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 Ca(2+)-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 La(3+)-dependent. Despite the absence of Ca(2+) in the medium strain AM1 was able to grow on methanol in the presence of La(3+). Addition of La(3+) 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 La(3+), and its N-terminal amino acid sequence corresponded to that of XoxF1. The enzyme contained La(3+) as a cofactor. The ΔmxaF mutant strain could not grow on methanol in the presence of Ca(2+), but was able to grow after supplementation with La(3+). Taken together, these results show that XoxF1 participates in methanol metabolism as a La(3+)-dependent MDH in strain AM1.

Molecular structure and gene analysis of Ce3+ -induced methanol dehydrogenase of Bradyrhizobium sp. MAFF211645.

The molecular structure and nucleotide sequence of Ce(3+)-induced methanol dehydrogenase (MDH) of Bradyrhizobium sp. MAFF211645 were investigated. The addition of 30 µM Ce(3+) to 1/10 nutrient broth containing 0.5% methanol remarkably increased MDH activity. Furthermore, La(3+) increased MDH activity, but other heavier rare earth and metal elements did not have the same effect. MDH increased by Ce(3+) was purified by sequential column chromatography, and the purified MDH migrated as a single band with an apparent molecular weight of 68 kDa on SDS-PAGE. The apparent molecular weight of native MDH was estimated to be 108,000 by gel chromatography. The MDH was comprised of two identical subunits. N-terminal 23-amino acid sequence, 1-NDELHKMAQNPKDWVMPAGDYAN-23, of the purified MDH exhibited 91.3% identity to that of the MDH large subunit-like protein encoded by mxaF' of Bradyrhizobium japonicum USDA110. Nucleotide sequencing of the MDH gene of strain MAFF211645 yielded a deduced amino acid sequence comprising 601 amino acid residues, an N-terminal signal peptide, and a mature MDH comprising 578 amino acid residues with a predicted molecular mass of 62,918 Da. Further analysis of the deduced amino acid sequence of mature MDH revealed that the functional amino acids in its active site, such as two adjacent Cys residues, and bacterial quinoprotein signatures 1 and 2 were conserved. These results indicate that Ce(3+)-induced MDH encoded by mxaF' may be involved in methanol metabolism in Bradyrhizobium sp. MAFF211645.

Molecular structure of La3+-induced methanol dehydrogenase-like protein in Methylobacterium radiotolerans.

La(3+) and not Ca(2+) increases methanol dehydrogenase (MDH) activity in Methylobacterium radiotolerans NBRC15690. La(3+)- and Ca(2+)-MDH-like proteins were found to be homodimeric (α(2)) and heterotetrameric (α(2)ß(2)), respectively. N-terminal amino acid sequences of these proteins revealed that La(3+)- and Ca(2+)-MDH-like proteins were encoded by xoxF and mxaFI, respectively.

Ce³+-induced exopolysaccharide production by Bradyrhizobium sp. MAFF211645.

Ce³+, a rare earth element (REE), has been widely used in high-technology industries. Despite the importance of Ce³+ in the fields of chemistry and physics, the role of Ce³+ in biology has been ignored. To investigate physiological effects of Ce³+ on microorganisms, we screened microorganisms that showed peculiar growth in the presence of Ce³+. We isolated a free-living soil bacterium that produced exopolysaccharide (EPS) around its colonies on 1/100 nutrient agar with 30 µM CeCl3 or 1.0% D-mannitol. The bacterium was identified as Bradyrhizobium sp. by morphological, biochemical, and physiological tests as well as 16S rDNA sequence analysis. La³+, Pr³+, and Nd³+ also induced EPS production in large quantities, while Sm³+ did in small amounts. However, other heavier REEs from Eu³+ to Lu³+, and metals such as Na+, Al³+, K+, Ca²+, V³+, Cr³+, Co²+, Ni²+, Sr²+, Ba²+, and Pb²+ did not induce EPS production. The mean molecular weight of EPS was estimated to be approximately 1 x 106 by Sepharose CL-4B column chromatography. TLC revealed that EPS was composed of L-rhamnose. Quantitative analysis of alditol acetate derivatives of acid hydrolyzate of EPS by GLC revealed that EPS was composed of more than 95% L-rhamnose, indicating that this EPS was a rhamnan. The spectrum of FT-IR of the rhamnan demonstrated that L-rhamnose residues in the rhamnan were α-linked. GC/MS analysis of methylated alditol acetate derivatives of the rhamnan demonstrated that it was composed of main chain α-(1→4)-linked L-rhamnopyranosyl residues. From spectral analyses of ¹H-NMR and FT-IR, EPS produced in the presence of 1.0% D-mannitol was found to be structurally similar to rhamnans.

Differential recognition of citrate and a metal-citrate complex by the bacterial chemoreceptor Tcp.

The chemoreceptor Tcp of Salmonella enterica serovar Typhimurium can sense citrate and a metal-citrate complex as distinct attractants. In this study, we tried to investigate the molecular mechanism of this discrimination. That citrate binds directly to Tcp was verified by the site-specific thiol modification assays using membrane fractions prepared from Escherichia coli cells expressing the mutant Tcp receptors in which single Cys residues were introduced at positions in the putative ligand-binding pocket. To determine the region responsible for the ligand discrimination, we screened for mutations defective in taxis to magnesium in the presence of citrate. All of the isolated mutants from random mutagenesis with hydroxylamine were defective in both citrate and metal-citrate sensing, and the mutated residues are located in or near the alpha1-alpha2 and alpha3-alpha4 loops within the periplasmic domain. Further analyses with site-directed replacements around these regions demonstrated that the residue Asn(67), which is presumed to lie at the subunit interface of the Tcp homodimer, plays a critical role in the recognition of the metal-citrate complex but not that of citrate. Various amino acids at this position differentially affect the citrate and metal-citrate sensing abilities. Thus, for the first time, the abilities to sense the two attractants were genetically dissected. Based on the results obtained in this study, we propose models in which the discrimination of the metal-citrate complex from citrate involves cooperative interaction at Asn(67) and allosteric switching.

Immobilization of Saccharomyces cerevisiae cystathionine gamma-lyase and application of the product to cystathionine synthesis.

Cystathionine gamma-lyase of Saccharomyces cerevisiae was immobilized to aminohexyl-Sepharose through the cofactor pyridoxal 5'-phosphate and was characterized with respect to its cystathionine gamma-synthase activity. The immobilized product was so stable that it repeatedly catalyzed as many as five cycles of the reaction without losing activity.

Comparative characterization of the oah2 gene homologous to the oah1 of Thermus thermophilus HB8.

The oah2 gene homologous to the oah1 of Thermus thermophilus HB8 was cloned and sequenced. It comprised 1,236 bp encoding a protein of 412 amino acid residues and was overexpressed. The gene product, also having O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity, was purified to homogeneity and characterized comparatively with the oah1 product. The two proteins shared many characteristics.

Distribution of ytterbium (Yb) in cells of Streptomyces sp. YB-1 which can accumulate Yb, and reusability of cells and cell membrane as bioadsorbent for Yb.

The cells of Streptomyces sp. YB-1 adsorbed 4-6 mg ytterbium (Yb) per g dry weight. The Yb contents of the cell wall fraction, cell-free extract, and cell membrane fraction were 11%, 2%, and 87%, respectively. The Yb content in the cell membrane fraction was 20-25 mg per g dry weight. The adsorbed Yb could be quantitatively desorbed by treating the cell membrane fraction with 1 mM EDTA and 1 M HCl at 37 degrees C for 4 h. Treatment with 1 M NaOH caused Yb desorption to some extent. Treatments with proteinase K, lysozyme, 0.5% Triton X-100, 0.4% sodium dodecyl sulfate, and 1 M NaCl did not cause Yb desorption. Elemental analysis of Yb-adsorbed materials after removal of proteins and then extraction of lipids from the membrane fraction revealed that the molar ratio of Yb and P in the materials was about 1:1. The cells and the membrane fraction could be used repeatedly as a bioadsorbent for Yb.

Conversion of the aminocrotonate intermediate limits the rate of gamma-elimination reaction catalyzed by L-cystathionine gamma-lyase of the yeast Saccharomyces cerevisiae.

L-Cystathionine gamma-lyase [EC 4.4.1.1] of Saccharomyces cerevisiae was shown to bind cofactor pyridoxal 5'-phosphate, up to 2 molecules/subunit. The association constants of the enzyme for the cofactor were estimated to be 3.67 x 10(5) M(-1) and 9.05 x 10(3) M(-1). However, the latter value was too small for the binding to play a catalytic role. Changes in the absorption spectra of the enzyme in gamma-elimination reaction mixtures with various amino acids as substrates were observed at 10 degrees C to elucidate the reaction mechanism of the enzyme. The enzyme formed a chromophore exhibiting absorption at approximately 480 nm, which is characteristic of an aminocrotonate intermediate with O-succinyl-L-homoserine, L-cystathionine, L-homoserine, or O-acetyl-L-homoserine, at rates in this order. The intermediate was consumed at much lower rates than those of formation. The order of the rates of consumption was the same as the order of the formation rates and the order of the gamma-elimination activity of the enzyme with the above-mentioned substrates. These results strongly suggested that the intermediate was essential for gamma-elimination and that the reaction was rate-limited by its conversion into the product alpha-ketobutyrate. L-Cysteine sensitively inhibited the alpha, gamma-elimination activity of the enzyme, and also retarded the formation of the chromophore when it was provided to the enzyme together with a substrate. The reason for these phenomena is discussed.

Cysteine synthase of an extremely thermophilic bacterium, Thermus thermophilus HB8.

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source. Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography). The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism. Molecular masses of 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure. The enzyme was stable at 70 degrees C at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80 degrees C with 10 mm dithiothreitol. The enzyme was also quite stable at pH 8-12 (50 degrees C, 30 min). It had an apparent Km of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a Vmax of 435 micromol/min/mg of protein. The apparent Km for sulfide was approximately 50 microM (with 20 mM acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine. The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5'-phosphate as a cofactor. The apo-enzyme showed an apparent Km of 29 microM for the cofactor at pH 8. Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme. The meaning of a very high enzyme content in the cell is discussed.

Substrate inhibition of L-cysteine alpha,beta-elimination reaction catalyzed by L-cystathionine gamma-lyase of Saccharomyces cerevisiae.

The alpha,beta-elimination of L-cysteine catalyzed by Saccharomyces cerevisiae L-cystathionine gamma-lyase (EC 4.4.1.1) was inhibited by the substrate. The absorption spectrum of the holoenzyme in the presence of L-cysteine showed that the substrate inhibition observed in this reaction was due mainly to removal of the cofactor.