_x)MoO₄:Tb³⁺ (Ln = Y³⁺, Gd³⁺ and Lu³⁺) phosphor is obviously higher than that of ZnMoO₄:Tb³⁺ phosphor. The energy transfer process between trivalent rare earth ions indicates that the inert earth ions can act as an energy bridge from MoO₄^2‐ to Tb³⁺. Copyright © 2013 John Wiley & Sons, Ltd.     

Luminescence in Ba2Sr2Al2O7:RE (RE = Tb3+,Eu3+ and Dy3+) novel aluminate phosphors

The luminescence of novel rare earth ( Tb ^{3 +} , Eu ^{3 +} and Dy ^{3 +} )‐activated Ba ₂ Sr ₂ Al ₂ O ₇ phosphors for solid‐state lighting is presented. The aluminate phosphors were synthesized using a one‐step combustion method. X‐Ray diffraction, scanning electron microscopy and photoluminescence characterizations were performed to understand the mechanism of excitation and the corresponding emission in the as‐prepared phosphor, as characterized the phase purity and microstructure. Improvements in the luminescence properties of the phosphors with rare earth concentration were observed. The phosphor hue could be tuned from blue, green and red by proper selection of rare earth ions in typical concentrations. Effective absorption in the near‐ultraviolet region was observed, which makes the phosphor a potential candidate for ultraviolet light‐emitting diodes. Copyright © 2016 John Wiley & Sons, Ltd.     

Antioxidation and DNA‐Binding Properties of Binuclear Lanthanide(III) Complexes with a Schiff Base Ligand Derived from 8‐Hydroxyquinoline‐7‐carboxaldehyde and Benzoylhydrazine

8‐Hydroxyquinoline‐7‐carboxaldehyde (8‐HQ‐7‐CA), Schiff‐base ligand 8‐hydroxyquinoline‐7‐carboxaldehyde benzoylhydrazone, and binuclear complexes [LnL(NO₃)(H₂O)₂]₂ were prepared from the ligand and equivalent molar amounts of Ln(NO₃)?6 H₂O (Ln=La³⁺, Nd³⁺, Sm³⁺, Eu³⁺, Gd³⁺, Dy³⁺, Ho³⁺, Er³⁺, Yb³⁺, resp.). Ligand acts as dibasic tetradentates, binding to Ln^III through the phenolate O‐atom, N‐atom of quinolinato unit, and C?N and ? O? C?N? groups of the benzoylhydrazine side chain. Dimerization of this monomeric unit occurs through the phenolate O‐atoms leading to a central four‐membered (LnO)₂ ring. Ligand and all of the Ln^III complexes can strongly bind to CT‐DNA through intercalation with the binding constants at 10⁵–10⁶ M ^?1. Moreover, ligand and all of the Ln^III complexes have strong abilities of scavenging effects for hydroxyl (HO^.) radicals. Both the antioxidation and DNA‐binding properties of Ln^III complexes are much better than that of ligand.     

Characterization of a group of pyrroloquinoline quinone‐dependent dehydrogenases that are involved in the conversion of L‐sorbose to 2‐Keto‐L‐gulonic acid in Ketogulonicigenium vulgare WSH‐001

Ketogulonicigenium vulgare WSH‐001 is an industrial strain used for vitamin C production. Based on genome sequencing and pathway analysis of the bacterium, some of its potential pyrroloquinoline quinone (PQQ)‐dependent dehydrogenases were predicted, including KVU_pmdA_0245, KVU_2142, KVU_2159, KVU_1366, KVU_0203, KVU_0095, and KVU_pmdB_0115. BLAST and function domain searches showed that enzymes encoded by these genes may act as putative PQQ‐dependent L ‐sorbose dehydrogenases (SDH) or L ‐sorbosone dehydrogenases (SNDH). To validate whether these dehydrogenases are PQQ‐dependent or not, these seven putative dehyrogenases were overexpressed in Escherichia coli BL21 (DE3) and purified for characterization. Biochemical and kinetic characterization of the purified proteins have led to the identification of seven enzymes that possess the ability to oxidize L ‐sorbose or L ‐sorbosone to varying degrees. In addition, the dehydrogenation of sorbose in K. vulgare is validated to be PQQ dependent, identification of these PQQ‐dependent dehydrogenases expanded the PQQ‐dependent dehydrogenase family. Besides, the optimal combination of enzymes that could more efficiently catalyze the conversion of sorbose to gulonic acid was proposed. These are important in supporting the development of metabolic engineering strategies and engineering of efficient strains for one‐step production of vitamin C in the future. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1398–1404, 2013     

Enhanced green emissions of Er3+/Yb3+ co‐doped Gd2(MoO4)3 by co‐excited up‐conversion processes

Improving the emission from rare earth ions doped materials is of great importance to broaden their application in bio‐imaging, photovoltaics and temperature sensing. The green emissions of Gd₂(MoO₄)₃:Er³⁺/Yb³⁺ powder upon co‐excitation with 980 and 808 nm lasers were investigated in this paper. Distinct enhancement of green emissions was observed compared with single laser excitation. Based on the energy level structure of Er³⁺, the enhancement mechanism was discussed. Moreover, the result of temperature‐dependent enhancement revealed that the enhancement factor reached its maximum (2.5) as the sample heated to 120°C, which is due to the competition of two major thermal effects acting in the co‐excited up‐conversion processes. In addition, the same enhancement of green emissions was also observed in Gd₂(MoO₄)₃:Er³⁺ powder and NaYF₄:Er³⁺/Yb³⁺ powder.     

Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of 2,3-DPG-hemoglobin’s oxygen affinity

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln³⁺ binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln³⁺ binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln³⁺ binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N_I), non-cooperative strong sites (N_S) and non-cooperative weak sites (N_W) with binding constants in decreasing order: K_I>K_S>K_W. The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln³⁺ concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln³⁺ binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln³⁺ concentration. At the range of [Ln³⁺]/[Hb]<2, the marked increase of oxygen affinity (P₅₀ decrease) with the Ln³⁺ concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln³⁺ concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln³⁺ binding. This was indicated by the changes in secondary structure characterized by the decrease of α-helix content.     

FUNCTIONAL ANALYSIS OF A MOSQUITO SHORT‐CHAIN DEHYDROGENASE CLUSTER

The short‐chain dehydrogenases (SDR) constitute one of the oldest and largest families of enzymes with over 46,000 members in sequence databases. About 25% of all known dehydrogenases belong to the SDR family. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, hormone, and xenobiotic metabolism as well as in redox sensor mechanisms. This family is present in archaea, bacteria, and eukaryota, emphasizing their versatility and fundamental importance for metabolic processes. We identified a cluster of eight SDRs in the mosquito Aedes aegypti (AaSDRs). Members of the cluster differ in tissue specificity and developmental expression. Heterologous expression produced recombinant proteins that had diverse substrate specificities, but distinct from the conventional insect alcohol (ethanol) dehydrogenases. They are all NADP⁺‐dependent and they have S‐enantioselectivity and preference for secondary alcohols with 8–15 carbons. Homology modeling was used to build the structure of AaSDR1 and two additional cluster members. The computational study helped explain the selectivity toward the (10S)‐isomers as well as the reduced activity of AaSDR4 and AaSDR9 for longer isoprenoid substrates. Similar clusters of SDRs are present in other species of insects, suggesting similar selection mechanisms causing duplication and diversification of this family of enzymes.     

Genomics and Biochemistry of Metabolic Pathways for the C<Subscript>1</Subscript> Compounds Utilization in Colorless Sulfur Bacterium <Emphasis Type="Italic">Beggiatoa leptomitoformis</Emphasis> D-402

The metabolic pathways of one-carbon compounds utilized by colorless sulfur bacterium Beggiatoa leptomitoformis D-402 were revealed based on comprehensive analysis of its genomic organization, together with physiological, biochemical and molecular biological approaches. Strain D-402 was capable of aerobic methylotrophic growth with methanol as a sole source of carbon and energy and was not capable of methanotrophic growth because of the absence of genes of methane monooxygenases. It was established that methanol can be oxidized to CO₂ in three consecutive stages. On the first stage methanol was oxidized to formaldehyde by the two PQQ (pyrroloquinolinequinone)-dependent methanol dehydrogenases (MDH): XoxF and Mdh2. Formaldehyde was further oxidized to formate via the tetrahydromethanopterin (H₄MPT) pathway. And on the third stage formate was converted to CO₂ by NAD⁺-dependent formate dehydrogenase Fdh2. Finally, it was established that endogenous CO₂, formed as a result of methanol oxidation, was subsequently assimilated for anabolism through the Calvin–Benson–Bassham cycle. The similar way of one-carbon compounds utilization also exists in representatives of another freshwater Beggiatoa species—B. alba.     

Characteristics of external and internal NAD(P)H dehydrogenases in Hoya carnosa mitochondria

This study aims at characterizing NAD(P)H dehydrogenases on the inside and outside of the inner membrane of mitochondria of one phosphoenolpyruvate carboxykinase??crassulacean acid metabolism plant, Hoya carnosa. In crassulacean acid metabolism plants, NADH is produced by malate decarboxylation inside and outside mitochondria. The relative importance of mitochondrial alternative NADH dehydrogenases and their association was determined in intact??and alamethicin??permeabilized mitochondria of H. carnosa to discriminate between internal and external activities. The major findings in H. carnosa mitochondria are: (i) external NADPH oxidation is totally inhibited by DPI and totally dependent on Ca²⁺, (ii) external NADH oxidation is partially inhibited by DPI and mainly dependent on Ca²⁺, (iii) total NADH oxidation measured in permeabilized mitochondria is partially inhibited by rotenone and also by DPI, (iv) total NADPH oxidation measured in permeabilized mitochondria is partially dependent on Ca²⁺ and totally inhibited by DPI. The results suggest that complex I, external NAD(P)H dehydrogenases, and internal NAD(P)H dehydrogenases are all linked to the electron transport chain. Also, the total measurable NAD(P)H dehydrogenases activity was less than the total measurable complex I activity, and both of these enzymes could donate their electrons not only to the cytochrome pathway but also to the alternative pathway. The finding indicated that the H. carnosa mitochondrial electron transport chain is operating in a classical way, partitioning to both Complex I and alternative Alt. NAD(P)H dehydrogenases.     

Pyrrole‐coupled salicylimine‐based fluorescence “turn on” probe for highly selective recognition of Zn2+ ions in mixed aqueous media: Application in living cell imaging

Cation sensing behaviour of a pyrrole‐based derivative (2‐hydroxyl 3 methyl 6 isopropyl benzaldehyde}‐3,4‐dimethyl‐1H‐pyrrole‐2‐carbohydrazide (receptor 3) has been explored and is found to be selective towards Zn²⁺ over a variety of tested cations. The receptor 3 has shown high selectivity and sensitivity towards Zn²⁺ over the other alkali, alkaline earth and transition metal ions. In the presence of Zn²⁺, absorption band of receptor 3 has shown the red shift. The sensing behaviour has been suggested to continue via enhancement process which has further been supported by UV‐vis absorption and theoretical density functional theory (DFT) calculations indicating the formation of a 1:1 complex between the pyrrole based receptor 3 and Zn²⁺. The present work is presenting a highly selective dual channel colorimetric sensor for zinc with great sensitivity. The developed sensor was successfully applied to image intracellular Zn²⁺ in living cells. Copyright © 2015 John Wiley & Sons, Ltd.     

The use of reconstitution and inhibitor/ion interaction assays to distinguish between Ca2+/H+and Cd2+/H+ antiporter activities of oat and tobacco tonoplast vesicles

Tonoplast, ion antiport activities are critical to ion homeostasis and sequestration in plants. The biochemical properties of these activities, and the enzymes that catalyse them, are little characterized. Here we applied biochemical approaches to study some characteristics and to distinguish between Ca²⁺/H⁺ and Cd²⁺/H⁺ antiporter activities of tonoplast vesicles from non‐transformed, wild‐type plants. Solubilization and reconstitution of oat‐seedling (Avena sativa L.) root tonoplast vesicles resulted in about a 6‐fold loss of protein, about a 6‐fold enhancement of Cd²⁺/H⁺ antiport specific activity (at 10 µM Cd²⁺), and almost complete loss of Ca²⁺/H⁺ antiport activity. Similar results were found for vesicles from mature tobacco (Nicotiana tabacum) roots. Cd²⁺ concentration‐dependent proton efflux was similar and linear with both oat vesicles and proteoliposomes. In contrast, Ca²⁺ concentration‐dependent proton efflux of oat vesicles was easily observed while that with proteoliposomes was minimal and non‐linear. Cd²⁺ pre‐treatment of oat vesicles reduced verapamil inhibition of Cd²⁺/H⁺ activity and verapamil binding to vesicles, while Ca²⁺ pre‐treatment was much less protective of Ca^2+/H⁺ activity and verapamil binding. Results show the usefulness of reconstitution, and also inhibitor/ion interaction assays for distinguishing between transporter activities in vitro, but they do not resolve the question of whether there are separate enzymes for Cd²⁺/H⁺ and Ca²⁺/H⁺. Our observation that solubilization and reconstitution have similar effects on both Cd²⁺/H⁺ and Ca²⁺/H⁺ activities of root tonoplast vesicles from immature oat and mature tobacco roots suggests that the transporters involved are similar in young and mature roots, and in roots of different species.