The 2.8 A crystal structure of hydroxylamine oxidoreductase of a nitrifying chemoautotrophic bacterium, Nitrosomonas europaea, is described. Twenty-four haems lie in the centre bottom of the trimeric molecule, localized in four clusters within each monomer. The haem clusters within the trimer are aligned to form a ring that has inlet and outlet sites. The inlet is occupied by a novel haem, P460, and there are two possible outlet sites per monomer formed by paired haems lying within a cavity or cleft on the protein surface. The structure suggests pathways by which electron transfer may occur through the precisely arranged haems and provides a framework for the interpretation of previous and future biochemical and genetic observations.

Catalase-peroxidase is a member of the class I peroxidase superfamily. The enzyme exhibits both catalase and peroxidase activities to remove the harmful peroxide molecule from the living cell. The 2.0 A crystal structure of the catalase-peroxidase from Haloarcula marismortui (HmCP) reveals that the enzyme is a dimer of two identical subunits. Each subunit is composed of two structurally homologous domains with a topology similar to that of class I peroxidase. The active site of HmCP is in the N-terminal domain. Although the arrangement of the catalytic residues and the cofactor heme b in the active site is virtually identical to that of class I peroxidases, the heme moiety is buried inside the domain, similar to that in a typical catalase. In the vicinity of the active site, novel covalent bonds are formed among the side chains of three residues, including that of a tryptophan on the distal side of the heme. Together with the C-terminal domain, these covalent bonds fix two long loops on the surface of the enzyme that cover the substrate access channel to the active site. These features provide an explanation for the dual activities of this enzyme.

A highly active nitric oxide reductase was purified from Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha, which was anaerobically cultivated in the presence of nitrate. The enzyme was composed of two subunits with molecular masses of 34 and 15 kDa and contained two hemes b and one heme c per molecule. Copper was not found in the enzyme. The spectral properties suggested that one of the two hemes b and heme c were in six-coordinated low-spin states and another heme b was in a five-coordinated high-spin state and reacted with carbon monoxide. The enzyme showed high cytochrome c-nitric oxide oxidoreductase activity and formed nitrous oxide from nitric oxide with the expected stoichiometry when P. denitrificans ATCC 35512 ferrocytochrome c-550 was used as the electron donor. The V max and K m values for nitric oxide were 84 mol of nitric oxide per min/mg of protein and 0.25 M, respectively. Furthermore, the enzyme showed ferrocytochrome c-550-O 2 oxidoreductase activity with a V max of 8.4 mol of O 2 per min/mg of protein and a K m value of 0.9 mM. Both activities were 50% inhibited by about 0.3 mM KCN. (4,5,20,22), the NO reductase of the bacterium remains poorly characterized. NO, which is one of the reaction intermediates in the denitrification process (15,29), is chemically active and toxic to living cells. However, the concentration of NO in denitrifiers is stabilized only at the nano-or subnanomolar level because of the active consumption by NO reductase (12,30). NO reductases have been purified from only two kinds of denitrifying bacteria, Pseudomonas stutzeri (14, 17) and P. denitrificans ATCC 19367 (NCIB 8944) (6, 9). Both enzyme molecules contain protoheme and heme c and catalyze the reduction of NO to N 2 O in artificial electron-donating assays using phenazine methosulfate-ascorbate or 2,3,5,6-tetramethylphenylenediamine-ascorbate-horse cytochrome c. However, the physiological electron pathway to NO has not been reconstituted in vitro. Paracoccus denitrificansWe describe here a simple purification of NO reductase from P. denitrificans ATCC 35512 by using sucrose monocaprate (SM-1080) as a solubilizing detergent. The purified enzyme showed a subunit structure and spectral properties similar to those of cb-type NO reductases. Furthermore, the enzyme was very stable in the presence of the detergent and oxygen and showed not only cytochrome c-NO reductase activity but also cytochrome c-O 2 reductase activity when P. denitrificans ATCC 35512 cytochrome c-550 was used as the electron donor. We succeeded in reconstituting the electron transport from physiological cytochrome c to NO catalyzed by NO reductase. MATERIALS AND METHODSOrganisms and cultivation. P. denitrificans ATCC 35512 was anaerobically cultivated as previously described by Robertson and Kuenen (21), with some modifications. The bacterium was grown at 37ЊC in a medium containing (per liter) 1.36 g of CH 3 COONa ⅐ 3H 2 O, 3.26 g of KNO 3 , 0.8 g of K 2 HPO 4 , 0.3 g of KH 2 PO 4 , and 0.4 g of MgSO 4 and a trace amount ...

The 2-deoxystreptamine aglycon is a commonstructural feature found in aminocyclitol antibiotics including neomycin, kanamycin, tobramycin, gentamicin, sisomicin, butirosin and ribostamycin. A key enzyme involved in the biosynthesis of the 2-deoxystreptamine moiety is 2-deoxy-1sic>'//<9-inosose (DOI) synthase which catalyses the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scy//<9-inosose. The recent success of isolating the 2-deoxy-,s'cy//6>-inosose synthase from Bacillus circulans prompted us to clone the gene responsible for this important enzyme by the use of reverse genetics approach. With the aid of DNAprobes constructed on the basis of the amino-terminal sequence of the purified 42kDa subunit of the enzyme, the responsible gene btrC was successfully cloned. Subsequently the btrC gene was heterologously expressed in Escherichia coll, and the 2-deoxy-scy//oinosose synthase activity of the recombinant polypeptide was confirmed by chemical analysis. The btrC gene encodes a protein composed of 368 amino acids with a molecular mass of 40.7 kDa. Our previous proposal for the similarity of 2-deoxy-scylloinosose synthase to dehydroquinate synthase has been confirmed on the basis of their amino acid sequences. Significant differences in the sequences can also be observed however, particularly in the crucial substrate recognition regions. Comparison of the BtrC sequence with those ofbiosynthetic enzymes for other related microbial products is also discussed.

Dissimilatory nitrate reductase was purified from a denitrifying halophilic archaeon, Haloarcula marismortui, to an electrophoretically homogeneous state. The purified enzyme was inferred to be a homotetramer composed of a 63 kDa polypeptide. The electron paramagnetic resonance spectrum of the purified enzyme revealed typical rhombic signals which were ascribed to Mo(V) in the Mo^molybdopterin complex. Like the bacterial membrane-bound (Nar-) enzyme, the purified enzyme supported the catalysis of chlorate. The enzyme was activated in extreme saline conditions and the values of k cat and K m toward nitrate were 145 s 31 and 79 W WM, respectively, in the presence of 2.0 M NaCl.z 2000 Federation of European Biochemical Societies.

Genes encoding the NarG and NarH subunits of the molybdo^iron^sulfur enzyme, a nitrate reductase from a denitrifying halophilic euryarchaeota Haloarcula marismortui, were cloned and sequenced. An incomplete cysteine motif reminiscent of that for a [4Fe^4S] cluster binding was found in the NarG subunit, and complete cysteine arrangements for binding one [3Fe^4S] cluster and three [4Fe^4S] clusters were found in the NarH subunit. In conjunction with chemical, electron paramagnetic resonance, and subcellular localization analyses, we firmly establish that the H. marismortui enzyme is a new archaeal member of the known membrane-bound nitrate reductases whose homologs are found in the bacterial domain. ß

Abstract. Nitrous oxide (N2O) is a potent greenhouse gas and produced in denitrification and nitrification by various microorganisms. Site preference (SP) of 15N in N2O, which is defined as the difference in the natural abundance of isotopomers 14N15NO and 15N14NO relative to 14N14NO, has been reported to be a useful tool to quantitatively distinguish N2O production pathways. To determine representative SP values for each microbial process, we firstly measured SP of N2O produced in the enzyme reaction of hydroxylamine oxidoreductase (HAO) purified from two species of ammonia oxidizing bacteria (AOB), Nitrosomonas europaea and Nitrosococcus oceani, and that of nitric oxide reductase (NOR) from Paracoccus denitrificans. The SP value for NOR reaction (−5.9 ± 2.1‰) showed nearly the same value as that reported for N2O produced by P. denitrificans in pure culture. In contrast, SP value for HAO reaction (36.3 ± 2.3‰) was a little higher than the values reported for N2O produced by AOB in aerobic pure culture. Using the SP values obtained by HAO and NOR reactions, we calculated relative contribution of the nitrite (NO2–) reduction (which is followed by NO reduction) to N2O production by N. oceani incubated under different O2 availability. Our calculations revealed that previous in vivo studies might have underestimated the SP value for the NH2OH oxidation pathway possibly due to a small contribution of NO2– reduction pathway. Further evaluation of isotopomer signatures of N2O using common enzymes of other processes related to N2O would improve the isotopomer analysis of N2O in various environments.

Tokyo Bay, a eutrophic bay in Japan, receives nutrients from wastewater plants and other urban diffuse sources via river input. A transect was conducted along a line from the Arakawa River into Tokyo Bay to investigate the ecological relationship between the river outflow and the distribution, abundance and population structure of ammonia-oxidizing bacteria (AOB). Five surficial marine sediments were collected and analysed with polyphasic approaches. Heterogeneity and genetic diversity of beta-AOB populations were examined using restriction fragment length polymorphism (RFLP) analysis of 16S rRNA and amoA genes. A shift of the microbial community was detected in samples along the transect. Both 16S rRNA and amoA genes generated polymorphisms in the restriction profiles that were distinguishable at each sampling site. Two 16S rRNA gene libraries were constructed using the reverse transcription polymerase chain reaction (RT-PCR) method to determine the major ammonia oxidizers maintaining high cellular rRNA content. Two major groups were observed in the Nitrosomonas lineage; no Nitrosospira were detected. The effort to isolate novel AOB was successful; the isolate dominated in the gene libraries. For quantitative analysis, a real-time PCR assay targeting the 16S rRNA gene was developed. The population sizes of beta-AOB ranged from 1.6 x 10(7) to 3.0 x 10(8) cells g(-1) in dry sediments, which corresponded to 0.1-1.1% of the total bacterial population. An immunofluorescence staining using anti-hydroxylamine oxidoreductase (HAO) antibody was also tested to obtain complementary data. The population sizes of ammonia oxidizers ranged between 2.4 x 10(8) and 1.2 x 10(9) cells g(-1) of dry sediments, which corresponded to 1.2-4.3% of the total bacterial fraction. Ammonia-oxidizing bacteria cell numbers deduced by the two methods were correlated (R = 0.79, P < 0.01). In both methods, the number of AOB increased with the distance from the river mouth; ammonia-oxidizing bacteria were most numerous at B30, where the ammonium concentration in the porewater was markedly lower and the nitrite concentration was slightly higher than nearby sites. These results reveal spatial distribution and shifts in the population structure of AOB corresponding to nutrients and organic inputs from the river run-off and phytoplankton bloom.