Novel substrate specificity of designer 3-isopropylmalate dehydrogenase derived from Thermus thermophilus HB8.

Redesigning of an enzyme for a new catalytic reaction and modified substrate specificity was exploited with 3-isopropylmalate dehydrogenase (IPMDH). Point-mutation on Gly-89, which is not in the catalytic site but near it, was done by changing it to Ala, Ser, Val, and Pro, and all the mutations changed the substrate specificity. The mutant enzymes showed higher catalytic efficiency (kcat/Km) than the native IPMDH when malate was used as a substrate instead of 3-isopropylmalate. More interestingly, an additional insertion of Gly between Gly-89 and Leu-90 significantly altered the substrate-specificity, although the overall catalytic activity was decreased. Particularly, this mutant turned out to efficiently accept D-lactic acid, which was not accepted as a substrate by wild-type IPMDH at all. These results demonstrate the opportunity for creating nove,enzymes by modification of amino acid residues that do not directly participate in catalysis, or by insertion of additional residues.

Pressure regulation of soluble cytochromes c in a deep-Sea piezophilic bacterium, Shewanella violacea.

Two c-type cytochromes from the soluble fraction of a deep-sea moderately piezophilic bacterium, Shewanella violacea, were purified and characterized, and the genes coding for these cytochromes were cloned and sequenced. One of the cytochromes, designated cytochrome c(A), was found to have a molecular mass of approximately 8.3 kDa, and it contained one heme c per molecule. The other, designated cytochrome c(B), was found to have a molecular mass of approximately 23 kDa, and it contained two heme c molecules per protein molecule. The amount of cytochrome c(B) expressed in cells grown at high hydrostatic pressure (50 MPa) was less than that in cells grown at atmospheric pressure, whereas cytochrome c(A) was constitutively expressed under all pressure conditions examined. The results of Northern blotting analysis were consistent with the above-mentioned observations and suggested that the pressure regulation of cytochrome c(B) gene expression occurred at the transcriptional level. These results suggest that the components of the respiratory chain of moderately piezophilic S. violacea could be exchanged according to the growth pressure conditions.

Butirosin-biosynthetic gene cluster from Bacillus circulans.

Butirosin is an interesting 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotic produced by non-actinomycete Bacilli. Recently we were successful in purification of 2-deoxy-scyllo-inosose synthase from butirosin-producer Bacillus circulans as the key enzyme for the biosynthesis of DOS, in cloning of the responsible gene (btrC), and in its overexpression in Escherichia coli. The present study involved gene-walking approach, which allowed us to find a gene cluster around btrC. The function of each gene was further investigated by gene disruption, and the disruptants of btrB, btrC, btrD and btrM showed no antibiotic producing activity. Therefore, the gene cluster found so far was determined to be a part of the butirosin biosynthetic gene cluster. Functions of some ORFs are also discussed in terms of butirosin biosynthesis on the basis of database search.

Molecular cloning of the gene for the key carbocycle-forming enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics and its comparison with dehydroquinate synthase.

The 2-deoxystreptamine aglycon is a common structural 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-scyllo-inosose (DOI) synthase which catalyses the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. The recent success of isolating the 2-deoxy-scyllo-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 DNA probes constructed on the basis of the amino-terminal sequence of the purified 42 kDa subunit of the enzyme, the responsible gene btrC was successfully cloned. Subsequently the btrC gene was heterologously expressed in Escherichia coli, and the 2-deoxy-scyllo-inosose 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-scyllo-inosose 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 of biosynthetic enzymes for other related microbial products is also discussed.

Purification and characterization of 2-deoxy-scyllo-inosose synthase derived from Bacillus circulans. A crucial carbocyclization enzyme in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics.

The biosynthesis of 2-deoxystreptamine, the central aglycon of a major group of clinically important aminoglycoside antibiotics, commences with the initial carbocycle formation step from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. This crucial step is known to be catalyzed by 2-deoxy-scyllo-inosose synthase, which has not yet been characterized so far. Reported in this paper is the first purification of 2-deoxy-scyllo-inosose synthase from butirosin-producing Bacillus circulans SANK 72073 to electrophoretic homogeneity. The enzyme was isolated as a heterodimeric protein comprising from a 23 kDa- and a 42 kDa polypeptide chains. The Km of the enzyme for D-glucose-6-phosphate was estimated to be 9.0 x 10(-4) M and that for NAD+ 1.7 x 10(-4) M, kcat for D-glucose-6-phosphate being 7.3 x 10(-2) s(-1). The presence of Co2+ was essential for the enzyme activity, but Zn2+ was totally inhibitory. While the reaction mechanisms are quite similar, 2-deoxy-scyllo-inosose synthase appears to be distinct from dehydroquinate synthase in the shikimate pathway, with respect to the quaternary structure, metal ion requirement, and the kinetic parameters.

A denitrifying bacterium from the deep sea at 11,000-m depth.

The denitrifying bacterium strain MT-1 was isolated from the mud of the Mariana Trench. The optimal temperature and pressure for growth of this bacterium were found to be 30 degrees C and 0.1 MPa, respectively. However, it showed greater tolerance to low temperature (4 degrees C) and high hydrostatic pressure (50 MPa) as compared with denitrifiers obtained from land. From the results, it can be said that this organism is adapted to the environment of the deep sea. Strain MT-1 was shown to belong to the genus Pseudomonas by analysis of its 16S rDNA. The cytochrome contents of the bacterium were similar to those of Ps. stutzeri in spectrophotometric studies.

Open reading frame 3 of the barotolerant bacterium strain DSS12 is complementary with cydD in Escherichia coli: cydD functions are required for cell stability at high pressure.

Escherichia coli strain JD518, a cydD-deficient mutant, displayed temperature-sensitive and pressure-sensitive growth. The defective cydD gene in this strain was complemented by open reading frame 3 (ORF3), previously identified in DNA from a barotolerant bacterium, strain DSS12, allowing growth of the cydD mutant under high temperature and high pressure conditions. Spectrophotometrical analysis indicated that the cytochrome bd complex which is assembled by the CydD protein was expressed in E. coli strain JD518 carrying the ORF3 gene at the same level as occurred in the wild-type strain. Our results indicate that the cydD gene functions are required for cell stability under the condition of high pressure stress in bacteria.

Purification, and some molecular and enzymatic features of a novel ccb-type cytochrome c oxidase from a microaerobic denitrifier, Magnetospirillum magnetotacticum.

A novel ccb-type cytochrome c oxidase was purified from the magnetic bacterium, Magnetospirillum magnetotacticum MS-1. The enzyme was composed of three subunits with M(r)'s of 43,000, 34,000 and 28,000, respectively, and contained 0.91 mol of protoheme, 2.0 mol of heme c and 0.70 g atom of copper per mol of minimal structural unit. One mol of enzyme oxidized 187 mol of horse heart ferrocytochrome c and 34.4 mol of M. magnetotacticum ferrocytochrome c550/s. The cytochrome c oxidase activity of the enzyme was 50% inhibited by 12 microM KCN. The enzyme seems to function as the terminal oxidase in microaerobic respiration.

Two membrane-bound c-type cytochromes of Thiobacillus ferrooxidans: purification and properties.

Membrane-bound cytochrome c, cytochrome c-552 (m) was purified from Thiobacillus ferrooxidans. It showed an absorption peak at 410 nm in the oxidized form, and peaks at 552, 523 and 416 nm in the reduced form. Its molecular mass, Em,7 and isoelectric point were 22,300, +0.336 volt and 9.1, respectively. Another membrane-bound cytochrome c, cytochrome c-550 (m) was also purified. It showed an absorption peak at 408 nm in the oxidized form, and peaks at 550, 523 and 418 nm in the reduced form. Its molecular mass was estimated to be 51,000. Ferrocytochromes c-552 (m) and c-550 (m) were oxidized by cytochrome c oxidase of the bacterium. The reactivity with the oxidase of cytochrome c-550 (m) was higher than that of cytochrome c-552 (s) (soluble cytochrome) of the bacterium, while the reactivity of cytochrome c-552 (m) was greatly lower than that of cytochrome c-552 (s).

Purification and properties of a 'cytochrome a1'-like hemoprotein from a magnetotactic bacterium, Aquaspirillum magnetotacticum.

A novel hemoprotein was purified from a magnetotactic bacterium, Aquaspirillum magnetotacticum MS-1. The protein showed absorption peaks at 437 nm in the oxidized form, and 592, 550 and 450 nm in the reduced form. Although the spectral properties of the hemoprotein were very similar to those of 'cytochrome a1', the hemoprotein contained no molecules of heme a. The protein contained two kinds of hemes; one was extracted with HCl-acetone and the other was covalently bound to the protein. The pyridine ferrohemochrome of the former heme showed absorption peaks at 440, 545 and 585 nm. The chromatographic behavior of the heme on reverse-phase HPLC was different from that of heme a. The pyridine ferrohemochrome of the covalently bound heme showed an alpha peak at 565 nm. On the basis of the iron analysis, the hemoprotein contained one molecule of each of the two kinds of heme in the holoprotein. The protein was composed of two kinds of subunit with molecular weights of 41,000 and 17,000 and showed very little cytochrome c oxidase activity. The amounts of the hemoprotein in the magnetic cells of A. magnetotacticum were larger than those in non-magnetic cells. These results suggest that the 'cytochrome a1'-like hemoprotein is not the terminal oxidase of the bacterium and may be related to the formation of magnetosome in the magnetic cells of A. magnetotacticum.

Thiobacillus ferrooxidans cytochrome c oxidase: purification, and molecular and enzymatic features.

Cytochrome c oxidase from Thiobacillus ferrooxidans was purified to homogeneity and some of its properties were studied. The oxidase was solubilized with n-octyl-beta-D-thioglucoside (OTG) under acidic conditions (pH 4.0) and purified by one step of ion-exchange chromatography with a CM-Toyopearl column. The absorption spectrum of the oxidase showed peaks at 420 and 595 nm in the oxidized form and at 440 and 595 nm in the reduced form. Its CO compound showed a novel absorption spectrum; a double-peaked gamma band appeared at 429 and 438 nm. The oxidase seemed to have CuA-like copper atom from its ESR and near-infrared spectra. The oxidase molecule consisted of three polypeptides with molecular weights of 53,000, 22,000, and 17,000, respectively, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme in a solution containing detergents was estimated to be 169,000 on the basis of the results obtained by gel filtration, while the molecular weight per heme alpha was estimated to be 83,700. The copper content of the oxidase was 1.01 g atom per mol of heme alpha. Therefore, the cytochrome seemed to contain one molecule of heme alpha and one atom of copper in the minimal structural unit consisting of one molecule each of the three subunits, and to occur as a dimer of the unit in the solution. The oxidase oxidized ferrocytochrome c-552 of the bacterium, and the optimal pH of the reaction was 3.5.(ABSTRACT TRUNCATED AT 250 WORDS)