Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters.

Artificial dye-coupled assays have been widely adopted as a rapid and convenient method to assess the activity of methanol dehydrogenases (MDH). Lanthanide(Ln)-dependent XoxF-MDHs are able to incorporate different lanthanides (Lns) in their active site. Dye-coupled assays showed that the earlier Lns exhibit a higher enzyme activity than the late Lns. Despite widespread use, there are limitations: oftentimes a pH of 9 and activators are required for the assay. Moreover, Ln-MDH variants are not obtained by isolation from the cells grown with the respective Ln, but by incubation of an apo-MDH with the Ln. Herein, we report the cultivation of Ln-dependent methanotroph Methylacidiphilum fumariolicum SolV with nine different Lns, the isolation of the respective MDHs and the assessment of the enzyme activity using the dye-coupled assay. We compare these results with a protein-coupled assay using its physiological electron acceptor cytochrome cGJ (cyt cGJ ). Depending on the assay, two distinct trends are observed among the Ln series. The specific enzyme activity of La-, Ce- and Pr-MDH, as measured by the protein-coupled assay, exceeds that measured by the dye-coupled assay. This suggests that early Lns also have a positive effect on the interaction between XoxF-MDH and its cyt cGJ thereby increasing functional efficiency.

Biotechnological potential of psychrotolerant methylobacteria isolated from biotopes of Antarctic oases.

Ten strains of psychrotolerant methylotrophic bacteria were isolated from the samples collected in Larsemann and Bunger Hills (Antarctica). Most of the isolates are assigned to the genus Pseudomonas, representatives of the genera Janthinobacterium, Massilia, Methylotenera and Flavobacterium were also found. Majority of isolates were able to grow on a wide range of sugars, methylamines and other substrates. Optimal growth temperatures for the isolated strains varied from 6 °C to 28 °C. The optimal concentration of NaCl was 0.5-2.0%. The optimal pH values of the medium were 6-7. It was found that three strains synthesized indole-3-acetic acid on a medium with L-tryptophan reaching 11-12 µg/ml. The values of intracellular carbohydrates in several strains exceeded 50 µg/ml. Presence of calcium-dependent and lanthanum-dependent methanol dehydrogenase have been shown for some isolates. Strains xBan7, xBan20, xBan37, xBan49, xPrg27, xPrg48, xPrg51 showed the presence of free amino acids. Bioprospection of Earth cryosphere for such microorganisms has a potential in biotechnology.

Identification of a TonB-Dependent Receptor Involved in Lanthanide Switch by the Characterization of Laboratory-Adapted Methylosinus trichosporium OB3b.

Two methanol dehydrogenases (MDHs), MxaFI and XoxF, have been characterized in methylotrophic and methanotrophic bacteria. MxaFI contains a calcium ion in its active site, whereas XoxF contains a lanthanide ion. Importantly, the expression of MxaFI and XoxF is inversely regulated by lanthanide bioavailability, i.e., the "lanthanide switch." To reveal the genetic and environmental factors affecting the lanthanide switch, we focused on two Methylosinus trichosporium OB3b mutants isolated during routine cultivation. In these mutants, MxaF was constitutively expressed, but lanthanide-dependent XoxF1 was not, even in the presence of 25 µM cerium ions, which is sufficient for XoxF expression in the wild type. Genotyping showed that both mutants harbored a loss-of-function mutation in the CQW49_RS02145 gene, which encodes a TonB-dependent receptor. Gene disruption and complementation experiments demonstrated that CQW49_RS02145 was required for XoxF1 expression in the presence of 25 µM cerium ions. Phylogenetic analysis indicated that CQW49_RS02145 was homologous to the Methylorubrum extorquens AM1 lanthanide transporter gene (lutH). These findings suggest that CQW49_RS02145 is involved in lanthanide uptake across the outer membrane. Furthermore, we demonstrated that supplementation with cerium and glycerol caused severe growth arrest in the wild type. CQW49_RS02145 underwent adaptive laboratory evolution in the presence of cerium and glycerol ions, resulting in a mutation that partially mitigated the growth arrest. This finding implies that loss-of-function mutations in CQW49_RS02145 can be attributed to residual glycerol from the frozen stock. IMPORTANCE Lanthanides are widely used in many industrial applications, including catalysts, magnets, and polishing. Recently, lanthanide-dependent metabolism was characterized in methane-utilizing bacteria. Despite the global demand for lanthanides, few studies have investigated the mechanism of lanthanide uptake by these bacteria. In this study, we identify a lanthanide transporter in Methylosinus trichosporium OB3b and indicate the potential interaction between intracellular lanthanide and glycerol. Understanding the genetic and environmental factors affecting lanthanide uptake should not only help improve the use of lanthanides for the bioconversion of methane into valuable products like methanol but also be of value for developing biomining to extract lanthanides under neutral conditions.

Multispecies Populations of Methanotrophic Methyloprofundus and Cultivation of a Likely Dominant Species from the Iheya North Deep-Sea Hydrothermal Field.

The Methyloprofundus clade is represented by uncultivated methanotrophic bacterial endosymbionts of deep-sea bathymodiolin mussels, but only a single free-living species has been cultivated to date. This study reveals the existence of free-living Methyloprofundus variants in the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. A clade-targeted amplicon analysis of the particulate methane monooxygenase gene (pmoA) detected 647 amplicon sequence variants (ASVs) of the Methyloprofundus clade in microbial communities newly formed in in situ colonization systems. Such systems were deployed at colonies of bathymodiolin mussels and a galatheoid crab in diffuse-flow areas. These ASVs were classified into 161 species-like groups. The proportion of the species-like groups representing endosymbionts of mussels was unexpectedly low. A methanotrophic bacterium designated INp10, a likely dominant species in the Methyloprofundus population in this field, was enriched in a biofilm formed in a methane-fed cultivation system operated at 10°C. Genomic characterization with the gene transcription data set of INp10 from the biofilm suggested traits advantageous to niche competition in environments, such as mobility, chemotaxis, biofilm formation, offensive and defensive systems, and hypoxia tolerance. The notable metabolic traits that INp10 shares with some Methyloprofundus members are the use of lanthanide-dependent XoxF as the sole methanol dehydrogenase due to the absence of the canonical MxaFI, the glycolytic pathway using fructose-6-phosphate aldolase instead of fructose-1,6-bisphosphate aldolase, and the potential to perform partial denitrification from nitrate under oxygen-limited conditions. These findings help us better understand the ecological strategies of this possibly widespread marine-specific methanotrophic clade. IMPORTANCE The Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough is characterized by abundant methane derived from organic-rich sediments and diverse chemosynthetic animal species, including those harboring methanotrophic bacterial symbionts, such as bathymodiolin mussels Bathymodiolus japonicus and "Bathymodiolus" platifrons and a galatheoid crab, Shinkaia crosnieri. Symbiotic methanotrophs have attracted significant attention, and yet free-living methanotrophs in this environment have not been studied in detail. We focused on the free-living Methyloprofundus spp. that thrive in this hydrothermal field and identified an unexpectedly large number of species-like groups in this clade. Moreover, we enriched and characterized a methanotroph whose genome sequence indicated that it corresponds to a new species in the genus Methyloprofundus. This species might be a dominant member of the indigenous Methyloprofundus population. New information on free-living Methyloprofundus populations suggests that the hydrothermal field is a promising locale at which to investigate the adaptive capacity and associated genetic diversity of Methyloprofundus spp.

Systems analysis of the effect of hydrogen sulfide on the growth of Methylococcus capsulatus Bath.

Methanotrophs are bacteria capable on growing on methane as their sole carbon source. They may provide a promising route for upgrading natural gas into more valuable fuels and chemicals. However, natural gas may contain significant quantities of hydrogen sulfide. Little is known about how hydrogen sulfide affects the growth and physiology of methanotrophs aside from a few studies showing that it is inhibitory. This study investigated how hydrogen sulfide affects the growth and physiology of the model methanotroph, Methylococcus capsulatus Bath. Growth studies demonstrated that hydrogen sulfide inhibits the growth of M. capsulatus Bath when the concentration exceeds 0.5% (v/v). To better understand how hydrogen sulfide is inhibiting the growth of M. capsulatus Bath, transcription and metabolite concentrations were profiled using RNA sequencing and gas chromatography-mass spectrometry, respectively. Our analysis of the differentially expressed genes and changes in metabolite concentrations suggests that hydrogen sulfide inhibits cellular respiration. The cells respond to sulfide stress in part by increasing the rate of sulfide oxidation and by increasing the expression of sulfide quinone reductase and a putative persulfide dioxygenase. In addition, they reduce the expression of the native calcium-dependent methanol dehydrogenase and increase the expression of XoxF, a lanthanide-dependent methanol dehydrogenase. While the reason of this switch in unknown, XoxF has previously been shown to be induced by lanthanides or nitric oxide in methanotrophs. Collectively, these results further our understanding of how methanotrophs respond to sulfide stress and may aid in the engineering of strains resistant to hydrogen sulfide. KEY POINTS: • Hydrogen sulfide inhibits growth of Methylococcus capsulatus Bath • Sulfide stress inhibits cellular respiration • Sulfide stress induces XoxF, a lanthanide-dependent methanol dehydrogenase.

Isolation and Characterization of Homologically Expressed Methanol Dehydrogenase from Methylorubrum extorquens AM1 for the Development of Bioelectrocatalytical Systems.

(Ca2+)-dependent pyrroloquinolinequinone (PQQ)-dependent methanol dehydrogenase (MDH) (EC: 1.1.2.7) is one of the key enzymes of primary C1-compound metabolism in methylotrophy. PQQ-MDH is a promising catalyst for electrochemical biosensors and biofuel cells. However, the large-scale use of PQQ-MDH in bioelectrocatalysis is not possible due to the low yield of the native enzyme. Homologously overexpressed MDH was obtained from methylotrophic bacterium Methylorubrum extorquens AM1 by cloning the gene of only one subunit, mxaF. The His-tagged enzyme was easily purified by immobilized metal ion affinity chromatography (36% yield). A multimeric form (α6ß6) of recombinant PQQ-MDH possessing enzymatic activity (0.54 U/mg) and high stability was demonstrated for the first time. pH-optimum of the purified protein was about 9-10; the enzyme was activated by ammonium ions. It had the highest affinity toward methanol (KM = 0.36 mM). The recombinant MDH was used for the fabrication of an amperometric biosensor. Its linear range for methanol concentrations was 0.002-0.1 mM, the detection limit was 0.7 µM. The properties of the invented biosensor are competitive to the analogs, meaning that this enzyme is a promising catalyst for industrial methanol biosensors. The developed simplified technology for PQQ-MDH production opens up new opportunities for the development of bioelectrocatalytic systems.

In Vitro One-Pot 3-Hydroxypropanal Production from Cheap C1 and C2 Compounds.

One- or two-carbon (C1 or C2) compounds have been considered attractive substrates because they are inexpensive and abundant. Methanol and ethanol are representative C1 and C2 compounds, which can be used as bio-renewable platform feedstocks for the biotechnological production of value-added natural chemicals. Methanol-derived formaldehyde and ethanol-derived acetaldehyde can be converted to 3-hydroxypropanal (3-HPA) via aldol condensation. 3-HPA is used in food preservation and as a precursor for 3-hydroxypropionic acid and 1,3-propanediol that are starting materials for manufacturing biocompatible plastic and polytrimethylene terephthalate. In this study, 3-HPA was biosynthesized from formaldehyde and acetaldehyde using deoxyribose-5-phosphate aldolase from Thermotoga maritima (DERATma) and cloned and expressed in Escherichia coli for 3-HPA production. Under optimum conditions, DERATma produced 7 mM 3-HPA from 25 mM substrate (formaldehyde and acetaldehyde) for 60 min with 520 mg/L/h productivity. To demonstrate the one-pot 3-HPA production from methanol and ethanol, we used methanol dehydrogenase from Lysinibacillus xylanilyticus (MDHLx) and DERATma. One-pot 3-HPA production via aldol condensation of formaldehyde and acetaldehyde from methanol and ethanol, respectively, was investigated under optimized reaction conditions. This is the first report on 3-HPA production from inexpensive alcohol substrates (methanol and ethanol) by cascade reaction using DERATma and MDHLx.

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

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.

Biosensor-Based Directed Evolution of Methanol Dehydrogenase from Lysinibacillus xylanilyticus.

Methanol dehydrogenase (Mdh), is a crucial enzyme for utilizing methane and methanol as carbon and energy sources in methylotrophy and synthetic methylotrophy. Engineering of Mdh, especially NAD-dependent Mdh, has thus been actively investigated to enhance methanol conversion. However, its poor catalytic activity and low methanol affinity limit its wider application. In this study, we applied a transcriptional factor-based biosensor for the direct evolution of Mdh from Lysinibacillus xylanilyticus (Lxmdh), which has a relatively high turnover rate and low KM value compared to other wild-type NAD-dependent Mdhs. A random mutant library of Lxmdh was constructed in Escherichia coli and was screened using formaldehyde-detectable biosensors by incubation with low methanol concentrations. Positive clones showing higher fluorescence were selected by fluorescence-activated cell sorting (FACS) system, and their catalytic activities toward methanol were evaluated. The successfully isolated mutants E396V, K318N, and K46E showed high activity, particularly at very low methanol concentrations. In kinetic analysis, mutant E396V, K318N, and K46E had superior methanol conversion efficiency, with 79-, 23-, and 3-fold improvements compared to the wild-type, respectively. These mutant enzymes could thus be useful for engineering synthetic methylotrophy and for enhancing methanol conversion to various useful products.

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

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster's blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented. Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.

How Lanthanide Ions Affect the Addition-Elimination Step of Methanol Dehydrogenases.

The recently discovered methanol dehydrogenase, XoxF, is a widespread enzyme used by methylotrophic bacteria to oxidize methanol for carbon and energy, and requires lanthanide ions for its activity. This enzyme represents an essential component of methanol utilization by both methanol- and methane-utilizing bacteria. The present investigation looks on the electronic, energetic and geometrical behavior of the methanol dehydrogenase from Methylacidiphilum fumariolicum SolV, which is strictly dependent on early lanthanide metals with +3 oxidation states, by examining enzyme-substrate complexes of all the lanthanides. We focus on the catalytic reaction mechanism of two methanol dehydrogenases having as cofactor europium and ytterbium belonging to the mid- and later- series of lanthanides, in comparison with the methanol dehydrogenase containing the cerium, one early lanthanide. Our results provide evidence for the influence of the lanthanide contraction effect in all the elementary steps of the catalytic reaction mechanism. This indication may prove useful for developing new catalytic machineries of enzymes that adopt new-to-nature transformations.

Methylotenera oryzisoli sp. nov., a lanthanide-dependent methylotrophic bacteria isolated from rice field soil.

A new lanthanide (Ln3+)-dependent methanol-utilizing bacterial strain, La3113T, was isolated from rice field soil and its taxonomic position was investigated using polyphasic approaches. The strain was aerobic, Gram-stain-negative, strongly motile, catalase-positive and cytochrome oxidase-positive. It could neither catalyse the hydrolysis of urea nor reduce nitrate to nitrite. Growth was observed within a temperature range of 10-40 °C and a pH range of 6-8, with optimum growth at 28 °C and pH 7. Methylamine was utilized as the single source of energy, carbon and nitrogen, and it was oxidized by methylamine dehydrogenase. C16 : 1 ω7c, C16 : 1 ω6c and C16 : 0 were the dominant cellular fatty acids. Its draft genome (2.67 Mbp and 44.9 mol% G+C content) encodes genes including three Ln3+-dependent methanol dehydrogenase (XoxF-type MDH) genes, those for formaldehyde assimilation (ribulose monophosphate pathway), formate dehydrogenases and methylamine dehydrogenases, but not Ca2+-dependent MDH (MxaFI-MDH), which characterizes the species as a Ln3+-dependent methylotroph. The 16S rRNA gene sequence showed that strain La3113T belongs to the genus Methylotenera and is closely related to Methylotenera mobilis JLW8T (98.29 % identity). The digital DNA-DNA hybridization (dDDH) values (less than 30 %) and average nucleotide identity (ANI) values (less than 85 %) between genomes of strain La3113T and related type strains were lower than the thresholds for species delineation (70 % for dDDH and 95-96 % for ANI). On the basis of these polyphasic approaches, we propose a novel Methylotenera species, Methylotenera oryzisoli sp. nov. (type strain La3113T=NBRC 111954T=DSM 103219T).

Identification of a transcriptional regulator for oligotrophy-responsive promoter in Rhodococcus erythropolis N9T-4.

Two genes, aldA, and mnoA, encoding an NAD-dependent aliphatic dehydrogenase and N,N'-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase, respectively, are strongly expressed when Rhodococcus erythropolis N9T-4 is grown under oligotrophic conditions. In this study, we found a transcriptional regulator required for the transcription of both aldA and mnoA. The transcriptional regulator was also found to be essential for the oligotrophic growth of N9T-4.

Methanol production by reversed methylotrophy constructed in Escherichia coli.

We constructed a reversed methylotrophic pathway that produces methanol, a promising feedstock for production of useful compounds, from fructose 6-phosphate (F6P), which can be supplied by catabolism of biomass-derived sugars including glucose, by a synthetic biology approach. Using Escherichia coli as an expression host, we heterologously expressed genes encoding methanol utilization enzymes from methylotrophic bacteria, i.e. the NAD+-dependent methanol dehydrogenase (MDH) from Bacillus methanolicus S1 and an artificial fusion enzyme of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase from Mycobacterium gastri MB19 (HPS-PHI). We confirmed that these enzymes can catalyze reverse reactions of methanol oxidation and formaldehyde fixation. The engineered E. coli strain co-expressing MDH and HPS-PHI genes produced methanol in resting cell reactions not only from F6P but also from glucose. We successfully conferred reversed methylotrophy to E. coli and our results provide a proof-of-concept for biological methanol production from biomass-derived sugar compounds.

A Mutagenic Screen Identifies a TonB-Dependent Receptor Required for the Lanthanide Metal Switch in the Type I Methanotroph "Methylotuvimicrobium buryatense" 5GB1C.

Several of the metabolic enzymes in methanotrophic bacteria rely on metals for both their expression and their catalysis. The MxaFI methanol dehydrogenase enzyme complex uses calcium as a cofactor to oxidize methanol, while the alternative methanol dehydrogenase XoxF uses lanthanide metals such as lanthanum and cerium for the same function. Lanthanide metals, abundant in the earth's crust, strongly repress the transcription of mxaF yet activate the transcription of xoxF This regulatory program, called the "lanthanide switch," is central to methylotrophic metabolism, but only some of its components are known. To uncover additional components of the lanthanide switch, we developed a chemical mutagenesis system in the type I gammaproteobacterial methanotroph "Methylotuvimicrobium buryatense" 5GB1C and designed a selection system for mutants unable to repress the mxaF promoter in the presence of lanthanum. Whole-genome resequencing for multiple lanthanide switch mutants identified several unique point mutations in a single gene encoding a TonB-dependent receptor, which we have named LanA. The LanA TonB-dependent receptor is absolutely required for the lanthanide switch and controls the expression of a small set of genes. While mutation of the lanA gene does not affect the amount of cell-associated lanthanum, it is essential for growth in the absence of the MxaF methanol dehydrogenase, suggesting that LanA is involved in lanthanum uptake to supply the XoxF methanol dehydrogenase with its critical metal ion cofactor. The discovery of this novel component of the lanthanide regulatory system highlights the complexity of this circuit and suggests that further components are likely involved.IMPORTANCE Lanthanide metals, or rare earth elements, are abundant in nature and used heavily in technological devices. Biological interactions with lanthanides are just beginning to be unraveled. Until very recently, microbial mechanisms of lanthanide metal interaction and uptake were unknown. The TonB-dependent receptor LanA is the first lanthanum receptor identified in a methanotroph. Sequence homology searches with known metal transporters and regulators could not be used to identify LanA or other lanthanide metal switch components, and this method for mutagenesis and selection was required to identify the receptor. This work advances the knowledge of microbe-metal interactions in environmental niches that impact atmospheric methane levels and are thus relevant to climate change.

Mycofactocin is essential for the establishment of methylotrophy in Mycobacterium smegmatis.

Mycobacterium smegmatis possesses (N,N-dimethyl-4-nitrosoaniline)-dependent (NDMA) methanol dehydrogenase (Mno) to establish methylotrophy by utilizing methanol as the source of both carbon and energy. In this study, we show that Mno forms decamer and has NADPH as the bound cofactor. Interestingly, Mno uses NDMA and not NADP+ as an electron acceptor in in vitro reactions. We further show that the operon mftAD required for the biosynthesis of mycofactocin, a ribosomally-synthesized electron carrier, is indispensable for the growth of M. smegmatis on methanol. Our data obtained from 2,6-Dichlorophenolindophenol reduction assays also suggest that Mno uses mycofactocin as an in vivo electron acceptor for the oxidation of methanol to formaldehyde. We thus provide here biochemical evidence for mycofactocin as an electron carrier in mycobacterial physiology.

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

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.

Characterization of the substrate scope of an alcohol dehydrogenase commonly used as methanol dehydrogenase.

Many alcohol dehydrogenases (ADHs) catalyze oxidation of a broad scope of alcohols. When an NAD-dependent ADH oxidizes methanol, albeit at a poor rate, it may be treated as methanol dehydrogenase (MDH). One ADH from Geobacillus stearothermophilus DSM 2334 (GsADH) has been widely used as MDH, but its actual substrate scope remains less characterized. Here we purified recombinant GsADH from Escherichia coli and determined its crystal structure. We collected kinetics data of this enzyme towards a number of short chain alcohols, and found that isopropanol is by far the most favorable substrate. Moreover, molecular docking analysis suggested that substrate preference is mainly attributed to the conformer energy of the protein-substrate complex. Our data clarified the substrate scope of GsADH and provided structural insights, which may facilitate more efficient cofactor regeneration and rational metabolic engineering.

Methylotrophy in Mycobacteria: Dissection of the Methanol Metabolism Pathway in Mycobacterium smegmatis.

The mycobacteria comprise both pathogenic and nonpathogenic bacteria. Although several features related to pathogenicity in various mycobacterial species, such as Mycobacterium tuberculosis, have been studied in great detail, methylotrophy, i.e., the ability of an organism to utilize single-carbon (C1) compounds as the sole source of carbon and energy, has remained largely unexplored in mycobacteria. Reports are available that suggest that mycobacteria, including M. tuberculosis and M. smegmatis, are capable of utilizing alternative C1 compounds to meet their carbon and energy requirements. However, physiological pathways that are functional in mycobacteria to utilize such carbon compounds are only poorly understood. Here we report the identification and characterization of the gene products required for establishing methylotrophy in M. smegmatis We present N,N-dimethyl-p-nitrosoaniline (NDMA)-dependent methanol oxidase (Mno) as the key enzyme that is essential for the growth of M. smegmatis on methanol. We show that Mno has both methanol and formaldehyde dehydrogenase activities in vitro Further, M. smegmatis is able to utilize methanol even in the absence of the major formaldehyde dehydrogenase MscR, which suggests that Mno is sufficient to dissimilate methanol and the resulting formaldehyde in vivo Finally, we show that M. smegmatis devoid of phosphoenolpyruvate carboxykinase, which has been shown to fix CO2 in M. tuberculosis, does not grow on methanol, suggesting that the final step of methanol utilization requires CO2 fixation for biomass generation. Our work here thus forms the first comprehensive report that explores methylotrophy in a mycobacterial species.IMPORTANCE Methylotrophy, the ability to utilize single-carbon (C1) compounds as the sole carbon and energy sources, is only poorly understood in mycobacteria. Both pathogenic and nonpathogenic mycobacteria, including Mycobacterium tuberculosis, are capable of utilizing C1 compounds to meet their carbon and energy requirements, although the precise pathways are not well studied. Here we present a comprehensive study of methylotrophy in Mycobacterium smegmatis With several genetic knockouts, we have dissected the entire methanol metabolism pathway in M. smegmatis We show that while methanol dissimilation in M. smegmatis differs from that in other mycobacterial species, the concluding step of CO2 fixation is similar to that in M. tuberculosis It is therefore both interesting and important to examine mycobacterial physiology in the presence of alternative carbon sources.

Engineering Artificial Fusion Proteins for Enhanced Methanol Bioconversion.

Methanol is a low-cost and abundantly available feedstock derived from natural gas and syngas. Although bioconversion holds promise for producing desired chemicals from methanol under economically viable operating conditions, the efficiency is limited by unfavorable kinetics of methanol oxidation and assimilation. Herein, artificial fusion proteins were engineered to enhance methanol bioconversion. Nicotinamide adenine dinucleotide (NAD)-dependent methanol dehydrogenase (Mdh), 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi) from different sources were first screened for catalytic activity. Next, we designed six fusion proteins using the best enzyme candidates and flexible linkers. Fusing Mdh with Hps or Hps-Phi increased the Vmax of methanol oxidation up to 5.8-fold, and enhanced methanol conversion to fructose-6-phosphate up to 1.3-fold. Interestingly, fusion engineering changed the polymerization states of proteins and produced larger multimers, which may be responsible for the changed catalytic characteristics. This fusion engineering approach can be coupled with other metabolic engineering strategies for enhanced methanol bioconversion to valuable chemicals.