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.

[Biosynthesis of secondary metabolites in methanotrophs: biochemical and genetic aspects (review)].

The review summarizes the data on the metabolic potential of methanotrophs as producers of biopolymers, alternative biofuel, bioprotectants, and other secondary metabolites. The work provides the examples of modern 'omic' technologies used for genetic engineering of efficient methanotrophic producers.

Role of EctR as transcriptional regulator of ectoine biosynthesis genes in Methylophaga thalassica.

In the halophilic aerobic methylotrophic bacterium Methylophaga thalassica, the genes encoding the enzymes for biosynthesis of the osmoprotectant ectoine were shown to be located in operon ectABC-ask. Transcription of the ect-operon was started from the two promoters homologous to the σ(70)-dependent promoter of Escherichia coli and regulated by protein EctR, whose encoding gene, ectR, is transcribed from three promoters. Genes homologous to ectR of methylotrophs were found in clusters of ectoine biosynthesis genes in some non-methylotrophic halophilic bacteria. EctR proteins of methylotrophic and heterotrophic halophiles belong to the MarR-family of transcriptional regulators but form a separate branch on the phylogenetic tree of the MarR proteins.

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.

Characterization of recombinant fructose-1,6-bisphosphate aldolase from Methylococcus capsulatus Bath.

The gene fba from the thermotolerant obligate methanotroph Methylococcus capsulatus Bath was cloned and expressed in Escherichia coli BL21(DE3). The fructose-1,6-bisphosphate aldolase (FBA) carrying six His on the C-end was purified by affinity metal chelating chromatography. The Mc. capsulatus FBA is a hexameric enzyme (240 kDa) that is activated by Co2+ and inhibited by EDTA. The enzyme displays low K(m) to fructose-1,6-bisphosphate (FBP) and higher K(m) to the substrates of aldol condensation, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The FBA also catalyzes sedoheptulose-1,7-bisphosphate cleavage. The presence of Co2+ in the reaction mixture changes the kinetics of FBP hydrolysis and is accompanied by inhibition of the reaction by 2 mM FBP. Phylogenetically, the Mc. capsulatus enzyme belongs to the type B of class II FBAs showing high identity of translated amino acid sequence with FBAs from autotrophic bacteria. The role of the FBA in metabolism of Mc. capsulatus Bath, which realizes simultaneously three C(1) assimilating pathways (the ribulose monophosphate, the ribulose bisphosphate, and the serine cycles), is discussed.

[Moderately haloalkaliphilic aerobic methylobacteria].

Aerobic methylobacteria utilizing oxidized and substituted methane derivatives as carbon and energy sources are widespread in nature and involved in the global carbon cycle, being a unique biofilter on the path of these C1 compounds from different ecosystems to the atmosphere. New data on the biological features of moderately halophilic, neutrophilic, and alkaliphilic methylobacteria isolated from biotopes with higher osmolarity (seas, saline and soda lakes, saline soils, and deteriorating marble) are reviewed. Particular attention is paid to the latest advances in the study of the mechanisms of osmoadaptation of aerobic moderately haloalkaliphilic methylobacteria: formation of osmolytes, in particular, molecular and genetic aspects of biosynthesis of the universal bioprotectant ectoine. The prospects for further studies of the physiological and biochemical principles of haloalkalophily and for the application of haloalkaliphilic aerobic methylobacteria in biosynthesis and biodegradation are discussed.

Cloning, purification, and characterization of diaminobutyrate acetyltransferase from the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z.

L-2,4-Diaminobutyrate (DAB) acetyltransferase (DABAcT) catalyzes one of the key reactions of biosynthesis of the bacterial osmoprotectant ectoine--acetylation of L-2,4-DAB yielding Ngamma-acetyl-2,4-DAB. Gene ectA encoding DABAcT was cloned from DNA of the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z and expressed in Escherichia coli with an additional six His residues at the C-terminus. Homogeneous enzyme preparation with specific activity 200 U/mg was obtained by affinity metal-chelating chromatography. DABAcT was found to be a homodimer with molecular mass 40 kD. The enzyme is most active at pH 9.5 and 20 degrees C, and its activity increased threefold in the presence of 0.1-0.2 M NaCl or 0.2 M KCl. The Km values of recombinant DABAcT measured at the optimal pH and temperature in the presence of 0.2 M KCl were 460 and 36.6 microM for L-2,4-DAB and acetyl-CoA, respectively. The enzyme is specific for L-2,4-DAB and acetyl-CoA and is also active against propionyl-CoA (20%). Zn2+ and Cd2+ at 1 mM concentration completely inhibit the recombinant enzyme; 10 mM ATP inhibits 26% of the enzyme activity, whereas EDTA, o-phenanthroline, ADP, NAD(P), and NAD(P)H do not significantly effect the enzyme activity. The possible participation of DABAcT in regulation of ectoine biosynthesis in M. alcaliphilum 20Z is discussed.

[Synthesis of osmoprotectors by halophilic and alkalophilic methanotrophs]. / Sintez osmoprotektorov galofil'nymi i aklalofil'nymi metanotrofami.

The 1H-NMR analysis of methanol extracts of halophilic and halotolerant alkaliphilic methanotrophs isolated from the soda lakes of Southern Transbaikal and Tuva showed that bacterial cells grown at an optimum salinity accumulated mainly sucrose and 5-oxo-1-proline, whereas cells adapted to 0.5-1.0 M NaCl additionally synthesized ectoine. A more detailed study showed that nitrogen deficiency in the growth medium of Methylobacter alcaliphilus 20Z decreased the synthesis of nitrogen-containing osmoprotectants, ectoine and 5-oxo-1-proline. M. alcaliphilus 20Z cells exhibited activities of UDP-glucose pyrophosphorylase and sucrose-phosphate synthase involved in sucrose synthesis. Glutamine synthetase in vitro did not require NH4+ ions, which implies that this enzyme is involved in 5-oxo-1-proline synthesis. Cells grown at high salinity exhibited elevated levels of aspartate kinase, aspartate-semialdehyde dehydrogenase, and ectoine synthase. This suggests that ectoine is synthesized via aspartate and aspartate-semialdehyde, i.e., via the route earlier established for extremely halophilic bacteria.