Malyl-CoA lyase provides glycine/glyoxylate synthesis in type I methanotrophs.

The biochemical routes for assimilation of one-carbon compounds in bacteria require many clarifications. In this study, the role of malyl-CoA lyase in the metabolism of the aerobic type I methanotroph Methylotuvimicrobium alcaliphilum 20Z has been investigated by gene inactivation and biochemical studies. The functionality of the enzyme has been confirmed by heterologous expression in Escherichia coli. The mutant strain lacking Mcl activity demonstrated the phenotype of glycine auxotrophy. The genes encoding malyl-CoA lyase are present in the genomes of all methanotrophs, except for representatives of the phylum Verrucomicrobium. We suppose that malyl-CoA lyase is the enzyme that provides glyoxylate and glycine synthesis in the type I methanotrophs supporting carbon assimilation via the serine cycle in addition to the major ribulose monophosphate cycle.

Properties of Malic Enzyme from the Aerobic Methanotroph Methylosinus trichosporium.

Recombinant malic enzyme from the aerobic methanotroph Methylosinus trichosporium was obtained by heterologous expression in Escherichia coli and purified by affinity metal-chelating chromatography. The homohexameric enzyme of 6×80 kDa catalyzed the reversible reaction of oxidative decarboxylation of malate to pyruvate in the presence of mono- and divalent cations and NADP+ as a cofactor. The kcat/Km ratio indicated much higher catalytic efficiency of the malate decarboxylation reaction as compared with the pyruvate carboxylation reaction. Analysis of the protein sequence revealed that the C-region of the enzyme contains a large domain homologous to phosphoacetyltransferase, but no phosphoacetyltransferase activity was detected either for a full chimeric malic enzyme or for the C-end fragment obtained as a separate protein. This C-end domain promoted activity of the malic enzyme.

Serine-glyoxylate aminotranferases from methanotrophs using different C1-assimilation pathways.

The indicator enzyme of the serine pathway of assimilation of reduced C1 compounds, serine-glyoxylate aminotransferase (Sga), has been purified from three methane-oxidizing bacteria, Methylomicrobium alcaliphilum 20Z, Methylosinus trichosporium OB3b and Methylococcus capsulatus Bath. The native enzymes were shown to be dimeric (80 kDa, strain 20Z), tetrameric (~ 170 kDa, strain OB3b) or trimeric (~ 120 kDa, strain Bath). Sga from the three methanotrophs catalyse the pyridoxal phosphate-dependent transfer of an amino group from serine to glyoxylate and pyruvate; the enzymes from strains 20Z and Bath also transfer an amino group from serine to α-ketoglutarate and from alanine to glyoxylate. No other significant differences between the Sga from the three methanotrophs were found. The three methanotrophic Sga have their highest catalytic efficiencies in the reaction between glyoxylate and serine, which is in agreement with their function to provide circulation of the serine assimilation pathway.The disruption of the sga gene in Mm. alcaliphilum resulted in retardation of growth rate of the mutant cells and in a prolonged lag-phase after passaging from methane to methanol. In addition, the growth of the mutant strain is accompanied by formaldehyde accumulation in the culture liquid. Hence, Sga is important in the serine cycle of type I methanotrophs and this pathway could be related to the removal of excess formaldehyde and/or energy regulation.

Biochemical Properties and Phylogeny of Hydroxypyruvate Reductases from Methanotrophic Bacteria with Different C1-Assimilation Pathways.

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C1-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C1-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.

Characterization of Two Recombinant 3-Hexulose-6-Phosphate Synthases from the Halotolerant Obligate Methanotroph Methylomicrobium alcaliphilum 20Z.

Two key enzymes of the ribulose monophosphate (RuMP) cycle for formaldehyde fixation, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexulose isomerase (PHI), in the aerobic halotolerant methanotroph Methylomicrobium alcaliphilum 20Z are encoded by the genes hps and phi and the fused gene hps-phi. The recombinant enzymes HPS-His6, PHI-His6, and the two-domain protein HPS-PHI were obtained by heterologous expression in Escherichia coli and purified by affinity chromatography. PHI-His6, HPS-His6 (2 × 20 kDa), and the fused protein HPS-PHI (2 × 40 kDa) catalyzed formation of fructose 6-phosphate from formaldehyde and ribulose-5-phosphate with activities of 172 and 22 U/mg, respectively. As judged from the kcat/Km ratio, HPS-His6 had higher catalytic efficiency but lower affinity to formaldehyde compared to HPS-PHI. AMP and ADP were powerful inhibitors of both HPS and HPS-PHI activities. The two-domain HPS-PHI did not show isomerase activity, but the sequences corresponding to its HPS and PHI regions, when expressed separately, were found to produce active enzymes. Inactivation of the hps-phi fused gene did not affect the growth rate of the mutant strain. Analysis of annotated genomes revealed the separately located genes hps and phi in all the RuMP pathway methylotrophs, whereas the hps-phi fused gene occurred only in several methanotrophs and was absent in methylotrophs not growing under methane. The significance of these tandems in adaptation and biotechnological potential of methylotrophs is discussed.

[Homo- and heterologous reporter proteins for evaluation of promoter activity in Methylomicrobium alcaliphilum 20Z].

A number of vectors were constructed based on the plasmid from the broad range of pMHA200 hosts. Also, the expression of some key genes of the haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z was studied. The activities of the promoter regions of genes for hexulose phosphate synthase, glutamine synthetase, and glucokinase, as well as the promoter of the ectABC-ask operon, which encodes enzymes for osmoprotectant ectoine biosynthesis, were evaluated with the use of the gfp gene; the evaluation was proven to be ineffective. Conversely, glucokinase and a heterologous enzyme of chloramphenicol acetyltransferase were useful for the evaluation of promoter activity. In M. alcaliphilum 20Z cells, the expression level of chloramphenicol acetyltransferase transcribed from the methanol dehydrogenase promoter was higher as compared with that of glucokinase. This seems to be due to a regulatory mechanism for homologous protein expression. The introduction of a synthetic nucleotide sequence forming the secondary structure in the 5' untranslated region of the glucokinase mRNA resulted in an increase of this enzyme level. This is the first attempt to use M. alcaliphilum 20Z for homo- and heterologous protein expression.

Properties of recombinant ATP-dependent fructokinase from the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z.

In the cluster of genes for sucrose biosynthesis and cleavage in Methylomicrobium alcaliphilum 20Z, a gene whose encoded sequence showed high similarity to sugar kinases of the ribokinase family was found. By heterologous expression of this gene in Escherichia coli cells and following metal chelate affinity chromatography, the electrophoretically homogenous recombinant enzyme with six histidine residues on the C-end was obtained. The enzyme catalyzes ATP-dependent phosphorylation of fructose into fructose-6-phosphate but is not active with other sugars as phosphoryl acceptors. The fructokinase of M. alcaliphilum 20Z is most active in the presence of Mn(2+) at pH 9.0 and 60°C, being inhibited by ADP (K(i) = 2.50 ± 0.03 mM). The apparent K(m) values for fructose and ATP are 0.26 and 1.3 mM, respectively; the maximal activity is 141 U/mg protein. The enzyme shows the highest similarity of translated amino acid sequence with putative fructokinases of methylotrophic and autotrophic proteobacteria whose fruK gene is located in the gene cluster of sucrose biosynthesis. The involvement of fructokinase in sucrose metabolism in M. alcaliphilum 20Z and other methanotrophs and autotrophs is discussed.

Simultaneous determination of molybdenum(VI) and tungsten(VI) and its application in elemental analysis of polyoxometalates.

Spectrophotometric determination of molybdenum(VI) and tungsten(VI) with application of Artificial Neural Networks is proposed and it was applied for elemental analysis of solid polyoxometalates. Better results in comparison with previously those achieved by previous published method were demonstrated. MALDI-TOF Mass Spectrometry was tested for possible determination of molecular weight of polyoxometalates utilizing different matrices. Phenomena observed during desorption-ionisation processes are discussed. LDI-TOF MS was found to be suitable for the determination of Mo:W ratio in polyoxometalates as a rapid screening method to follow synthetic procedure.

Chemometric and statistical analysis of polyoxometalate interaction with lanthanide(III) ions.

Application of CAT (computer-assisted target factor analysis) for quality assessment and quality control in spectroscopy is demonstrated. As a working example, interaction of polyoxometalates (POM) with neodymium in aqueous solutions is followed by UV-Vis spectroscopy. CAT is combined with Monte Carlo and resampling techniques (i.e. threshold bootstrap methods) in order to capture the complete distribution of suitable interpretations of the systems under study. Decisions during the evaluation process are based on statistical criteria conditional on the empirical distributions obtained from CAT. Single component spectra of the Nd-POM solution species are extracted and species concentrations in the experimental samples are obtained together with their respective confidence limits.