Purification and properties of histidinol dehydrogenases from psychrophilic, mesophilic and thermophilic bacilli.

As a first step in elucidating one molecular mechanism of adaptation to life at extreme temperatures, we purified and characterized the enzyme histidinol dehydrogenase (EC 1.1.1.23) from a number of bacilli whose growth temperatures range from 5 degrees t to 90 degrees C. The enzymes were purified by (NH4)2SO4 precipitation, ion-exchange chromatography on Sephadex, affinity chromatography on histamine- or histidine-Sepharose and preparative gradient gel electrophoresis. All had similar mol.wts. (29200), sedimentation coefficients (S20,w 2.56S), affinities for histidinol and NAD+ (Km = 48 micron and 0.2 mM respectively) and all had pH optima at 9.6. Marked differences were observed in stability with respect to temperature and the temperature at which the initial velocity for histidinol dehydrogenation was optimal. These optima range from 25 degrees C for the enzyme from the psychrophilic species through to 41 degrees C for the mesophiles to 85-92 degrees C for the extreme thermophiles. It is concluded that the ability of the enzymes to operate at their various optimum temperatures is an intrinsic property of their amino acid sequences.     

Purification and characterization of histidinol dehydrogenase from cabbage

Histidinol dehydrogenase (EC 1.1.1.23) activity was determined in several plant species and in cultured plant cell lines. The enzyme was purified from cabbage (Brassica oleracea) to apparent homogeneity. To render complete purification, a new, specific histidinol-Sepharose 4B affinity chromatography was developed. The apparent molecular mass of the protein is 103 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein migrated as a single band with a molecular mass of 52 kDa, giving evidence for a dimeric quaternary structure. By isoelectric focusing, the enzyme was separated into six protein bands, five of which possessed the dehydrogenase activity when examined by an activity staining method. The Km values for L-histidinol and NAD+ were 15.5 and 42 microM, respectively. Enzyme activity was stimulated by addition of Mn2+, but was inhibited in the presence of Ba2+, Mg2+, Ni2+, Ca2+, Zn2+, or Cu2+. Histidinol dehydrogenase is the first histidine enzyme that has been purified to homogeneity and characterized from plants. This plant enzyme catalyzes the NAD-linked four-electron dehydrogenase reaction leading from histidinol to His. The results indicate a similar pathway of His in plants and show furthermore the last two reaction steps to be identical to those in microorganisms.     

Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties.

Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydrogenase were purified by cholate extraction, (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The properties of D-lactate dehydrogenase and D-mandelate dehydrogenase were similar in several respects: they had relative molecular masses of 62 800 and 59 700 respectively, pI values of 5.8 and 5.5, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH. Both enzymes appeared to contain non-covalently bound FAD as cofactor.     

Purification and properties of sheep-liver aldehyde dehydrogenases.

Sheep liver cytoplasmic aldehyde dehydrogenase was purified to homogeneity to give a sample with a specific activity of 380 nmol NADH min(-1) mg(-1). An amino acid analysis of the enzyme gave results similar to those reported for aldehyde dehydrogenases from other sources. The isoelectric point was at pH 5.25 and the enzyme contained no significant amounts of metal ions. On the binding of NADH to the enzyme there is a shift in absorption maximum of NADH to 344 nm, and a 5.6-fold enhancement of nucleotide fluorescence. The protein fluorescence (lambdaexcit = 290 nm, lambdaemisson = 340 nm) is quenched on the binding of NAD+ and NADH. The enhancement of nucleotide fluorescence on the binding of NADH has been utilised to determine the dissociation constant for the enzyme . NADH complex (Kd = 1.2 +/- 0.2 muM). A Hill plot of the data gave a straight line with a slope of 1.0 +/- 0.3 indicating the absence of co-operative effects. Ellman's reagent reacted only slowly with the enzyme but in the presence of sodium dodecylsulphate complete reaction occurred within a few minutes to an extent corresponding to 36 thiol groups/enzyme. Molecular weights were determined for both cytoplasmic and mitochondrial aldehyde dehydrogenases and were 212 000 +/- 8 000 and 205 000 respectively. Each enzyme consisted of four subunits with molecular weight of 53 000 +/- 2 000. Properties of the cytoplasmic and mitochondrial aldehyde dehydrogenases from sheep liver were compared with other mammalian liver aldehyde dehydrogenases.     

Amino acid substitutions in mutant forms of histidinol dehydrogenase from Neurospora crassa

Amino acid changes in the enzyme l-histidinol dehydrogenase (l-histidinol–NAD oxidoreductase, EC 1.1.1.23) have been determined between the wild-type Neurospora crassa and two temperature-sensitive mutants. Comparison was made between amino acid analyses of peptides of differing electrophoretic and chromatographic mobilities resulting from tryptic and chymotryptic digestion of protein from wild-type and mutant K26, and wild-type and mutant K445 strains, respectively. The analyses demonstrate the substitution of aspartic acid for alanine in mutant K26, and leucine for histidine in mutant K445. The effects of the resulting changes in polarity and charge are discussed in relation to the catalytic functioning of the proteins.     

Mapping the functional topology of the animal fatty acid synthase by mutant complementation in vitro

An in vitro mutant complementation approach has been used to map the functional topology of the animal fatty acid synthase. A series of knockout mutants was engineered, each mutant compromised in one of the seven functional domains, and heterodimers generated by hybridizing all possible combinations of the mutated subunits were isolated and characterized. Heterodimers comprised of a subunit containing either a beta-ketoacyl synthase or malonyl/acetyltransferase mutant, paired with a subunit containing mutations in any one of the other five domains, are active in fatty acid synthesis. Heterodimers in which both subunits carry a knockout mutation in either the dehydrase, enoyl reductase, keto reductase, or acyl carrier protein are inactive. Heterodimers comprised of a subunit containing a thioesterase mutation paired with a subunit containing a mutation in either the dehydrase, enoyl reductase, beta-ketoacyl reductase, or acyl carrier protein domains exhibit very low fatty acid synthetic ability. The results are consistent with a model for the fatty acid synthase in which the substrate loading and condensation reactions are catalyzed by cooperation of an acyl carrier protein domain of one subunit with the malonyl/acetyltransferase or beta-ketoacyl synthase domains, respectively, of either subunit. The beta-carbon-processing reactions, responsible for the complete reduction of the beta-ketoacyl moiety following each condensation step, are catalyzed by cooperation of an acyl carrier protein domain with the beta-ketoacyl reductase, dehydrase, and enoyl reductase domains associated exclusively with the same subunit. The chain-terminating reaction is carried out most efficiently by cooperation of an acyl carrier protein domain with the thioesterase domain of the same subunit. These results are discussed in the context of a revised model for the fatty acid synthase.     

Purification and comparative studies of alcohol dehydrogenases

Alcohol dehydrogenases from various animal and plant sources were purified by a common procedure which employed DEAE, Sephadex-G100 and affinity chromatographies. The procedure achieves an 80-130 fold purification for animal enzymes. However, only a 5-15 fold purification for plant enzymes was attained because of the instability of these enzymes. Purified alcohol dehydrogenases from animal and plant sources differ in coenzyme and substrate specificities. The enzymes from mammalian, avian and fish livers display aldehyde oxidizing and esterolytic activities in addition to alcohol oxidizing activity. However, the enzymes from plants and yeast show only the oxidative activity toward alcohols. Chemical modifications have been performed to identify amino acid residues which are essential to the oxidative and esterolytic activities of alcohol dehydrogenases.     

Isolation and genetic complementation of a sulfolipid-deficient mutant of Rhodobacter sphaeroides.

All photosynthetic organisms are thought to contain the sulfolipid 6-sulfo-alpha-D-quinovosyl diacylglycerol. However, the pathway of sulfolipid biosynthesis has not been elucidated, and the functional or structural significance of this lipid is not known. Mutants of Rhodobacter sphaeroides deficient in sulfolipid accumulation were isolated by directly screening for altered sulfolipid content. The mutants had no apparent phenotype except for the sulfolipid deficiency. A gene, designated sqdA, which complemented one of the mutations was isolated and characterized. The putative sqdA gene product is a protein with a molecular mass of 33.6 kDa that has no sequence similarity to any enzyme of known function.     

Purification and characterization of two aldehyde dehydrogenases from Pseudomonas aeruginosa.

Two soluble aldehyde dehydrogenases isoenzymes have been purified and separated from extracts of a paraffin-assimilating bacterium, Pseudomonas aeruginosa. The first one, obtained at an estimated purity of 20% (spec. act. with butanal 0.33 kat/kg) was NAD-dependent. It was rapidly inactivated at pH 8.6 but was efficiently protected by NAD. It had a molecular weight of 225000 and presented a high affinity for aldehydes of short and middle chain lengths. The second enzyme, obtained in a nearly homogenous state (spec. act. with pentanal 0.62 kat/kg) was NADP-dependent. It was activated by ions, in particular potassium ions, and had a good affinity for aldehydes of higher chain lengths. Both enzymes were stabilized by thiols and glycerol and were inactivated by reagents of sulfhydryl groups. These enzymes are 'constitutive' and their physiological function is uncertain. When the bacteria were grown on n-paraffin a new membrane-bound NAD-dependent aldehyde dehydrogenase activity was produced.     

In vitro and in vivo complementation of the Helicobacter pylori arginase mutant using an intergenic chromosomal site

BACKGROUND: Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects. MATERIALS AND METHODS: A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203-hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF). RESULTS: A rocF mutant unable to hydrolyze or consume l-arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3' ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice. CONCLUSIONS: This complementation strategy should greatly facilitate genetic experiments in H. pylori.     

Infectious center assay for complementation and recombination between mutant of reovirus.

An infectious center assay has been developed to measure recombination and complementation in L cells mixedly infected with ts mutants of reovirus. The mutants studied so far fall into complementation groups that correspond to the recombination groups previously defined by other laboratories (B. N. Fields, 1971; B. N. Filds and W. K. Joklik, 1969).