Characterization of 3-Guanidinopropionate Amidinohydrolase from Pseudomonas aeruginosa and a Comparative Study with 4-Guanidinobutyrate Amidinohydrolase from another Pseudomonas

3-Guanidinopropionate amidinohydrolase, a new enzyme (EC class 3.5.3), was purified 220-fold from Pseudomonas aeruginosa PAO 1 grown on 3-guanidinopropionate. The enzyme was found to be essentially homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was estimated to be 195,000-215,000. The subunit molecular weight was estimated to be 36,000. The optimal pH was 9.0. The Km value for 3-guanidinopropionate was 45 mm. Incubation of the enzyme with EDTA in potassium phosphate buffer, pH 7.0, at 40°C resulted in almost complete inactivation, and the inactive enzyme was specifically reactivated by Mn²⁺. Taurocyamine (11%) and 4-guanidinobutyrate (3%) were hydrolyzed as fast as 3-guanidinopropionate at the relative rates indicated. The enzyme was inactivated by p-chloromercuribenzoic acid and the inactive enzyme was reactivated by incubation with 2-mercaptoethanol. Coelectrophoresis of the enzyme with 4-guanidinobutyrate amidinohydrolase purified from Pseudomonas sp. ATCC 14676 in polyacrylamide gels in the presence and absence of sodium dodecyl sulfate demonstrated their close mobilities. 4-Aminobutyrate, propionate, and n-butyrate were common competitive inhibitors of these enzymes. The evolutionary relationship between the two enzymes was discussed.     

Crystal structures of Pseudomonas aeruginosa guanidinobutyrase and guanidinopropionase, members of the ureohydrolase superfamily

Pseudomonas aeruginosa guanidinobutyrase (GbuA) and guanidinopropionase (GpuA) catalyze the hydrolysis of 4-guanidinobutyrate and 3-guanidinopropionate, respectively. They belong to the ureohydrolase superfamily, which includes arginase, agmatinase, proclavaminate amidinohydrolase, and formiminoglutamase. In this study, we have determined the crystal structures of GbuA and GpuA from P. aeruginosa to provide a structural insight into their substrate specificity. Although GbuA and GpuA share a common structural fold of the typical ureohydrolase superfamily, they exhibit significant variations in two active site loops. Mutagenesis of Met161 of GbuA and Tyr157 of GpuA, both of which are located in the active site loop 1 and predicted to be involved in substrate recognition, significantly affected their enzymatic properties, implying their important roles in catalysis.     

Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from aKlebsiella pneumoniae mutant strain

Unlike the parent wild-type strain, theKlebsiella pneumoniae mutant strain MAO4 has a 4-HBA⁺ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when theAcinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoateK. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.     

Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t _1/2 values of 7.0 and 1.5 min at 55°C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instabiliby due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M _r value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.     

Glycerol catabolism in wild-type and mutant strains ofPseudomonas aeruginosa

Glycerol uptake, glycerol kinase (EC 2.7.1.30) and glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) activities are specifically induced during growth ofPseudomonas aeruginosa PAO on either glycerol or glycerol-3-phosphate. Mutants of strain PAO unable to grow on both glycerol and glycerol-3-phosphate were isolated. Mutant PFB 121 was deficient in an inducible, membrane-bound, pyridine nucleotide-independent, glycerol-3-phosphate dehydrogenase activity and PFB 82 was deficient in glycerol uptake and glycerol kinase and glycerol-3-phosphate dehydrogenase activities. Each mutant spontaneously reverted to wild phenotype, which indicates that each contained a single genetic lesion. These results demonstrate that membrane-bound, inducible glycerol-3-phosphate dehydrogenase is required for catabolism of both glycerol and glycerol-3-phosphate and provide suggestive evidence for a single regulatory locus that controls the synthesis of glycerol uptake, glycerol kinase, and glycerol-3-phosphate dehydrogenase inP. aeruginosa.     

D-arginase of Arthrobacter sp. KUJ 8602: characterization and its identity with Zn(2+)-guanidinobutyrase

D-Arginase activity was found in the cells of an isolate, Arthrobacter sp. KUJ 8602, grown in the L-arginine medium, and the enzyme was purified and characterized. Its molecular weight was estimated to be about 232,000 by gel filtration, and that of the subunit was approximately 40,000 by SDS-PAGE, suggesting that the enzyme is a homohexamer. The enzyme acted on not only D-arginine but also 4-guanidinobutyrate, 3-guanidinopropionate and even L-arginine. The V(max)/K(m) values for 4-guanidinobutyrate and D-arginine were determined to be 87 and 0.81 micro mol/min/mg/mM, respectively. Accordingly, the enzyme is regarded as a kind of guanidinobutyrase [EC 3.5.3.7]. The pH optima for 4-guanidinobutyrate and D-arginine were 9.0 and 9.5, respectively. The enzyme was inhibited competitively by 5-aminovalerate, and thiol carboxylates such as mercaptoacetate served as strong mixed-type inhibitors. The enzyme contained about 1 g-atom of firmly bound Zn(2+) per mol of subunit, and removal of the metal ions by incubation with 1,10-phenanthroline resulted in loss of activity. The inactivated enzyme was reactivated markedly by incubation with either Zn(2+) or Co(2+), and slightly by incubation with Mn(2+). The nucleotide sequence of enzyme contains an open reading frame that encodes a polypeptide of 353 amino acid residues (M(r): 37,933). The predicted amino acid sequence contains sequences involved in the binding of metal ions and the guanidino group of the substrate, which show a high homology with corresponding sequences of Mn(2+)-dependent amidinohydrolases such as agmatinase from Escherichia coli and L-arginase from rat liver, though the homology of their entire sequences is relatively low (24-43%).     

The Effect of the Plant Growth Stimulant Bactozole on Rhizobium leguminosarum bv. viciae 250a and Its Nitrogen-Tolerant Mutant M-71 under Different Nitrogen Supply Conditions

The effect of the plant growth stimulant bactozole on the growth of Rhizobium leguminosarum bv. viciae 250a and its nitrogen-tolerant mutant M-71 and the synthesis of extracellular carbohydrates was studied. At a low content of nitrate (6 mM) in the medium, all three bactozole concentrations tested (0.001, 0.01, and 0.1%) exerted similar stimulating effects on the growth of the parent strain 250a (about 1.5-fold) and the synthesis of extracellular carbohydrates (about 2-fold). At a high content of nitrate (20 mM) in the medium, when the growth of the parent strain and the synthesis of extracellular carbohydrates were inhibited, bactozole at all three concentrations exerted only a growth-stimulating effect. At the same time, mutant M-71 showed better growth at higher concentrations of bactozole, whereas the ability of the mutant to synthesize extracellular carbohydrates decreased with increasing bactozole concentration. The cell biomass of the mutant accumulated at 20 mM nitrate was 1.8–2.5 times greater than it was at 6 mM nitrate. Bactozole enhanced the symbiosis of legume plants with both parent and mutant strains, raising the mass of plants and enhancing nodulation and the nitrogen-fixing activity of root nodules. The symbiotic parameters of mutant M-71 were better (irrespective of whether bactozole was present or not) when its inoculum was grown at a high nitrogen content (20 mM nitrate), whereas the respective parameters of the parent strain were better when it was grown at 6 mM nitrate. The inference is made that the better physiological characteristics of the mutant in the high-nitrate medium are due to its higher nitrate reductase activity (as compared with the parent strain) in both the free-living state and in legume nodules.     

Alternation of the Dissimilation Pathway for Glycerol and Amplification of Glycerol Dehydrogenase in Mutant Strains of Cellulomonas sp. NT3060

Mutant strain ME544, which is able to grow on glycerol slowly, was derived from glycerol-negative mutant strain G011, which is a derivative strain of Cellulomonas sp. NT3060 and is defective in both the enzyme activities of glycerol kinase and glycerol 3-phosphate dehydrogenase. The mutant strain still lacked both the enzyme activities involved in the dissimilation of glycerol and had the same level of glycerol dehydrogenase activity as the parent strain. Dihydroxyacetone kinase activity in mutant strain ME544 was inducibly formed, reaching 4-fold the level in mutant strain G011 in glycerol medium. Thus, the mutant strain seemed to dissimilate glycerol by means of glycerol dehydrogenase followed by an increase in dihydroxyacetone kinase. Subsequently, a mutant strain, GP1807, which was resistant to the inhibition of growth on glycerol by 1,2-propanediol, was derived from mutant strain ME544. Glycerol dehydrogenase activity of the mutant strain was amplified about 6-fold compared to that of the wild type strain.     

Kinase replacement by a dehydrogenase for Escherichia coli glycerol utilization.

A mutant of Escherichia coli that employs a glycerol:nicotinamide adenine dinucleotide 2-oxidoreductase (EC 1.1.1.6), instead of adenosine 5'-triphosphate:glycerol 3-phosphotransferase (EC 2.7.1.30), as the first enzyme for the dissimilation of glycerol was constructed. This mutant, like the wild-type strain, still cannot grow anaerobically on glycerol without an exogenous hydrogen acceptor.     

Glucose-1-Phosphate-Negative Mutant of Agrobacterium tumefaciens

Glucose-1-phosphate-negative mutants that are unable to grow in a synthetic medium containing glucose-1-phosphate (G-1-P) as a sole carbon source were isolated by treatment of Agrobacterium tumefaciens IAM 1525 with N-methyl-N'-nitro-N-nitrosoguanidine. All of the enzymes involved in G-1-P metabolism (glucoside-3-dehydrogenase, 3-ketoglucose-1-phosphate-degrading enzyme, alpha-glucosidase, and phosphatases) were detected in the sonic extract prepared from resting cells of one of the mutants, strain M-24, in approximately equal levels to those in the parent strain. Resting cells of the mutant oxidized G-1-P to 3-ketoglucose-1-phosphate (3KG-1-P), the first product in G-1-P metabolism by the bacterium, with little subsequent degradation, whereas the parent showed further degradation of G-1-P via 3KG-1-P. Glucoside-3-dehydrogenase catalyzing 3-ketoglucoside formation was readily released from cells by osmotic shock, whereas the 3KG-1-P-degrading enzyme was not released. Thus, the former and the latter enzymes might be at different intracellular loci, such as periplasm and cytoplasm, respectively. It is suggested that the mutant strain M-24 is a G-1-P-negative mutant deficient in a 3KG-1-P transport system located on the cytoplasmic membrane.     

Comparison of Alkaline Proteinase from Hyperproductive Mutants and Parent Strain of Aspergillus sojae

Physico-chemical properties of alkaline proteinase from the parent strain were compared with those from hyperproductive mutants of Aspergillus sojae. All the results on behavior of enzyme protein to ion exchange resin and celluloses, gel filtration, ultracentrifugal sedimentation, disc electrophoresis and isoelectrofocusing on polyacrylamide gel column, specific activity, substrate specificity, and kinetic constants provided evidence in favor of the conclusion that the parent and mutant strains produced the chemically identical enzymes and that superactivity of alkaline proteinase in culture extracts or filtrates of mutant strains was not attributed to alteration of catalytic property of the enzyme, but to hyperproduction of the identical enzyme resulting from the genetic change in the regulatory mechanism of enzyme synthesis.     

Role of amino-acid residue 95 in substrate specificity of phosphagen kinases

The purpose of this study is to elucidate the mechanisms of guanidine substrate specificity in phosphagen kinases, including creatine kinase (CK), glycocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and arginine kinase (AK). Among these enzymes, LK is unique in that it shows considerable enzyme activity for taurocyamine in addition to its original target substrate, lombricine. We earlier proposed several candidate amino acids associated with guanidine substrate recognition. Here, we focus on amino-acid residue 95, which is strictly conserved in phosphagen kinases: Arg in CK, Ile in GK, Lys in LK and Tyr in AK. This residue is not directly associated with substrate binding in CK and AK crystal structures, but it is located close to the binding site of the guanidine substrate. We replaced amino acid 95 Lys in LK isolated from earthworm Eisenia foetida with two amino acids, Arg or Tyr, expressed the modified enzymes in Escherichia coli as a fusion protein with maltose-binding protein, and determined the kinetic parameters. The K95R mutant enzyme showed a stronger affinity for both lombricine (Km=0.74 mM and kcat/Km=19.34 s(-1) mM(-1)) and taurocyamine (Km=2.67 and kcat/Km=2.81), compared with those of the wild-type enzyme (Km=5.33 and kcat/Km=3.37 for lombricine, and Km=15.31 and kcat/ Km=0.48for taurocyamine). Enzyme activity of the other mutant, K95Y, was dramatically altered. The affinity for taurocyamine (Km=1.93 and kcat/Km=6.41) was enhanced remarkably and that for lombricine (Km=14.2 and kcat/Km=0.72) was largely decreased, indicating that this mutant functions as a taurocyamine kinase. This mutant also had a lower but significant enzyme activity for the substrate arginine (Km=33.28 and kcat/Km=0.01). These results suggest that Eisenia LK is an inherently flexible enzyme and that substrate specificity is strongly controlled by the amino-acid residue at position 95.     

Poly β-hydroxybutyrate depolymerase (PhaZ) in<Emphasis Type="Italic"> Azospirillum brasilense</Emphasis> and characterization of a<Emphasis Type="Italic"> phaZ</Emphasis> mutant

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly--hydroxybutyrate (PHB) under sub-optimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments. PHB depolymerase (PhaZ) is an essential enzyme in PHB degradation. The phaZ gene was identified in Azospirillum brasilense, cloned, sequenced, and shown to be located on the chromosome. Insertion of a kanamycin-resistant cassette within phaZ of A. brasilense resulted in a phaZ mutant that was unable to degrade PHB; however, carbon source utilization was similar in both the wild-type and the mutant strain. The ability of the wild-type to endure starvation conditions, ultraviolet irradiation, heat, and osmotic shock, and to grow in the presence of hydrogen peroxide was higher than that of the mutant strain. By contrast, the ability of the phaZ mutant strain to endure desiccation was higher than that of the wild-type strain. No differences between the strains were seen in their ability to endure sonication, or to survive in carrier materials used for soil inoculants. In addition, motility was the same between the two strains, whereas cell aggregation and exopolysaccharide production were higher in the wild-type than in the phaZ mutant strain.     

Regulatory changes in the fucose system associated with the evolution of a catabolic pathway for propanediol in Escherichia coli.

Wild-type strains of Escherichia coli are unable to use L-1,2-propanediol as a carbon and energy source. Strain 3, a mutant selected for the ability to grow on this compound at progressively more rapid rates, synthesizes constitutively a nicotinamide adenine dinucleotide-linked propanediol oxidoreductase. This enzyme is normally synthesized during anaerobic growth on L-fucose when it functions as a lactaldehyde reductase. Propanediol, the end product of this fermentation process, escapes irretrievably into the medium. The propanediol-utilizing mutant can no longer grow on fucose in either the presence or absence of molecular oxygen. In the present study nine independent lines of propanediol-positive mutants were characterized. One mutant, strain 418, attained a propanediol growth rate close to that of strain 3 without loss of the ability to grow on fucose. In all cases examined, however, prolonged selection on propanediol did result in the emergence of fucose-negative mutants. All of these mutants had enzyme patterns similar to that of strain 3; namely, fucose permease, fucose isomerase, and fuculose kinase were noninducible, whereas fuculose 1-phosphate aldolase was constitutive. In strain 418 and in the fucose-positive predecessors of the other mutants, the first four enzymes in the pathway remained inducible, as in the wild-type strain. Improvements in the growth rate on propanediol appeared to reflect principally the increased activity level of the oxidoreductase during the early stages of evolution. According to transductional analysis, the mutations affecting the ability to grow on propanediol and those that affect the expression of the first enzymes in the fucose pathway were very closely linked. The loss of the ability to grow on fucose is thought to be a mechanistic consequence incidental to the remodeling of the regulatory system in favor of the utilization of the novel carbon source.     

Significance of Pyruvate Carboxylase in Sugar Metabolism of Agrobacterium tumefaciens

Mutants, which fail to grow on glucose medium but can grow on succinate medium, were isolated by treatment with N-methyl-N′-nitro-N-nitrosoganidine from the wild-type strain of Agrobacterium tumefaciens, and were found to lose growth on several hexoses and three-carbon intermediates. The revertant mutants, which recovered the ability to grow on glucose medium, simultaneously regained the ability to grow on hexoses and three-carbon intermediates. By comparison of biochemical properties of the wild-type, the mutants and the revertant mutants, two mutant strains were characterized to be pyruvate carboxylase-deficient. Then, we concluded that these mutants might be induced by a single mutation at a genetic locus of pyruvate carboxylase and that the deficiency in the enzyme gave a pleiotropic effect on the ability to grow on hexoses and three-carbon intermediates. Some properties of pyruvate carboxylase of this bacterium were also presented.     

D-Glucose Isomerase: Constitutive and Catabolite Repression-Resistant Mutants of Streptomyces phaeochromogenes

As in other Streptomyces species, the enzymatic conversion of D-glucose to D-fructose is carried out in Streptomyces phaeochromogenes NRRL B-3559 by the inducible enzyme, D-xylose keto isomerase (EC 5.3.1.5). Mutants of this microorganism were selected for their ability to grow on D-lyxose (2-epimer of D-xlose). As a result of the mutational event, the microorganism constitutively produced D-xylose isomerase. As in the parent strain, the constitutive formation of the isomerase was repressed by D-glucose. The fact that this mutant was unable to grow in low D-xylose concentrations in the presence of the D-glucose analogue, 3-O-methylglucose, permitted the isolation of D-xylose isomerase constitutive mutants which were insensitive to D-glucose repression.     

Physiological and enzymatic properties of a thymidine-requiring Pediococcus cerevisiae mutant.

We describe the isolation and characterization of a Pediococcus cerevisiae thymidine-requiring mutant and its thymidine-independent revertant. The mutant strain lacked thymidylate synthetase activity and had an absolute requirement for low concentrations (2 micrograms/ml) of thymidine in addition to a requirement for N-5-formyl tetrahydrofolic acid (folinate). Even at high concentrations (up to 500 micrograms/ml), thymine could not replace thymidine. In contrast to its wild-type parent, which grows only on folinate, the thymidine-requiring mutant (Thy- Fol+) was able to take up and grow on picogram quantities of unreduced folic acid. When both strains were grown on folinate, the Thy- Fol+ strain was at least 10(3)-fold more resistant to the folic acid analogs aminopterin and methotrexate than the wild-type strain. On the other hand, when grown on folic acid, the Thy- Fol+ strain was as sensitive to the folic acid analogs as the Thy+ Fol+ strain and was 10(2)-fold more sensitive than the wild-type strain grown on folinate. The thymidine-independent revertant (Thy+ Fol+) regained the wild-type level of thymidylate synthetase activity, but maintained the ability to take up and grow on unreduced folic acid like its Thy- Fol+ parent.     

Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum.

The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.     

Pathways leading to and from serine during growth of Pseudomonas AM1 in C1 compounds or succinate

1. The following enzymes of the phosphorylated pathway of serine biosynthesis have been found in methanol- and succinate-grown Pseudomonas AM1: phosphoglycerate dehydrogenase, phosphoserine-alpha-oxoglutarate aminotransferase and phosphoserine phosphohydrolase. Their specific activities were similar in the organism grown on either substrate. 2. A procedure for preparation of auxotrophic mutants of Pseudomonas AM1 is described involving N-methyl-N'-nitro-N-nitrosoguanidine as mutagen and a penicillin enrichment step. 3. A mutant, M-15A, has been isolated that is unable to grow on methanol and that lacks phenazine methosulphate-linked methanol dehydrogenase. The mutant is able to grow on methylamine, showing that the amine is not oxidized by way of methanol. 4. Loss of methanol dehydrogenase activity in mutant M-15A led to loss of phenazine methosulphate-linked formaldehyde dehydrogenase activity showing that the same enzyme is probably responsible for both activities. 5. A mutant, 20B-L, has been isolated that cannot grow on any C(1) compound tested but can grow on succinate. 6. Mutant 20B-L lacks hydroxypyruvate reductase, and revertants that regained the ability to grow on methanol, methylamine and formate contained hydroxypyruvate reductase activity at specific activities similar to that of the wild-type organism. This shows that hydroxypyruvate reductase is necessary for growth on methanol, methylamine and formate but not for growth on succinate. 7. The results suggest that during growth of Pseudomonas AM1 on C(1) compounds, serine is converted into 3-phosphoglycerate by a non-phosphorylated pathway, whereas during growth on succinate, phosphoglycerate is converted into serine by a phosphorylated pathway.     

Substitution of Glu-59 by val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme

A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase. The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene. The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography. The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method. The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70–80%. The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles. The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography. On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a M _r of 38,000 and 78,000 Dalton, respectively. These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme. Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase.