Genetic mapping of katG, a locus that affects synthesis of the bifunctional catalase-peroxidase hydroperoxidase I in Escherichia coli.

A locus unlinked to either katE or katF that affected catalase levels in Escherichia coli was identified and localized between metB and ppc at 89.2 min on the genome. The locus was named katG. Mutations in katG which prevented the formation of both isoenzyme forms of the bifunctional catalase-peroxidase HPI were created both by nitrosoguanidine and by transposon Tn10 insertions. All katG+ recombinants and transductants contained both HPI isoenzymes. Despite the common feature of little or no catalase activity in four of the catalase-deficient strains, subtle differences in the phenotypes of each strain resulted from the different katG mutations. All three mutants caused by nitrosoguanidine produced a protein with little or no catalase activity but with the same subunit molecular weight and with similar antigenic properties to HPI, implying the presence of missense mutations rather than nonsense mutations in each strain. Indeed one mutant produced an HPI-like protein that retained peroxidase activity, whereas the HPI-like protein in a second mutant exhibited no catalase or peroxidase activity. The third mutant responded to ascorbate induction with the synthesis of near normal catalase levels, suggesting a regulatory defect. The Tn10 insertion mutant produced no catalase and no protein that was antigenically similar to HPI.     

Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: mutational analysis of catalytic residues

We engineered an acetyl xylan esterase (AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser(119), Ser(146), Asp(168) and Asp(202), were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser(119) and Asp(202) are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased K(m) and decreased k(cat). The catalytic efficiency of S146A was 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold than that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.     

Site-directed mutagenesis and virulence assessment of the katG gene of Mycobacterium intracellulare

Mycobacterial catalases have been suggested as acting as virulence factors by protecting intracellular mycobacteria from reactive oxidative metabolites produced by host phagocytes. Mycobacterium intracellulare , like many other mycobacteria, produces two proteins with catalase activity: a heat-stable catalase (KatE) and an inducible, heat-labile catalase peroxidase (KatG). The M. intracellulare katG gene was cloned, and a plasmid derivative with a 4 bp insertion in the katG coding sequence was constructed and used for site-directed mutagenesis of M. intracellulare 1403 (ATCC 35761). The resulting katG mutant was highly resistant to isoniazid (INH), showed an increased sensitivity to H₂O₂ and had lost peroxidase and heat-sensitive catalase activity but retained heat-stable catalase activity. The plasmid carrying the katG frameshift allele was also used for mutagenesis of the mouse virulent M. intracellulare isolate D673. After intravenous injection into BALB/c mice, D673 and the isogenic katG mutant showed the same growth kinetics in the spleen, liver and lungs of the infected mice. Our results demonstrate that the KatG catalase peroxidase mediates resistance to H₂O₂ and susceptibility to INH but is not an essential virulence factor for the survival and growth of M. intracellulare in the mouse.     

Nucleotide sequence of katG, encoding catalase HPI of Escherichia coli.

The gene katG, encoding catalase HPI of Escherichia coli, was sequenced, predicting a 726-amino-acid protein. The sequence was confirmed by identification of potential regulatory elements and amino acid sequencing of peptides. HPI shows no homology to other catalases. The distances between katG, metF, and ppc were defined.     

Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

Despite the structural similarities between cholesterol oxidase from Streptomyces and that from Brevibacterium, both enzymes exhibit different characteristics, such as catalytic activity, optimum pH and temperature. In attempts to define the molecular basis of differences in catalytic activity or stability, substitutions at six amino acid residues were introduced into cholesterol oxidase using site-directed mutagenesis of its gene. The amino acid substitutions chosen were based on structural comparisons of cholesterol oxidases from Streptomyces and BREVIBACTERIUM: Seven mutant enzymes were constructed with the following amino acid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357N and S379T. All the mutant enzymes exhibited activity with the exception of that with the L117P mutation. The resulting V145Q mutant enzyme has low activities for all substrates examined and the S379T mutant enzyme showed markedly altered substrate specificity compared with the wild-type enzyme. To evaluate the role of V145 and S379 residues in the reaction, mutants with two additional substitutions in V145 and four in S379 were constructed. The mutant enzymes created by the replacement of V145 by Asp and Glu had much lower catalytic efficiency for cholesterol and pregnenolone as substrates than the wild-type enzyme. From previous studies and this study, the V145 residue seems to be important for the stability and substrate binding of the cholesterol oxidase. In contrast, the catalytic efficiencies (k(cat)/K(m)) of the S379T mutant enzyme for cholesterol and pregnenolone were 1.8- and 6.0-fold higher, respectively, than those of the wild-type enzyme. The enhanced catalytic efficiency of the S379T mutant enzyme for pregnenolone was due to a slightly high k(cat) value and a low K(m) value. These findings will provide several ideas for the design of more powerful enzymes that can be applied to clinical determination of serum cholesterol levels and as sterol probes.     

Oxidative stress effects on conjugational recombination and mutation in catalase-deficient Escherichia coli

The objective of the present investigation was to determine the effects on genetic recombination and mutation in Escherichia coli of either endogenous increases in oxygen radicals resulting from catalase deficiencies, or exogenous increases resulting from H2O2 treatment. Using the classical paradigm of Escherichia coli bacterial conjugation, strains deficient in the production of hydroperoxidase I (HPI) and/or hydroperoxidase II (HPII) were used as recipients in Hfr x F- matings. 'Background' recombination rates, measured by the rate of appearance of threonine prototrophs, was similar to wild-type levels in the HPI-deficient (katG) strain, but were significantly decreased in HPII- (katE) mutants. The addition of relatively nontoxic H2O2 concentrations (0.25 mmoles dm-3) to the mating mixtures stimulated recombination rates in wild-type and katE strains, but decreased rates in katG and katEkatG strains. A 0.5 mmoles dm-3 concentration of H2O2 inhibited recombination rates in all strains. In order to gauge the level of recA-dependent 'SOS' processes occurring under the experimental conditions, 'background' mutation rates were determined in both fluctuation and forward mutation (thyA) assays. Mutation rates in aerobically-grown cultures were increased up to 2.2-fold in katG and katEkatG strains. Treatment with relatively nontoxic H2O2 concentrations elevated the thyA mutagenesis up to 8-fold in catalase-deficient cultures. Furthermore, these studies along with data presented elsewhere show that the SOS phenotype of katEkatG is more resistant than that of katG strains. These studies clearly show that cellular oxidative stress occurring from catalase deficiency interferes with normal DNA metabolism.     

Activity and spectroscopic properties of bacterial D-amino acid transaminase after multiple site-directed mutagenesis of a single tryptophan residue

One of the three tryptophan residues per subunit of thermostable D-amino acid transaminase, Trp-139, is close to the active-site Lys-145 in the sequence of the protein. This tryptophan has been changed to several other types of residues by site-directed mutagenesis. The only mutant protein that was sufficiently active and stable for study had Phe substituted for Trp (W139F). The spectroscopic properties of this mutant enzyme differed from those of the wild-type transaminase. For example, denatured W139F showed the expected decrease in fluorescence emission intensity at 350 nm due to the deletion of one Trp residue, but the fluorescence emission of the wild-type and W139F enzymes in the native state did not differ in intensity. This result suggests that the fluorescence of Trp-139 in the native, wild-type enzyme is not manifested perhaps due to its proximity to the coenzyme, pyridoxal phosphate. Results of energy-transfer studies at several wavelengths could also be interpreted as due to the proximity of Trp-139 and the coenzyme. Circular dichroism studies indicated that the negative Cotton effect at 420 nm due to the coenzyme was still present in W139F. However, the 280-nm optically active band present in the wild-type enzyme was greatly diminished in W139F. The mutant protein with Asp at position 139 (W139D) could not be isolated presumably because it was degraded. The other mutant enzymes, W139P, W139A, and W139H, were isolated with partial activities (15-35%) that were slowly lost upon storage at 4 degrees C. Overall, these results indicate the importance of Trp-139 in the thermostable D-amino acid transaminase.     

Thr373, Asp375, and Lys260 are in the coenzyme site of porcine NADP-dependent isocitrate dehydrogenase

Thr(373), Lys(374), Asp(375), and Lys(260) were chosen as site-directed mutagenesis targets within porcine NADP-dependent isocitrate dehydrogenase based on structurally corrected sequence alignment among prokaryotic and eukaryotic NADP-isocitrate dehydrogenases. Wild-type and all mutant enzymes were expressed in Escherichia coli and purified to homogeneity. These mutations do not alter the secondary structure or dimerization state of the mutants. The D375N and K260Q mutants exhibit, respectively, a 15- and 28-fold increase in K(m) for NADP, along with marked decreases in V(max) as compared to wild-type enzyme. In contrast, replacing Lys(374), which was previously proposed to contribute to apparent coenzyme affinity, does not change the enzyme's kinetic parameters. T373S exhibits similar kinetic parameters to those of wild-type while T373A and T373V mutations reduce the V(max) values of the resulting enzymes to 1 and 20%, respectively of that of wild-type. We conclude that a hydroxyl group at position 373 is required for effective enzyme function and that Asp(375) and Lys(260) are critical amino acids contributing to coenzyme affinity as well as catalysis by porcine NADP-isocitrate dehydrogenase.     

Effect of distal cavity mutations on the formation of compound I in catalase-peroxidases

Catalase-peroxidases have a predominant catalase activity but differ from monofunctional catalases in exhibiting a substantial peroxidase activity and in having different residues in the heme cavity. We present a kinetic study of the formation of the key intermediate compound I by probing the role of the conserved distal amino acid triad Arg-Trp-His of a recombinant catalase-peroxidase in its reaction with hydrogen peroxide, peroxoacetic acid, and m-chloroperbenzoic acid. Both the wild-type enzyme and six mutants (R119A, R119N, W122F, W122A, H123Q, H123E) have been investigated by steady-state and stopped-flow spectroscopy. The turnover number of catalase activity of R119A is 14.6%, R119N 0.5%, H123E 0.03%, and H123Q 0.02% of wild-type activity. Interestingly, W122F and W122A completely lost their catalase activity but retained their peroxidase activity. Bimolecular rate constants of compound I formation of the wild-type enzyme and the mutants have been determined. The Trp-122 mutants for the first time made it possible to follow the transition of the ferric enzyme to compound I by hydrogen peroxide spectroscopically underlining the important role of Trp-122 in catalase activity. The results demonstrate that the role of the distal His-Arg pair in catalase-peroxidases is important in the heterolytic cleavage of hydrogen peroxide (i.e. compound I formation), whereas the distal tryptophan is essential for compound I reduction by hydrogen peroxide.     

Growth of Escherichia coli in iron-enriched medium increases HPI catalase activity

Escherichia coli has two catalases, HPI and HPII. HPI is induced during logarithmic growth in response to low concentrations of hydrogen peroxide. This induction is OxyR-dependent. On the other hand, HPII is not peroxide-inducible but is induced in entry to the stationary phase. We demonstrate here that E. coli displayed higher HPI catalase activity when compared to the cultures that were grown in a normal medium, if grown in a medium supplemented with iron-citrate. Iron supplementation had no effect on HPII catalase. This increase of HPI activity was OxyR-independent and not observed in a Deltafur mutant. The physiological significance of the increase of HPI activity is unclear, but it appears that the katG gene that codes for HPI catalase is among the genes that are regulated by Fur.     

Identification of the principal catalytically important acidic residue of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Kinetic analysis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has implicated a glutamate or aspartate residue in (i) formation of mevaldate thiohemiacetal by proton transfer to the carbonyl oxygen of mevaldate and (ii) enhanced ionization of CoASH by the resulting enzyme carboxylate anion, facilitating attack by CoAS- on the carbonyl carbon of mevaldate (Veloso, D., Cleland, W. W., and Porter, J. W. (1981) Biochemistry 81, 887-894). Although neither the identity of this acidic residue nor its location is known, the catalytic domains of 11 sequenced HMG-CoA reductases contain only 3 conserved acidic residues. For HMG-CoA reductase of Pseudomonas mevalonii, these residues are Glu52, Glu83, and Asp183. To identify the acidic residue that functions in catalysis, we generated mutants having alterations in these residues. The mutant proteins were expressed, purified, and characterized. Mutational alteration of residues Glu52 or Asp183 of P. mevalonii HMG-CoA reductase yielded enzymes with significant, but in some cases reduced, activity (Vmax = 100% Asp183----Ala, 65% Asp183----Asn, and 15% Glu52----Gln of wild-type activity, respectively). Although the activity of mutant enzymes Glu52----Gln and Asp183----Ala was undetectable under standard assay conditions, their Km values for substrates were 4-300-fold higher than those for wild-type enzyme. Km values for wild-type enzyme and for mutant enzymes Glu52----Gln and Asp183----Ala were, respectively: 0.41, 73, and 120 mM [R,S)-mevalonate); 0.080, 4.4, and 2.0 mM (coenzyme A); and 0.26, 4.4, and 1.0 mM (NAD+). By these criteria, neither Glu52 nor Asp183 is the acidic catalytic residue although each may function in substrate recognition. During chromatography on coenzyme A agarose or HMG-CoA agarose, mutant enzymes Asp183----Asn and Glu83----Gln behaved like wild-type enzyme. By contrast, and in support of a role for these residues in substrate recognition, mutant enzymes Glu52----Gln and Asp183----Ala exhibited impaired ability to bind to either support. Despite displaying Km values for substrates and chromatographic behavior on substrate affinity supports comparable to wild-type enzyme, only mutant enzyme Glu83----Gln was essentially inactive under all conditions studied (Vmax = 0.2% that of wild-type enzyme). Glutamate residue 83 of P. mevalonii HMG-CoA reductase, and consequently the glutamate of the consensus Pro-Met-Ala-Thr-Thr-Glu-Gly-Cys-Leu-Val-Ala motif of the catalytic domains of eukaryotic HMG-CoA reductases, is judged to be the acidic residue functional in catalysis.     

Isolation of a Bacillus stearothermophilus mutant exhibiting increased thermostability in its restriction endonuclease.

A procedure was developed for the selection of spontaneous mutants of Bacillus stearothermophilus NUB31 that are more efficient than the wild type in the restriction of phage at elevated temperatures. Inactivation studies revealed that two mutants contained a more thermostable restriction enzyme and one mutant contained three times more enzyme than the wild type. The restriction endonucleases from the wild type and one of the mutants were purified to apparent homogeneity. The mutant enzyme was more thermostable than the wild-type enzyme. The subunit molecular weight, amino acid composition, N-terminal and C-terminal amino acid residues, tryptic peptide map, and catalytic properties of the two enzymes were determined. The two enzymes have similar catalytic properties, but the molecular size of the mutant enzyme is approximately 6 to 7 kilodaltons larger than that of the wild-type enzyme. The mutant enzyme contains 54 additional amino acid residues, of which 26 to 28 are aspartate/asparagine, 8 to 15 are glutamate/glutamine, and 8 to 9 are tyrosine residues. The two enzymes contained similar amounts of the other amino acids, identical N-terminal residues, and different C-terminal residues. Tryptic peptide analyses revealed a high degree of homology between the two enzymes. The increased thermostability observed in the mutant enzyme appears to have been achieved by a mutation that resulted in the addition of amino acid residues to the wild-type enzyme. A number of mechanisms are discussed that could account for the observed difference between the mutant and wild-type enzymes.     

The role of antioxidant systems in response of bacteria Escherichia coli to heat shock]

Shifting the temperature from 30 to 45 degrees C in an aerobic Escherichia coli culture inhibited the expression of the antioxidant genes katG, katE, sodA, and gor. The expression was evaluated by measuring beta-galactosidase activity in E. coli strains that contained fusions of the antioxidant gene promoters with the lacZ operon. Heat shock inhibited catalase and glutathione reductase, lowered the intracellular level of glutathione, and increased its extracellular level. It also suppressed the growth of mutants deficient in the katG-encoded catalase HPI, whereas the sensitivity of the wild-type and sodA sodB mutant cells to heat shock was almost the same. In the E. coli culture adapted to growth at 42 degrees C, the content of both intracellular and extracellular glutathione was two times higher than in the culture grown at 30 degrees C. The temperature-adapted cells grown aerobically at 42 degrees C showed an increased ability to express the fused katG-lacZ genes.     

Catalase-peroxidase of Caulobacter crescentus: function and role in stationary-phase survival.

Caulobacter crescentus is an obligate aerobe which is exposed to high concentrations of photosynthetic oxygen and low levels of nutrients in its aquatic environment. Physiological studies of oxidative and starvation stresses in C. crescentus were undertaken through a study of lacZ fusion and null mutant strains constructed from the cloned 5' end of katG, encoding a catalase-peroxidase. The katG gene was shown to be solely responsible for catalase and peroxidase activity in C. crescentus. Like the katG of Escherichia coli, C. crescentus katG is induced by hydrogen peroxide and is important in sustaining the exponential growth rate. However, dramatic differences are seen in growth stage induction. E. coli KatE catalase and KatG catalase-peroxidase activities are induced 15- to 20-fold during exponential growth and then approximately halved in the stationary phase. In contrast, C. crescentus KatG activity is constant throughout exponential growth and is induced 50-fold in the stationary phase. Moreover, the survival of a C. crescentus katG null mutant is reduced by more than 3 orders of magnitude after 24 h in stationary phase and more than 6 orders of magnitude after 48 h, a phenotype not seen for E. coli katE and katG null mutants. These results indicate a major role for C. crescentus catalase-peroxidase in stationary-phase survival and raise questions about whether the peroxidatic activity as well as the protective catalatic activity of the dual-function enzyme is important in the response to starvation stress.     

Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat.

The likelihood for improvement in the catalytic properties of Escherichia coli alkaline phosphatase was examined using site-directed mutagenesis. Mutants were constructed by introducing sequence changes into nine preselected amino acid sites within 10 A of the catalytic residue serine 102. When highly conserved residues in the family of alkaline phosphatases were mutated, many of the resulting enzymes not only maintained activity, but also exhibited greatly improved kcat. Of approximately 170 mutant enzymes screened, 5% (eight mutants) exhibited significant increases in specific activity. In particular, a substitution by serine of a totally invariant Asp101 resulted in a 35-fold increase of specific activity over wild-type at pH 10.0. Up to 6-fold increases of the kcat/Km ratio were observed.     

Site-directed mutagenesis of the katG gene of Mycobacterium tuberculosis: effects on catalase–peroxidase activities and isoniazid resistance

Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug-resistant strains are mutated in the katG gene which encodes a catalase–peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug-resistant phenotype have been identified by evaluating INH-resistant clinical isolates and in vitro mutants. In this study, site-directed mutagenesis techniques were utilized to alter the wild-type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG -defective, INH-resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase–peroxidase which reduce catalase activity also decrease peroxidase activity.     

Physical characterization of katG, encoding catalase HPI of Escherichia coli

The gene encoding the bifunctional catalase-peroxidase HPI from Escherichia coli was located on a 3.8-kb HindIII fragment of the Clarke and Carbon plasmid pLC36-19 using transposon Tn5 insertions. This fragment was subcloned into the HindIII site of pAT153 to create pBT22. The size of the insert was reduced by BAL 31 digestion of one end to an apparent minimum size for catalase expression of approx. 2.5 kb as determined by complementation and expression in maxicell strains. Further reduction in size or digestion from the opposite end inactivated the gene. The location and orientation of the promoter at the 0 kb end of the insert in pBT22 was confirmed by cloning a 320-bp BglII fragment into the promoter-cloning vector pKK232-8. Differences in the Southern blots of genomic DNA from a wild-type strain and a katG17::Tn10 mutant digested with HincII and probed with pBT22 confirmed that the transposon previously mapped in katG was located in the 2.5-kb coding region for HPI.     

Mutations in Escherichia coli that effect sensitivity to oxygen

Fifteen oxygen-sensitive (Oxys) mutants of Escherichia coli were isolated after exposure to UV light. The mutants did not form macroscopic colonies when plated aerobically. They did form macroscopic colonies anaerobically. Oxygen, introduced during log phase, inhibited the growth of liquid cultures. The degree of inhibition was used to separate the mutants into three classes. Class I mutants did not grow after exposure to oxygen. Class II mutants were able to grow, but at a reduced rate and to a reduced final titer, when compared with the wild-type parent. Class III mutants formed filaments in response to oxygen. Genetic experiments indicated that the mutations map to six different chromosomal regions. The results of enzymatic assays indicated that 7 of the 10 class I mutants have low levels of catalase, peroxidase, superoxide dismutase, and respiratory enzymes when compared with the wild-type parent. Mutations in five of the seven class I mutants which have the low enzyme activities mapped within the region 8 to 13.5 min. P1 transduction data indicated that mutations in three of these five mutants, Oxys-6, Oxys-14, and Oxys-17, mapped to 8.4 min. The correlation of low enzyme levels and mapping data suggests that a single gene may regulate several enzymes in response to oxygen. The remaining three class I mutants had wild-type levels of catalase, peroxidase, and superoxide dismutase, but decreased respiratory activity. The class II and III mutants had enzyme activities similar to those of the wild-type parent. Our results demonstrate that mutations in at least six genes can be expressed as oxygen sensitivity. Some of these genes may be involved in respiration or cell division or may regulate the expression of several enzymes.     

Modulation of activity and substrate specificity by modifying the backbone length of the distant interdomain loop of D-amino acid aminotransferase.

The activity and substrate specificity of D-amino acid aminotransferase (D-AAT) (EC 2.6.1.21) can be rationally modulated by replacing the loop core (P119-R120-P121) with glycine chains of different lengths: 1, 3, or 5 glycines. The mutant enzymes were much more active than the wild-type enzyme in the overall reactions between various amino acids and pyruvate. The presteady-state kinetic analyses of half-reactions revealed that the 5-glycine mutant has the highest affinity (Kd) among all mutant enzymes and the wild-type enzyme towards various amino acids except D-aspartate. The 5-glycine mutant was much more efficient as a catalyst than the wild-type enzyme because the mutant enzyme showed the highest value of specificity constant (kmax/Kd) for all amino acids except D-aspartate and D-glutamate. The kmax/Kd values of the three mutants decreased with decrease in glycine chain length for each amino acid examined. Our findings may provide a new approach to rational modulation of enzymes.