scholarly journals Ribitol dehydrogenase of Klebsiella aerogenes. Sequence and properties of wild-type and mutant strains

1985 ◽  
Vol 230 (3) ◽  
pp. 569-578 ◽  
Author(s):  
J M Dothie ◽  
J R Giglio ◽  
C B Moore ◽  
S S Taylor ◽  
B S Hartley
Keyword(s):  
Amino Acids ◽  
Continuous Culture ◽  
Specific Activity ◽  
Klebsiella Aerogenes ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Mutant Strains ◽  
Ribitol Dehydrogenase

Evidence is presented for the sequence of 249 amino acids in ribitol dehydrogenase-A from Klebsiella aerogenes. Continuous culture on xylitol yields strains that superproduce ‘wild-type’ enzyme but mutations appear to have arisen in this process. Other strains selected by such continuous culture produce enzymes with increased specific activity for xylitol but without loss of ribitol activity. One such enzyme, ribitol dehydrogenase-D, has Pro-196 for Gly-196. Another, ribitol dehydrogenase-B, has a different mutation.

scholarly journals Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance

2006 ◽  
Vol 397 (2) ◽  
pp. 305-312 ◽  
Author(s):  
G. H. Erica Law ◽  
Olga A. Gandelman ◽  
Laurence C. Tisi ◽  
Christopher R. Lowe ◽  
James A. H. Murray
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Green Light ◽  
Firefly Luciferase ◽  
Wild Type ◽  
Photinus Pyralis ◽  
Wild Type Enzyme ◽  
Ph Tolerance

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow–green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 °C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

Enzymes of the Entner-Doudoroff and pyruvate decarboxylation pathways in Zymomonas mobilis wild-type CP4 and mutant strains grown in continuous culture

2001 ◽  
Vol 80 (3) ◽  
pp. 287-295
Author(s):  
Alexander L. Sawides ◽  
Kalliopi I. Kalliopi ◽  
milton A. Typas ◽  
Amalia D. Karagouni
Keyword(s):  
Continuous Culture ◽  
Zymomonas Mobilis ◽  
Wild Type ◽  
Mutant Strains

scholarly journals Structural identification of the myo-inositol 1,4,5-trisphosphate-binding domain in rat brain inositol 1,4,5-trisphosphate 3-kinase

1997 ◽  
Vol 326 (1) ◽  
pp. 221-225 ◽  
Author(s):  
Shinji TOGASHI ◽  
Kazunaga TAKAZAWA ◽  
Toyoshi ENDO ◽  
Christophe ERNEUX ◽  
Toshimasa ONAYA
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Apparent Homogeneity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Charged Amino Acid Residue

A series of key amino acids involved in Ins(1,4,5)P3 (InsP3) binding and catalytic activity of rat brain InsP3 3-kinase has been identified. The catalytic domain is at the C-terminal end and restricted to a maximum of 275 amino acids [Takazawa and Erneux (1991) Biochem. J. 280, 125–129]. In this study, newly prepared 5′-deletion and site-directed mutants have been compared both for InsP3 binding and InsP3 3-kinase activity. When the protein was expressed from L259 to R459, the activity was lost but InsP3 binding was conserved. Another deletion mutant that had lost only four amino acids after L259 had lost InsP3 binding, and this finding suggests that these residues (i.e. L259DCK262) are involved in InsP3 binding. To further support the data, we have produced two mutants by site-directed mutagenesis on residues C261 and K262. The two new enzymes were designated M4 (C261S) and M5 (K262A). M4 showed similar Vmax and Km values for InsP3 and ATP to wild-type enzyme. In contrast, M5 was totally inactive but had kept the ability to bind to calmodulin–Sepharose. C-terminal deletion mutants that had lost five, seven or nine amino acids showed a large decrease in InsP3 binding and InsP3 3-kinase activity. One mutant that had lost five amino acids (M2) was purified to apparent homogeneity: Km values for both substrates appeared unchanged but Vmax was decreased approx. 40-fold compared with the wild-type enzyme. The results indicate that (1) a positively charged amino acid residue K262 is essential for InsP3 binding and (2) amino acids at the C-terminal end of the protein are necessary to act as a catalyst in the InsP3 3-kinase reaction.

scholarly journals Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

2012 ◽  
Vol 78 (11) ◽  
pp. 3880-3884 ◽  
Author(s):  
Yu-Ri Lim ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

scholarly journals Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

1979 ◽  
Vol 179 (1) ◽  
pp. 99-107 ◽  
Author(s):  
Jeffrey D. Hillman
Keyword(s):  
Escherichia Coli ◽  
Simple Procedure ◽  
Rabbit Antiserum ◽  
Double Diffusion ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Glycolytic Intermediate ◽  
E Coli ◽  
Catalytic Function ◽  
Mutant Strains

NAD+-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD+ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD+ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.

scholarly journals Amino Acid Residue 147 of Human Aldosterone Synthase and 11β-Hydroxylase Plays a Key Role in 11β-Hydroxylation*

2000 ◽  
Vol 85 (3) ◽  
pp. 1261-1266
Author(s):  
Angela Fisher ◽  
Robert Fraser ◽  
John MC Connell ◽  
Eleanor Davies
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Affinity ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Active Centers ◽  
Hormone Production ◽  
Aldosterone Synthase ◽  
Linear Distance ◽  
Aldosterone Production

Abstract A number of amino acids differ between aldosterone synthase and 11β-hydroxylase. To assess their importance in determining the different functional specificities, we substituted aldosterone synthase-specific (aspartate D147, isoleucine I248, glutamine Q43, and threonine T493) with 11β-hydroxylase-specific amino acids (glutamate E147, threonine T248, arginine R43, and methionine M493), respectively. I248T, Q43R, and T493M had no effect on steroid production compared to wild-type aldosterone synthase. However, CYP11B2-D147E caused a significant increase in corticosterone production and a smaller increase in aldosterone production from 11-deoxycorticosterone (DOC). This appeared to be predominantly due to an increase in the 11β-hydroxylation of DOC to corticosterone mediated by a decrease in Km, which was 1.4 μmol/L for the mutant compared with 5μ mol/L for the wild-type enzyme. CYP11B2-D147E had no effect on the conversion of 11-deoxycortisol to cortisol. The reverse construct (CYP11B1-E147D), substituting the 11β-hydroxylase residue with the aldosterone synthase equivalent, decreased the conversion of DOC to corticosterone, which was mediated by an increase in Km that was 7.5 μmol/L for the mutant compared with 2.5 μmol/L for the wild-type enzyme. Again, the conversion of 11-deoxycortisol to cortisol was unimpaired. Thus, amino acid 147 is involved in the transformation of the 17-deoxysubstrate, but not the 17α-hydroxysubstrate. The results demonstrate that a conservative change in amino acid, even at some linear distance from known active centers, can significantly affect enzyme substrate affinity and subsequent steroid hormone production.

scholarly journals THE INFLUENCE OF PRECURSOR POOL SIZE ON MITOCHONDRIAL COMPOSITION IN NEUROSPORA CRASSA

1965 ◽  
Vol 24 (3) ◽  
pp. 445-460 ◽  
Author(s):  
David J. L. Luck
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Wild Type ◽  
Difference Spectra ◽  
Leucine Supplementation ◽  
Protein Ratio ◽  
Kinetic Measurements ◽  
Precursor Pool ◽  
Mutant Strains ◽  
Isopycnic Centrifugation

The chemical composition of mitochondria obtained from exponentially growing Neurospora can be varied by addition of choline or amino acids to the culture medium. The variation affects the phospholipid to protein ratio, and the density of mitochondria as determined by isopycnic centrifugation in sucrose gradients. These variations have been observed in biochemical mutant strains as well as wild type cultures. In a choline-requiring strain, two levels of choline supplementation to the medium have been defined: a low choline concentration just adequate to support maximal logarithmic growth, and a high choline concentration which permits maximal incorporation of radioactive choline into cellular lipids. Mitochondria isolated from cultures growing at the low choline concentration have one-half the phospholipid to protein ratio of those from high choline cultures, and their density is significantly higher. Artificial mixtures of the two types of mitochondria can be resolved into two populations by isopycnic centrifugation. The concentration of cytochromes (measured by mitochondrial difference spectra) and of malate and succinate dehydrogenases (measured by enzyme activity) were the same in both types of mitochondria, on a protein basis. The results suggest that during growth of the mitochondrial mass, the incorporation of phospholipid and protein components can vary independently. Direct kinetic measurements did indeed show that choline, added to a culture growing at low choline concentration, was incorporated into mitochondrial lipids at a rate faster than the incorporation of protein. The mitochondrial phospholipid to protein ratio can also be influenced by the level of leucine supplementation to a leucine-requiring mutant, so that with leucine concentrations above those required for maximal exponential growth, mitochondria of increasing density and decreasing phospholipid to protein ratio are produced. Additions of choline or amino acids to the minimal medium of wild type cultures influence mitochondrial composition in a manner directly comparable to that observed in biochemical mutant strains. The results suggest that mitochondrial composition, in general, is determined by rates of incorporation of the two major components, phospholipid and protein; that these rates can vary independently in response to precursor concentration in the culture medium; and that they normally operate at a precursor (substrate) concentration below saturation level.

Site-directed mutagenesis of farnesyl diphosphate synthase; effect of substitution on the three carboxyl-terminal amino acids

1994 ◽  
Vol 72 (1) ◽  
pp. 75-79 ◽  
Author(s):  
Tanetoshi Koyama ◽  
Kazuhiro Saito ◽  
Kyozo Ogura ◽  
Shusei Obata ◽  
Ayumi Takeshita
Keyword(s):  
Amino Acids ◽  
Bacillus Stearothermophilus ◽  
Farnesyl Diphosphate ◽  
Site Directed Mutagenesis ◽  
Farnesyl Diphosphate Synthase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Directed Mutation ◽  
Catalytic Function ◽  
Terminal Amino

Site-directed mutation was introduced into the gene for the farnesyl diphosphate synthase of Bacillus stearothermophilus. To investigate the significance of the three C-terminal amino acids, where arginine is completely conserved throughout the farnesyl diphosphate synthases of prokaryotes and eukaryotes, three kinds of mutant enzymes, R295V, D296G, and H297L, which have replacements of arginine-295 with valine, aspartate-296 with glycine, and histidine-297 with leucine, respectively, were overproduced and purified to homogeneity. All of the three mutant enzymes showed similar catalytic activities to that of the wild-type enzyme, indicating that the basic amino acids including the conserved arginine in the C-terminal region are not essential for catalytic function. They were also similar to the wild-type enzyme with respect to pH optima, thermostability, reaction product, and kinetic parameters for allylic substrates. However, their Km values for isopentenyl diphosphate are approximately twice that of the wild type.

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