Taketomo Fujiwara

Occurrence of peptidyl d-amino acids in soluble fractions of several eubacteria, archaea and eukaryotes

The occurrence of peptidyl D-amino acids in the aqueous soluble fractions was investigated in various eubacteria, some archaea and some eukaryotes. The contents of the D-enantiomers of serine, alanine, proline, glutamate (glutamine), aspartate (asparagine) and phenylalanine were determined with cell- and tissue-extracts, by means of acid hydrolysis and high-performance liquid chromatography. The rate of D-enantiomer (%, the ratio in molar concentration of a D-amino acid to the total of the D-amino acid and the corresponding L-amino acid) of alanine and glutamate were high in some Gram-positive eubacteria: 11.7% in Staphylococcus epidermidis and 10.3% in Streptococcus pyogenes for alanine, and 22.3% for glutamate in Bacillus YN-1. The D-glutamate content was also high (8.0%) in the Gram-negative eubacterium, Thiobacillus ferrooxidans. D-Aspartate was common, as was D-glutamate: the highest D-aspartate content was detected in an archaeum, Pyrobaculum islandicum (4.0%). However, the content of D-aspartate was low, 0.2-1.8% in most other bacteria. The presence of D-serine was shown in some organisms, but that of D-proline was scarce. The D-enantiomer of phenylalanine was not detected in any of the organisms examined. These results indicate that of the bacteria examined herein most Gram-negative and some Gram-positive eubacteria, as well as archaea contain only low levels of D-amino acids in the soluble peptidyl fraction, and the levels were comparable to those in eukaryotes examined. To our knowledge, the general presence of peptidyl D-amino acids in these organisms, especially archaea and eukaryotic cells including those from rat liver tissues, has been shown here for the first time.

Occurrence of D-amino acids in a few archaea and dehydrogenase activities in hyperthermophile Pyrobaculum islandicum

The contents of D-enantiomers of serine, alanine, proline, glutamate (glutamine) and aspartate (asparagine) were examined in the membrane fractions, soluble proteins and free amino acids from some species of archaea, Pyrobaculum islandicum, Methanosarcina barkeri and Halobacterium salinarium. Around 2% (D/D+L) of D-aspartate was found in the membrane fractions. In the soluble proteins, the D-amino acid content was higher in P. islandicum than that in the other archaeal cells: the concentrations in P. islandicum were 3 and 4% for D-serine and D-aspartate, respectively. High concentrations of free D-amino acids were found in P. islandicum and H. salinarium; the concentrations of D-serine (12-13%), D-aspartate (4-7%) and D-proline (3-4%) were higher than those of D-alanine and D-glutamate. This result showed a resemblance between these archaea and not bacterial, but eukaryotic cells. The presence of D-amino acids was confirmed by their digestion with D-amino acid oxidase and D-aspartate oxidase. The occurrence of D-amino acids was also confirmed by the presence of activities catalyzing catabolism of D-amino acids in the P. islandicum homogenate, as measured by 2-oxo acid formation. The catalytic activities oxidizing D-alanine, D-aspartate and D-serine at 90 degrees C were considerably high. Under anaerobic conditions, dehydrogenase activities of the homogenate were 69, 84 and 30% of the above oxidase activities toward D-alanine, D-aspartate and D-serine, respectively. Comparable or higher dehydrogenase activities were also detected with these D-amino acids as substrate by the reduction of 2, 6-dichlorophenolindophenol. No D-amino acid oxidase activity was detected in the homogenates of M. barkeri and H. salinarium.

Molecular cloning of the cytochrome aa3 gene from the archaeon (Archaebacterium) Halobacterium halobium

A novel aa3-type cytochrome oxidase from the extremely halophilic archaeon, Halobacterium halobium, differs significantly from those of other prokaryotic and eukaryotic cytochrome oxidases (Fujiwara, T., Fukumori, Y., and Yamanaka, T. (1989) J. Biochem. 105, 287-292). In the present study, we cloned and sequenced the gene which encodes the cytochrome aa3 by using the polymerase chain reaction methods. The deduced amino acid sequence of subunit I of H. halobium cytochrome aa3 was more similar to that of subunit I of the eukaryotic cytochrome (44%, maize mitochondria) than that of the cytochrome from other bacteria (36%, Paracoccus denitrificans). The consensus sequence in putative metal binding residues is well-conserved also in H. halobium cytochrome aa3.

Studies on the respiratory system in alkaliphilic Bacillus; a proposed new respiratory mechanism

Abstract Respiratory electron transfer systems in two alkaliphilic Bacillus species, YN-1 and YN-2000, were investigated. In the cyanide-sensitive pathway of the obligate alkaliphilic Bacillus YN-1, the terminal enzyme was a caa3-type cytochrome c oxidase constituting up to just 10% of the total oxygen-reducing activity, while 90% of the respiratory activity was due to cyanide-insensitive, nonproteinaceous material with a molecular weight of 662. These results were consistent with the cyanide-tolerant growth of the bacterium. The molecular and catalytic properties of the nonproteinaceous material were not identical with those of menaquinones extracted from the bacterium. Furthermore, the nonproteinaceous material was also found in the facultative alkaliphilic Bacillus YN-2000, when that bacterium was cultivated in alkaline conditions. A new respiratory oxygen-reducing mechanism comprising a nonproteinaceous component and a catalase is proposed for these alkaliphilic Bacillus species.

The amino acid sequence of Nitrosomonas europaea cytochrome c-552

The complete amino acid sequence of cytochrome c-552 derived from the chemoautotrophic ammonia-oxidizing bacterium Nitrosomonas europaea was determined. The cytochrome consisted of 81 amino acid residues, and its molecular weight was calculated to be 9098 including heme c. Although the sequence of cytochrome c-552 was highly homologous to those of cytochromes c-551, which were known as the electron-donating components to dissimilatory nitrite reductase in pseudomonads, cytochrome c-552 differed from cytochrome c-551 in two points: (1) the sequence of cytochrome c-552 was shorter by two amino acid residues than that of cytochrome c-551 at the N-terminus and (2) one amino acid insertion was present in cytochrome c-552.

Purification, primary structure, and evolution of cytochrome c-550 from the magnetic bacterium, Magnetospirillum magnetotacticum

Cytochrome c-550 was purified from Magnetospirillum magnetotacticum to an electrophoretically homogeneous state, and some of its properties were determined. The cytochrome showed absorption peaks at 528 and 409 nm in the oxidized form, and at 550, 521, and 414 nm in the reduced form. Its midpoint redox potential at pH 7.0 was determined to be +289 mV. The primary structure of cytochrome c-550 was determined. Cytochrome c is composed of 97 amino acid residues, and its molecular weight was calculated to be 10,873, including heme c. Its primary structure is very similar to those of Rhodospirillum fulvum and Rhodospirillum molischianum cytochromes c2, suggesting that M. magnetotacticum is phylogenetically related to photosynthetic bacteria.

Reactivity of the co-type and baa3-type cytochrome c oxidases from Pseudomonas aeruginosa with different endogenous cytochromes c.

The reactivity between different cytochromes c purified from Pseudomonas aeruginosa cells grown aerobically in the absence of nitrate and isolated cytochromes co and baa3 was determined. The P. aeruginosa cytochrome co reacted most rapidly with the membrane-bound cytochrome c-551 among three c-type cytochromes analyzed, whereas the cytochrome baa3 reacted best with the membrane-bound cytochrome c-555. The results indicated that two terminal electron transfer systems are present in aerobic P. aeruginosa: one contains the cytochrome c-551 and cytochrome co, and the other contains the cytochrome c-555 and cytochrome baa3.

Cytochromebc purified from the methanogenMethanosarcina barkeri

Cytochromebc was partially purified from the methanogen,Methanosarcina barkeri. The cytochrome was composed of three subunits with molecular masses of 23.4, 20.9, and 9.1 kDa, respectively, and the 23.4 kDa subunit contained haemc. The absorption spectrum of cytochromebc showed a peak at 411 nm in the oxidized form, and peaks at 554, 524, and 422 nm in the reduced form. The cytochrome reacted with CO, and its low temperature absorption spectrum showed the α peak at 552 nm with a shoulder at 557 nm.

The Separation between Cytochrome A and Cytochrome A 3 in the Absolute Spectrum

aa3-Type cytochrome was purified from Halobacterium halobium (1). The cytochrome contained two heme a molecules per molecule but no copper. It did not show cytochrome c oxidase activity. One of the two heme a molecules in the cytochrome was reduced with ascorbate + TMPD, while the other was not reduced with this reducing reagents. The heme a molecule reducible with ascorbate + TMPD did not react with CO, while the heme a molecule reducible only with Na2S2O4 reacted with CO. Therefore, cytochrome a. or heme aA in the cytochrome was separated from cytochrome a3 or heme aB on the reduction with ascorbate + TMPD; the γ peaks of ferrocytochrome a and ferricytochrome a3 were observed spectrophotometrically in the absolute spectrum. As CuA is known to be unnecessary for cytochrome aa3 to oxidize ferrocytochrome c (2), these results mentioned above show that copper atom, CuB mediate electrons between heme aA and heme aB.

Structure and Function of NO Reductase with Oxygen-Reducing Activity

We succeeded in large-scale purification of NO reductase from Paracoccus denitrificans ATCC 35512 (formerly named Thiosphaera pantotropha) by using hydroxylapatite column chromatography. The spectral and enzymatic properties were the same as those of the enzyme purified by the previous method reported by Fujiwara and Fukumori. The enzyme was composed of two kinds of subunits with molecular masses of 34 and 15 kDa, respectively, and contained two hemes b and one heme c per molecule. We analyzed the metal content of NO reductase: the results suggested that NO reductase has no copper but does have 1–2 g · atoms of nonheme iron per mole. We also determined the structural gene of the enzyme. The norC and norB genes encoding the cytochrome c and cytochrome b subunits, respectively, showed considerable homology with those of Pseudomonas stuzeri NO reductase. Furthermore, the six invariant histidines in subunit I of the heme-copper oxidase superfamily were also conserved in the cytochrome b subunit of NO reductase.