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The indicator enzyme of the serine pathway of assimilation of reduced C 1 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.

In methylotrophic bacteria, which use one-carbon (C1) compounds as a carbon source, methanol is oxidized by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) enzymes. Methylotrophic genomes generally encode two distinct MDHs, MxaF and XoxF. MxaF is a well-studied, calcium-dependent heterotetrameric enzyme whereas XoxF is a lanthanide-dependent homodimer. Recent studies suggest that XoxFs are likely the functional MDHs in many environments. In methanotrophs, methylotrophs that utilize methane, interactions between particulate methane monooxygenase (pMMO) and MxaF have been detected. To investigate the possibility of interactions between pMMO and XoxF, XoxF was isolated from the methanotroph Methylomicrobium buryatense 5GB1C (5G-XoxF). Purified 5G-XoxF exhibits a specific activity of 0.16 μmol DCPIP reduced min -1  mg -1 . The 1.85 Å resolution crystal structure reveals a La(III) ion in the active site, in contrast to the calcium ion in MxaF. The overall fold is similar to other MDH structures, but 5G-XoxF is a monomer in solution. An interaction between 5G-XoxF and its cognate pMMO was detected by biolayer interferometry, with a K D value of 50 ± 17 μM. These results suggest an alternative model of MDH-pMMO association, in which a XoxF monomer may bind to pMMO, and underscore the potential importance of lanthanide-dependent MDHs in biological methane oxidation.

Four enzymes involved in sucrose metabolism: sucrose phosphate synthase (Sps), sucrose phosphate phosphatase (Spp), sucrose synthase (Sus) and fructokinase (FruK), were obtained as his-tagged proteins from the moderately thermophilic methanotroph Methylocaldum szegediense O12. Sps, Spp, FruK and Sus demonstrated biochemical properties similar to those of other bacterial counterparts, but the translated amino acid sequences of Sps and Spp displayed high divergence from the respective microbial enzymes. The Sus of M. szegediense O12 catalyzed the reversible reaction of sucrose cleavage in the presence of ADP or UDP and preferred ADP as a substrate, thus implying a connection between sucrose and glycogen metabolism. Sus-like genes were found only in a few methanotrophs, whereas amylosucrase was generally used in sucrose cleavage in this group of bacteria. Like other microbial fructokinases, FruK of M. szegediense O12 showed a high specificity to fructose.

Aerobic bacteria utilizing methane as the carbon and energy source do not use sugars as growth substrates but possess the gene coding for glucokinase (Glk), an enzyme converting glucose into glucose 6-phosphate. Here we demonstrate the functionality and properties of Glk from an obligate methanotroph Methylomicrobium alcaliphilum 20Z. The recombinant Glk obtained by heterologous expression in Escherichia coli was found to be close in biochemical properties to other prokaryotic Glks. The homodimeric enzyme (2 × 35 kDa) catalyzed ATP-dependent phosphorylation of glucose and glucosamine with nearly equal activity, being inhibited by ADP (K i  = 2.34 mM) but not affected by glucose 6-phosphate. Chromosomal deletion of the glk gene resulted in a loss of Glk activity and retardation of growth as well as in a decrease of intracellular glycogen content. Inactivation of the genes encoding sucrose phosphate synthase or amylosucrase, the enzymes involved in glycogen biosynthesis via sucrose as intermediate, did not prevent glycogen accumulation. In silico analysis revealed glk orthologs predominantly in methanotrophs harboring glycogen synthase genes. The data obtained suggested that Glk is implicated in the regulation of glycogen biosynthesis/degradation in an obligate methanotroph.

Recombinant acetate kinase (AcK) was obtained from the aerobic haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z by heterologous expression in Escherichia coli and purification by affinity chromatography. The substrate specificity, the kinetics and oligomeric state of the His6-tagged AcK were determined. The M. alcaliphilum AcK (2 × 45 kDa) catalyzed the reversible phosphorylation of acetate into acetyl phosphate and exhibited a dependence on Mg(2+) or Mn(2+) ions and strong specificity to ATP/ADP. The enzyme showed the maximal activity and high stability at 70 °C. AcK was 20-fold more active in the reaction of acetate synthesis compared to acetate phosphorylation and had a higher affinity to acetyl phosphate (K m 0.11 mM) than to acetate (K m 5.6 mM). The k cat /K m ratios indicated that the enzyme had a remarkably high catalytic efficiency for acetate and ATP formation (k cat/K m = 1.7 × 10(6)) compared to acetate phosphorylation (k cat/K m = 2.5 × 10(3)). The ack gene of M. alcaliphilum 20Z was shown to be co-transcribed with the xfp gene encoding putative phosphoketolase. The Blast analysis revealed the ack and xfp genes in most genomes of the sequenced aerobic methanotrophs, as well as methylotrophic bacteria not growing on methane. The distribution and metabolic role of the postulated phosphoketolase shunted glycolytic pathway in aerobic C1-utilizing bacteria is discussed.

In methanol-limited continuous cultures of the obligate methylotrophic bacterium Methylobacillus flagellatum grown at rates from 0.05 to 0.63 h-1, and also in an oxyturbidostat culture of M. flagellatum growing at the rate of 0.73 h-1, levels of methanol dehydrogenase, enzymes of formaldehyde oxidation (both linear and cyclic) and assimilation (RuMP cycle), a number of intermediary metabolism and TCA cycle enzymes and also 'dye-linked' formaldehyde dehydrogenase were determined. It was shown that the activities of dissimilatory enzymes, with the exception of 'dye-linked' formaldehyde dehydrogenase, decreased with increasing growth rate. Activities of assimilative enzymes and activities of the TCA cycle enzymes detected as well as the 'dye-linked' formaldehyde dehydrogenase activity, increased with increasing growth rate. A periplasmic location was shown for the latter enzyme and a role in formaldehyde detoxification was proposed.

Using 13C labeling and NMR spectroscopy we have determined biosynthetic precursors of pyrroloquinoline quinone (PQQ) in two closely related serine-type methylotrophs, Methylobacterium AM1 and Hyphomicrobium X. Analysis of the 13C-labeling data revealed that PQQ is constructed from two amino acids: the portion containing N-6,C-7, 8, 9 and the two carboxylic acid groups, C-7' and 9', is derived-intact -from glutamate. The remaining portion is derived from tyrosine; the phenol side chain provides the six carbons of the ring containing the orthoquinone, whereas internal cyclization of the amino acid backbone forms the pyrrole-2-carboxylic acid moiety. This is analogous to the cyclization of dopaquinone to form dopachrome. Dopaquinone is a product of the oxidation of tyrosine (via dopa) in reactions catalyzed by monophenol monooxygenase (EC 1.14.18.1). Starting with tyrosine and glutamate, we will discuss possible biosynthetic routes to PQQ.

Hydrogenase activity in cells of the nitrogen-fixing methane-oxidizing bacterium strain 41 of the Methylosinus type increased markedly when growth was dependent upon the fixation of gaseous nitrogen. A direct relationship may exist between hydrogenase and nitrogenase in this bacterium. Acetylene reduction was supported by the presence of hydrogen gas.

The phenotype of mutants unable to synthesize PQQ is analyzed for different categories of methylotrophic bacteria. The advantages offered by strains dissimilating methylamine through methylated amino-acids are discussed. In M.organophilum, 40% of the mutants unable to grow in methanol medium but with normal methylamine utilization, were affected in PQQ metabolism. The genetic properties of M. organophilum useful to study PQQ mutants are discussed, mainly the use of pSUP106 to create insertion mutations in the bacterial chromosome and to replace wild-type genes by modified genes. An example is given of the possibility to create R' plasmids containing large fragments of M.organophilum DNA. Some physiological properties of a PQQ mutant are described, regarding growth kinetics, PQQ uptake and accumulation.