Comparison of the membrane-bound hydrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain

Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus H16 is an integral membrane protein and can only be solubilized by detergent treatment, the membrane-bound hydrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption. For the enzyme of A. eutrophus H16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose. The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45%. During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl2. The hydrogenase of A. eutrophus type strain was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield. Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits (Mr = 62,000, 31,000) and both have an isoelectric point near pH 7.0. They have the same electron acceptor specificity reacting with similar high rates and similar Km values. The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide. Ubiquinones and NAD were not reduced. The two hydrogenases were shown to be immunologically identical and both have identical electrophoretic mobility. For the membrane-bound hydrogenase of A. eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H2 evolution from reduced methyl viologen.     

Structural organization of the membrane-bound hydrogenase isolated from Alcaligenes eutrophus as revealed by electron microscopy

Electron microscopy of negatively stained samples of the membrane-bound hydrogenase isolated from Alcaligenes eutrophus was used to obtain enzyme images with an estimated resolution of 2.5 nm. The two subunits with shapes similar to the letter 'U' making up the enzyme could be seen to be joined in two planes orthogonal to each other, making contact with their concave sides. In face-on view, the particle exhibited bilateral symmetry.     

Structural organization of the membrane-bound hydrogenase isolated from Alcaligenes eutrophus as revealed by electron microscopy

Abstract Electron microscopy of negatively stained samples of the membrane-bound hydrogenase isolated from Alcaligenes eutrophus was used to obtain enzyme images with an estimated resolution of 2.5 nm. The two subunits with shapes similar to the letter 'U' making up the enzyme could be seen to be joined in two planes orthogonal to each other, making contact with their concave sides. In face-on view, the particle exhibited bilateral symmetry.     

An oxygen-binding flavohemoprotein from Alcaligenes eutrophus.

A procedure is described for the purification of a soluble flavohemoprotein from the hydrogen bacterium Alcaligenes eutrophus. The isolated protein exists as a monomer with a molecular weight of approx. 43,000. The molecule contains two prosthetic groups, 1 mol each of noncovalently bound FAD and protoheme per monomer. The absorption spectra of the protein in its ferric, ferrous-deoxy and ferrous-carboxy forms are similar to those of hemoglobins, with the exception of the flavin contribution (absorption maxima--ferric form: 395, 456, 483, 645 nm; ferrous-deoxy form: 436, 560 nm; ferrour-CO form: 423, 539, 569 nm). The flavohemoprotein when reduced by NADH in aerobic solution is capable of binding oxygen reversibly. The stable oxygenated complex exhibits absorption maxima at 414, 541, and 576 nm. The protein catalyzes the reduction of various dyes and cytochrome c by NADH.     

Phospholipid bound to the flavohemoprotein from Alcaligenes eutrophus.

The structurally characterized flavohemoprotein from Alcaligenes eutrophus (FHP) contains a phospholipid-binding site with 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-ethanolamine and 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-glycerol as the major occupying compounds. The structure of the phospholipid is characterized by its compact form, due to the -sc/beta/-sc conformation of the glycerol and the nonlinear arrangement of the sn-1- and sn-2-fatty acid chains. The phospholipid-binding site is located adjacent to the heme molecule at the bottom of a large cavity. The fatty acid chains form a large number of van der Waal's contacts with nonpolar side chains, whereas the glycerophosphate moiety, which points towards the entrance of the channel, is linked to the protein matrix by polar interactions. The thermodynamically stable globin module of FHP, obtained after cleaving off the oxidoreductase module, also contains the phospholipid and can therefore be considered as a phospholipid-binding protein. Single amino acid exchanges designed to decrease the lipid-binding site revealed both the possibility of blocking incorporation of the phospholipid and its capability to evade steric barriers. Conformational changes in the phospholipid can also be induced by binding heme-ligating compounds. Phospholipid binding is not a general feature of flavohemoproteins, because the Escherichia coli and the yeast protein exhibit less and no lipid affinity, respectively.     

Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34.

From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2. This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-free A. eutrophus hosts, strains AE104 and H16. Resistances were not expressed in Escherichia coli. The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E. coli under the control of the bacteriophage T7 promoter. The genes cnrC (44 kDa), cnrB (40 kDa), and cnrA (115.5 kDa) are probably structural genes; the gene loci cnrH (11.6 kDa), cnrR (tentatively assigned to open reading frame 1 [ORF]; 15.5 kDa), and cnrY (tentatively assigned to ORF0ab; ORF0a, 11.0 kDa; ORF0b, 10.3 kDa) are probably involved in the regulation of expression. ORF0ab and ORF1 exhibit a codon usage that is not typical for A. eutrophus. The 8.5-kb EcoRI-PstI fragment was mapped by Tn5 transposon insertion mutagenesis. Among 72 insertion mutants, the majority were nickel sensitive. The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two- to fivefold-increased nickel resistance in both hosts. These genes are considered to be involved in the regulation of cnr. Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights. The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon). The other putative gene products, CnrB and CnrC, share 28 and 30% similarity, respectively, with the corresponding proteins of czc.     

An immobilized hydrogenase from Alcaligenes eutrophus H-16

Alcaligenes Eutrophus H-16 was grown in continuous culture under conditions which induced hydrogenase production. The hydrogenase enzyme was extracted, partially purified and immobilized on porous glass. This enzyme was then studied both in solution and in immobilized form as a possible candidate for a number of industrial applications. It proved to have a stability (storage and operational) which was highly temperature dependent. Temperatures near freezing caused the enzyme to retain its activity for long periods of time. Although its kinetics were more favorable at elevated temperatures of up to 40 degrees C, the loss of stability outweighed this gain substantially. The effects of buffer type and pH on enzyme activity were also studied. This enzyme has only a modest sensitivity to destruction by oxygen during storage, in contrast to hydrogenases produced by several other microorganisms.     

DNA topoisomerase I from Alcaligenes eutrophus H16

Molecular and functional properties of DNA topoisomerase I isolated from a hydrogen-oxidizing bacterium, Alcaligenes eutrophus H16, were investigated. Under native conditions the enzyme forms a monomer with a relative molar mass of 98.500. A rod-like shape of the molecule was derived from the calculated frictional coefficient. The isoelectric point of the enzyme was determined to be in the range of 7.6–8.0. The enzyme activity is strictly Mg²⁺ dependent with an optimum at 3 mM Mg²⁺. The pH optimum ranges within 7.5–9.0. A. eutrophus DNA topoisomerase I activity is inhibited by M13 ssDNA, high ionic strength, polyamines, heparin and by a number of intercalating drugs.Abbreviations DTT dithiothreitol - BSA bovine serum albumin - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane - PMSF phenylmethanesulfonyl fluoride - PAGE polyacrylamide gel electrophoresis     

Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus

Biophysical and genetic properties of six independently isolated plasmids encoding the degradation of the herbicides 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid are described. Four of the plasmids, pJP3, pJP4, pJP5, and pJP7, had molecular masses of 51 megadaltons, belonged to the IncP1 incompatibility group, and transferred freely to strains of Escherichia coli, Rhodopseudomonas sphaeroides, Rhizobium sp., Agrobacterium tumefaciens, Pseudomonas putida, Pseudomonas fluorescens, and Acinetobacter calcoaceticus. In addition, these four plasmids conferred resistance to merbromin, phenylmercury acetate, and mercuric ions, had almost identical restriction endonuclease cleavage patterns, and encoded degradation of m-chlorobenzoate. The two other plasmids, pJP2 and pJP9, did not belong to the IncP1 incompatibility group, had molecular masses of 37 megadaltons, encoded the degradation of phenoxyacetic acid, and possessed identical restriction endonuclease cleavage patterns.     

Redox-dependent inactivation of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1

A novel inactivation mechanism of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1 comprising redox-dependent steps is described. The model of the hydrogenase inactivation process is proposed which implies that the enzyme may exist in several forms which differ in their stability and spectral properties. One of these forms, existing within a limited (approximately -200 +/- 30 mV) potential range, undergoes a rapid and irreversible inactivation. The dissociation of the FMN prosthetic group from the apohydrogenase appears to be the main reason for the enzyme inactivation. The rationale for the enzyme stabilization under real operational conditions based on the chemical modification of the hydrogenase molecule is suggested.     

Formation of Dimethylmuconolactones from Dimethylphenols by Alcaligenes eutrophus JMP 134

2,3-, 2,4-, 2,5-, 3,4-, and 3,5-dimethylphenols were cometabolized by 2,4-dichlorophenoxyacetate-grown Alcaligenes eutrophus JMP 134 or the constitutive derivative JMP 134-1 via the ortho pathway into dimethylmuconolactones as dead-end products. Formation of two distinct lactones from 3,4-dimethylphenol is indicative of 2- as well as 6-hydroxylation. Induction of the meta-cleavage pathway by 2,3- and 3,4-dimethylphenols resulted in growth and no accumulation of products. In contrast, 3,5-dimethylphenol is not metabolized by the meta-cleavage pathway.     

The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus.

The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and O2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be -445 mV and -325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.     

The lysis of Gram-negative Alcaligenes eutrophus by enzymes from Cytophaga

Summary The use of Cytophaga lysing enzymes was investigated for the liberation of poly--hydroxybutyrate (PHB) granules from the Gram-negative bacterium Alcaligenes eutrophus. Complete cell lysis was approached within a 60 minute period. Contrary to previous findings for the lysis of Gram-negative bacteria, prior removal of the outer membrane was not essential for enzymic lysis. The destabilisation of the outer membrane by the removal of divalent cations resulted in no significant improvement in the disruption process.     

Translational system of the hydrogen-oxidizing bacterium Alcaligenes eutrophus

Some structural and functional properties of ribosomes from the hydrogen-oxidizing bacterium Alcaligenes eutrophus were studied in order to investigate the background of expression of genetic information at the translational level. Ribosomal proteins from 30S subunits of A. eutrophus H16 were separated by two-dimensional gel electrophoresis into 21 spots, those from 50S subunits into 32 spots. While electrophoretic mobilities of several ribosomal proteins differed markedly from those of Escherichia coli, proteins sharing common immunological determinants with E. coli ribosomal proteins S1 and L7/L12 were found in A. eutrophus. Shifting from heterotrophic to autotrophic conditions of growth had no influence on the ribosomal protein pattern. Ribosomes of A. eutrophus had similar requirements for Mg²⁺ and poly(U) concentrations for optimum polyphenylalanine synthesis as those of E. coli. Protein synthesis elongation factors Tu from A. eutrophus and E. coli were immunologically similar. Efficiency of the A. eutrophus polyphenylalanine-synthesizing system was comparable to that of an analogous system derived from E. coli. This suggests that A. eutrophus could be employed for efficient expression of recombinant DNA.     

Cloning of the Alcaligenes eutrophus alcohol dehydrogenase gene

Mutants of Alcaligenes eutrophus which are altered with respect to the utilization of 2,3-butanediol and acetoin were isolated after transposon mutagenesis. The suicide vehicle pSUP5011 was used to introduce the drug resistance transposable element Tn5 into A. eutrophus. Kanamycin-resistant transconjugants of the 2,3-butanediol-utilizing parent strains CF10141 and AS141 were screened for mutants impaired in the utilization of 2,3-butanediol or acetoin. Eleven mutants were negative for 2,3-butanediol but positive for acetoin; they were unable to synthesize active fermentative alcohol dehydrogenase protein (class 1). Forty mutants were negative for 2,3-butanediol and for acetoin (class 2). Tn5-mob was also introduced into a Smr derivative of the 2,3-butanediol-nonutilizing parent strain H16. Of about 35,000 transconjugants, 2 were able to grow on 2,3-butanediol. Both mutants synthesized the fermentative alcohol dehydrogenase constitutively (class 3). The Tn5-labeled EcoRI fragments of genomic DNA of four class 1 and two class 3 mutants were cloned from a cosmid library. They were biotinylated and used as probes for the detection of the corresponding wild-type fragments in a lambda L47 and a cosmid gene bank. The gene which encodes the fermentative alcohol dehydrogenase in A. eutrophus was cloned and localized to a 2.5-kilobase (kb) SalI fragment which is located within a 11.5-kb EcoRI-fragment. The gene was heterologously expressed in A. eutrophus JMP222 and in Pseudomonas oxalaticus. The insertion of Tn5-mob in class 3 mutants mapped near the structural gene for alcohol dehydrogenase on the same 2.5-kb SalI fragment.