Crystallization and preliminary X-ray study of iso-2 azurin from the methylotrophic bacterium, Methylomonas J.

The obligate methylotroph Methylomonas J possesses two distinct azurins. The iso-2 azurin, which functions as an electron acceptor for methylamine dehydrogenase, has been crystallized using two kinds of precipitants: PEG 4000 and ammonium sulfate. The crystals precipitated with PEG belong to the monoclinic system, space group P21, with unit-cell parameters a = 32.96, b = 33.67, c = 47.34 A and beta = 101.35 degrees. The crystals precipitated with ammonium sulfate belong to the orthorhombic system, space group C2221, with unit-cell parameters a = 31.52, b = 62.49 and c = 135.41 A. The crystals diffract to 1.6 and 1.9 A resolution, respectively, and were suitable for X-ray crystallographic studies. A Patterson search is being conducted using the recently reported structure of Alcaligenes xylosoxidans NCIMB 11015 as a starting model.

Spectroscopic and electrochemical properties of two azurins (Az-iso1 and Az-iso2) from the obligate methylotroph Methylomonas sp. strain J and the structure of novel Az-iso2.

Two azurin-type blue copper proteins, which are related to the electron-transfer processes involving methylamine/methanol oxidation, have been spectroscopically and electrochemically characterized. The obligate methylotroph Methylomonas sp. strain J gives rise to two azurins (Az-isol and Az-iso2) with methylamine dehydrogenase (MADH-Mj). The intense blue bands characteristic of Az-iso1 and Az-iso2 are observed at 621 and 616 nm in the visible absorption spectra respectively, being revealed at 620-630 nm in those of usual azurins. The EPR signal of Az-iso1, similar to usual azurins, shows axial symmetry, while the axial EPR signal of Az-iso2 involves a slightly rhombic character. The half-wave potentials (E1/2) of the two azurins and the intermolecular electron-transfer rate constants (kET) from MADH-Mj to each azurin were determined by cyclic voltammetry. The E1/2 values of Az-iso1 and Az-iso2 are +321 and +278 mV vs NHE at pH 7.0, respectively. The kET value of Az-iso2 is larger than that of Az-iso1 by a factor of 5. However, the electron-transfer rate of Az-iso2 is interestingly slower than those of the azurins from a denitrifying bacterium, Alcaligenes xylosoxidans NCIB 11015, and the amicyanin from a different methylotroph, Methylobacterium extorquens AM1. The structure of Az-iso2 has been determined and refined against 1.6 A X-ray diffraction data. The whole structure of Az-iso2 is quite similar to those of azurins reported already. The Cu(II) site of Az-iso2 is a distorted trigonal bipyramidal geometry like those of other azurins, but some of the Cu-ligand distances and ligand-Cu-ligand bond angle parameters are slightly different. These findings suggest that Az-iso2 is a novel azurin and perhaps functions as an electron acceptor for MADH.

Cloning and characterization of the azurin iso-1 gene, concerned with the electron transport chain involved in methylamine/methanol oxidation in the obligate methylotroph Methylomonas sp. strain J.

Two azurin-type blue copper proteins, which is concerned with the electron transport chain involved in methylamine/methanol oxidation, have been found in the obligate methylotroph Methylomonas sp. strain J. The azurin iso-1 gene was cloned and sequenced to analyze the role in the electron transport chain. PCR products synthesized with primers based on the N- and C-terminal amino acid sequences of azurin iso-1 were used as probes for cloning. One complete open reading frame (the azurin iso-1 gene) and one partial orf (orf1) were found in a cloned Eco105I-HindIII fragment, pMAZ3, with a total of 1066 bp. The gene encoded 148 amino acid residues. The amino acid sequence after Ala-21, deduced from the nucleotide sequence, was identical to that of the azurin iso-1 protein. The gene was in a region separate from the mau gene cluster in the chromosome. Escherichia coli expressed azurin iso-1. The results of northern blotting analysis suggested that expression of the azurin iso-1 gene is regulated by a complex regulatory network controlling oxidation of methylamine or methanol in this strain; for example, copper ions affected the expression of the azurin iso-1 gene.

Mycobacterium smegmatis malate dehydrogenase: activation of the lipid-depleted enzyme by anionic phospholipids and phosphatidylethanolamine.

Phospholipid-protein interactions have been investigated in a phospholipid-requiring enzyme, FAD-dependent malate dehydrogenase isolated from Mycobacterium smegmatis membranes, to correlate these interactions with enzyme function. The ability of several natural and synthetic phospholipids including CL and PE, which are major phospholipids in M. smegmatis membranes, to activate purified, lipid-depleted, enzymatically inactive malate dehydrogenase was examined. Anionic phospholipids and PE activated the enzyme, while zwitterionic phospholipids did not. A PE/PC mixture activated the enzyme in the form of both bilayer and non-bilayer structure. CL/PE mixtures activated malate dehydrogenase much more than each single phospholipid species. All anionic phospholipids used stabilized the enzyme, while PE and zwitterionic phospholipids did not. CL and a CL/PE mixture protected malate dehydrogenase from proteinase digestion, while PE did not. All phospholipids and phospholipid mixtures tested caused little secondary structural change in malate dehydrogenase. The results obtained in this study suggest that CL and CL/PE mixtures could form stable, enzymatically active complexes with malate dehydrogenase which might be similar to the native complex in M. smegmatis membranes. Although PE could activate malate dehydrogenase in both bilayer and non-bilayer form, it formed a complex with malate dehydrogenase which was inferior in terms of stability and susceptibility to proteinases, indicating that PE alone poorly reconstitutes the active enzyme-phospholipid complex.

Preparation of high-molecular-weight DNA: application to mycobacterial cells.

A method for isolating high-molecular-weight DNA from bacteria is described. A special feature of the method is the treatment of whole bacterial cells with an organic solvent (chloroform-methanol (2:1, v/v) or ethanol-ether (1:1, v/v)) prior to DNA extraction from the cells. The DNA preparations obtained from organic solvent-pretreated bacterial cells such as Mycobacterium smegmatis, M. phlei, and Escherichia coli contained highly polymerized DNA, as revealed by pulse-field gel electrophoresis. The size and yield of the DNA obtained from E. coli pretreated with the organic solvent were quite similar to that of the DNA obtained from protoplasts. The results strongly suggest that the organic solvent pretreatment is effective for extracting very large DNA from bacterial cells and especially from bacteria whose protoplasts cannot be easily formed.

Refined crystal structure of pseudoazurin from Methylobacterium extorquens AM1 at 1.5 A resolution.

The crystal structure of pseudoazurin from Methylobacterium extorquens AM1 (PAZAM1) has been solved by the molecular replacement method using copper-copper distances as translation parameters, which were obtained from difference Patterson maps calculated with the synchrotron radiation data containing the multiwavelength anomalous-dispersion effect. The structure refinement was carried out by the use of molecular dynamics optimization and the restrained least-squares method. The final crystallographic R factor was 19.9% for the 14 365 reflections greater than 3sigma between 1.5 and 8.0 A resolution. This report describes the characteristic features of the structure of PAZAM 1 as well as the effectiveness of synchrotron radiation for structure analysis of metalloproteins. The environment of the metal active site and the structural differences among blue-copper proteins are discussed.

Participation of poly(ADP-ribose) synthetase in the process of norepinephrine-induced inhibition of major histocompatibility complex class II antigen expression in human astrocytoma cells.

We previously demonstrated that the expression of a transfected poly(ADP-ribose) synthetase cDNA into macrophage tumor cells inhibited interferon-gamma-dependent induction of major histocompatibility complex(MHC) class II antigens. In the present study, we found that addition of norepinephrine to the cultured human astrocytoma STTG1 cells induced mRNA of poly(ADP-ribose) synthetase in 6-14h. Thus, we cultured the cells in the presence of norepinephrine for 24h, and then induced the MHC class II antigen by the addition of interferon-gamma. The expression of MHC class II antigen was inhibited, whereas it was not inhibited when norepinephrine and interferon-gamma were simultaneously added into the culture medium. These results suggest that an increase of poly(ADP-ribose) synthetase by norepinephrine cause the inhibition of interferon-gamma-mediated MHC class II antigen expression.

Preliminary crystallographic study of a pseudoazurin from methylotrophic bacterium, Methylobacterium extorquens AM1.

Single crystals of pseudoazurin, one of the blue copper proteins produced by methylotrophic bacterium Methylobacterium extorquens AM1, have been obtained by the method of vapor diffusion with ammonium sulfate as a precipitant at pH 8.0. Crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 52.619(4) A, b = 63.280(6) A, c = 35.133(4) A. The asymmetric unit includes one molecule of pseudoazurin (Vm = 2.18 A3/dalton). The crystals are so stable against X-ray irradiation that diffraction intensities of the native crystal up to 1.68 A resolution could be collected from only one crystal. Among the many heavy-metal reagents examined, uranyl acetate gave an effective isomorphous derivative.

Two distinct azurins function in the electron-transport chain of the obligate methylotroph Methylomonas J.

Methylomonas J is an obligate methylotroph although it is unable to grow on methane. Like Pseudomonas AM1, it produces two blue copper proteins when growing on methylamine, one of which is the recipient of electrons from the methylamine dehydrogenase. When grown on methanol, only the other blue copper protein is produced. We have determined the amino acid sequences of these blue copper proteins, and show that they are both true azurins. The sequences are clearly homologous to those of the proteins characterized from fluorescent pseudomonads and various species of Alcaligenes, and can be aligned with them and with each other without the need to postulate any internal insertions or deletions in the sequences. The iso-1 azurin, the one produced during both methanol and methylamine growth, shows 59-65% identity with these other azurins, whereas the iso-2 protein shows only 47-53% identity. The proteins show 52% identity with each other. The two functionally equivalent blue copper proteins from Pseudomonas AM1 belong to two sequence classes that are quite distinct from the true azurins. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50151 (23 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257, 5.

The primary structures of Pseudomonas AM1 amicyanin and pseudoazurin. Two new sequence classes of blue copper proteins.

The amino acid sequences of two blue copper proteins from the pink facultative methylotroph Pseudomonas AM1 (N.C.I.B. 9133) were determined. They each consist of a single polypeptide chain and bind one copper atom. Amicyanin contains 99 and pseudoazurin 123 residues. Copper-binding sites, consisting of the side chains of two histidine, one cysteine and one methionine residues, can be recognized in each protein by analogy with azurin and plastocyanin, but the spacings of the ligand residues are different, and other sequence similarity is limited. Proteins that are in the pseudoazurin sequence class can be recognized in some strains of Alcaligenes, and probably also in Paracoccus denitrificans. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50130 (23 pp.) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1985) 225, 5.

FAD-dependent malate dehydrogenase from Mycobacterium smegmatis: activation of the lipid-depleted enzyme by incorporation into cardiolipin liposome.

The lipid-depleted, enzymatically inactive malate dehydrogenase isolated from Mycobacterium smegmatis membrane was found to be incorporated spontaneously into cardiolipin liposome, but not into phosphatidylcholine liposome, as was revealed by electron spin resonance spectra with the use of 5-doxylstearic acid as a spin probe in the phospholipid liposomes. In addition, sucrose density gradient centrifugation in 0.5 M KCl showed hydrophobic interaction of the enzyme with cardiolipin liposome and further proved that the enzyme thus interacted hydrophobically with cardiolipin liposome became enzymatically active. From the results obtained above, it was concluded that the lipid-depleted, enzymatically inactive malate dehydrogenase isolated from M. smegmatis membrane was found to be activated by incorporating into the hydrophobic region of cardiolipin liposome.

Amino acid sequence studies of the light subunit of methylamine dehydrogenase from Pseudomonas AM1: existence of two residues binding the prosthetic group.

The methylamine dehydrogenase from Pseudomonas AM1 is a tetramer composed of two subunits, light(L)- and heavy-subunits. The amino acid sequence of the L-subunit was determined by sequence analyses of trypsin, chymotrypsin, staphylococcal protease, and thermolysin peptides of Cm-protein. The subunit consisted of a single polypeptide chain of 129 amino acid residues, with alanine and serine at the amino(N)- and carboxyl(C)-terminus, respectively. Yellow-colored peptides containing a prosthetic group were composed of two polypeptide chains and the prosthetic group was covalently bound to two residues at positions 55 and 106, which could not be identified yet. The molecular weight of the subunit was 13,500 excluding the binding residues and the prosthetic group. Various structural features are discussed.

Methanol dehydrogenase of Methylomonas J: purification, crystallization, and some properties.

A methanol dehydrogenase [EC 1.1.99.8] was purified and crystallized from methanol-grown Methylomonas J (formerly Pseudomonas sp. J), an obligate methylotroph. Its molecular weight was estimated to be 135,000 by gel chromatography. The sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed two bands and their molecular weights were approximately 60,000 and 10,000. The enzyme was relatively stable at pH 6 and 10, and was unstable at pH 8. The enzyme activity was lost at pH 4.0; however, the prosthetic group was not liberated from the enzyme. Its isoelectric point was pH 9.3. The visible-ultraviolet absorption, fluorescence, CD, and ESR spectra were measured. The amino acid composition was analyzed after separation of the two components. Primary alcohols and formaldehyde served as substrates. Phenazine methosulfate (PMS) was an effective electron acceptor of the enzyme in the presence of either NH4Cl or methylamine. The enzyme was inhibited partially by metal chelators and completely by Mn2+ and Co2+.

Two cytochromes c of Methylomonas J.

Two kinds of c-type cytochromes, cytochrome c-551 (I), and cytochrome c-551 (II), were highly purified and crystallized from cell-free extract of methanol-grown Methylomonas J (formerly Pseudomonas sp. J) and their physiochemical and biochemical properties were studied. Cytochrome c-551 (I) had an absorption peak at 409 nm in the oxidized form and peaks at 417, 523, 551 nm, and a shoulder at 532 nm in the reduced form. The millimolar extinction coefficient of the alpha-peak of the reduced form was 25.3. The isoelectric point was at pH 5.3 and its standard redox potential was 0.29 V at pH 7.0. The molecular weight was estimated to be 16,000. Cytochrome c-551 (II) had absorption maxima at 409 nm in the oxidized form, and at 416, 521, and 551 nm in the reduced form. The millimolar extinction coefficient of the alpha-peak of the reduced form was 22.4. The isoelectric point was at pH 4.3 and its standard redox form was 22.4. The isoelectric point was at pH 4.3 and its standard redox potential was 0.24 V at pH 7.0. The molecular weight was estimated to be 12,500. The two cytochromes were reduced by methanol dehydrogenase [EC 1.1.99.8] of this bacterium, and formaldehyde was detected as an oxidation product. Ammonium chloride was not essential for reduction of the cytochromes. No significant reduction of the cytochromes was observed by methylamine dehydrogenase isolated from methylamine-grown cells or by 2,6-dichlorophenol-indophenol (DCPIP)-dependent aldehyde dehydrogenase of the methanol-grown cells. The reduced forms of the cytochromes were oxidized by blue copper protein of the methanol-grown cells.

Methylamine dehydrogenases of Methylomonas J and Pseudomonas AM1. Hybridization of light and heavy subunits and some properties of an isolated hybrid enzyme.

Hybridization reactions were carried out in vitro between the light and heavy subunits of two methylamine dehydrogenases obtained from Methylomonas J (formerly Pseudomonas sp. J) and Pseudomonas AM1. Enzymatic activity was restored with a combination of L3(MW: 13,000) and HA(MW: 40,000) but not with LA(MW: 13,000) and HJ(NW: 40,000). This active hybrid enzyme was isolated with a Sephadex G-150 column after incubation of LJ and HA. Its molecular weight was nearly identical to that of the two native enzymes (MW: 105,000). The electrophoretic pattern of proteins cross-linked by dimethyl suberimidate showed the formation of a tetrameric structure. Its absorption spectrum was similar to that of the native enzymes. The specific activity was 4.3 and the substrate specificity resembled that of the Methylomonas J enzyme rather than that of the Pseudomonas AM1 enzyme. Michaelis constants were 290 microM for methylamine and 14 microM for phenazine methosulfate. The circular dichroism spectrum and thermal stability resembled those of the Pseudomonas AM1 enzyme. No appreciable amount of an inactive hybrid enzyme was formed in the reaction between LA and HJ, there being neither restoration of the enzymatic activity nor formation of cross-linked tetrameric proteins with dimethyl suberimidate. Hybridization of LJ and HA was further confirmed by the appearance of three enzymatic active bands on a gel after electrophoresis of an incubation mixture of LJ, HJ, and HA. The results are consistent with a previous observation; the two methylamine dehydrogenases from either obligate of facultative metaylotrophs are the alpha 2 beta 2-type and homologous enzymes. It is also suggested that substrate specificity and catalytic activity are directed by the light subunit, and binding affinity of the substrate to the active site, electrophoretic mobility, and thermal stability of the enzyme are endowed by the heavy subunit.

Methylamine dehydrogenases of Pseudomonas sp. J and Pseudomonas AM1. Study on subunit structure by dimethyl suberimidate.

Methylamine dehydrogenase (MW: 105,000) of Pseudomonas sp. J was treated with a bifunctional cross-linking reagent, dimethyl suberimidate. Cross-linked proteins having different molecular -eights of 53,000, 64,000, 80,000, 93,000, and 103,000 were found in addition to 13,000 (light subunit) and 40,000 (heavy subunit) by SDS polyacrylamide gel electrophoresis. Isolated light and heavy subunits were separately treated with the reagent. The product having a molecular weight of 80,000 was found to be a major cross-linked protein for the heavy subunit but no product was found for the light subunit. A similar electrophoretic pattern was also obtained for the reconstituted enzyme from the subunits of Pseudomonas sp. J and for methylamine dehydrogenase of Pseudomonas AM1. These results suggest that methylamine dehydrogenases obtained from these two bacteria are both the alpha2beta2-type subunit enzyme and have a geometrically analogous subunit structure.

Methylamine dehydrogenase of Pseudomonas sp. J. Reconstitution from its subunits.

Conditions for the restoration of catalytic activity from heavy and light subunits which had been isolated from methylamine dehydrogenase of Pseudomonas sp. J were investigated in vitro. Maximal restoration of the activity was obtained in 0.8 M potassium phosphate buffer, pH 7.0-9.3, at 30 degrees C with equimolar concentrations of the two subunits. Under the optimal conditions, the recovery of enzyme activity was 86% and the time required for half-maximal recovery was about 3 min. Addition of bovine serum albumin or p-chloromercuribenzoate to the incubation mixture had no effect on the rate or extent of recovery of the enzyme activity. When the heavy subunit was added to the light subunit, the absorption spectrum of the light subunit changed to a form similar to that observed for the native enzyme. The concentration of methylamine required to change the spectrum of the light subunit was greatly decreased in the presence of the heavy subunit. Reconstituted enzyme was prepared from the isolated subunits and purified by gel chromatography. The reconstituted enzyme resembled the native enzyme in specific activity, molecular weight, substrate specificity, reaction mechanism, Michaelis constants for methylamine and phenazine methosulfate, susceptibility to inhibitor, and absorption, fluorescence and ESR spectra. However, it was less stable than the native enzyme to thermal and pH treatments. The CD spectrum was also slightly different from that of the native enzyme.