Improving low-temperature activity ofSulfolobus acidocaldarius2-keto-3-deoxygluconate aldolase

Sulfolobus acidocaldarius2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95°C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 °C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40–60 °C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.

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

Biochemical Journal ◽

10.1042/bj2770647 ◽

1991 ◽

Vol 277 (3) ◽

pp. 647-652 ◽

Cited By ~ 10

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.

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

Biochemistry and Cell Biology ◽

10.1139/o90-153 ◽

1990 ◽

Vol 68 (7-8) ◽

pp. 1037-1044 ◽

Cited By ~ 14

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.

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Elimination of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activity from human N-acetylneuraminic acid 9-phosphate synthase by a single mutation

Biochemical Journal ◽

10.1042/bj20052034 ◽

2006 ◽

Vol 397 (1) ◽

pp. 195-201 ◽

Cited By ~ 9

Author(s):

Jijun Hao ◽

Willie F. Vann ◽

Stephan Hinderlich ◽

Munirathinam Sundaramoorthy

Keyword(s):

Aldol Condensation ◽

Saturation Mutagenesis ◽

Single Mutation ◽

Wild Type ◽

Wild Type Enzyme ◽

High Sequence Identity ◽

Acetylneuraminic Acid ◽

Nonulosonic Acid ◽

The Impact ◽

Phosphate Synthase

The most commonly occurring sialic acid Neu5Ac (N-acetylneuraminic acid) and its deaminated form, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid), participate in many biological functions. The human Neu5Ac-9-P (Neu5Ac 9-phosphate) synthase has the unique ability to catalyse the synthesis of not only Neu5Ac-9-P but also KDN-9-P (KDN 9-phosphate). Both reactions are catalysed by the mechanism of aldol condensation of PEP (phosphoenolpyruvate) with sugar substrates, ManNAc-6-P (N-acetylmannosamine 6-phosphate) or Man-6-P (mannose 6-phosphate). Mouse and putative rat Neu5Ac-9-P synthases, however, do not show KDN-9-P synthase activity, despite sharing high sequence identity (>95%) with the human enzyme. Here, we demonstrate that a single mutation, M42T, in human Neu5Ac-9-P synthase can abolish the KDN-9-P synthase activity completely without compromising the Neu5Ac-9-P synthase activity. Saturation mutagenesis of Met42 of the human Neu5Ac-9-P synthase showed that the substitution with all amino acids except leucine retains only the Neu5Ac-9-P synthase activity at levels comparable with the wild-type enzyme. The M42L mutant, like the wild-type enzyme, showed the additional KDN-9-P synthase activity. In the homology model of human Neu5Ac-9-P synthase, Met42 is located 22 Å (1 Å=0.1 nm) away from the substrate-binding site and the impact of this distant residue on the enzyme functions is discussed.

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

Applied and Environmental Microbiology ◽

10.1128/aem.07012-11 ◽

2012 ◽

Vol 78 (11) ◽

pp. 3880-3884 ◽

Cited By ~ 17

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.

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Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N-glycosylation and inactivation by deamidation

Biochemical Journal ◽

10.1042/bj3010275 ◽

1994 ◽

Vol 301 (1) ◽

pp. 275-281 ◽

Cited By ~ 35

Author(s):

H M Chen ◽

C Ford ◽

P J Reilly

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Membrane ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Aspergillus Awamori ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Recognition Sites ◽

Membrane Components

Aspergillus awamori glucoamylase is a secreted glycoprotein containing N-linked carbohydrate recognition sites at Asn-171, Asn-182 and Asn-395. Site-directed mutagenesis was performed at Asn-182 and Asn-395 to determine whether these residues were N-glycosylated by Saccharomyces cerevisiae, to investigate the function of any glycans linked to them, and to determine the effect of their deamidation on glucoamylase thermostability. Asn-171 and Asn-395, but not Asn-182, were N-glycosylated. Deletion of the glycan N-linked to Asn-395 did not affect specific activity, but greatly decreased enzyme secretion and thermostability. The mutant lacking the N-glycan linked to Asn-395 was synthesized very slowly, and was more associated with cell membrane components and susceptible to proteinase degradation than were wild-type or other mutant glucoamylases. Its secreted form was 30-fold less thermostable than wild-type enzyme at pH 4.5. Replacement of Asn-182 by Gln to eliminate deamidation at this site did not change glucoamylase specific activity or thermostability, while replacement by Asp decreased specific activity about 25%, but increased thermostability moderately at pH 4.5 below 70 degrees C. Both mutations of Asn-182 increased glucoamylase production.

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Enhancement of the Stability of a Prolipase from Rhizopus oryzae toward Aldehydes by Saturation Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.01176-07 ◽

2007 ◽

Vol 73 (22) ◽

pp. 7291-7299 ◽

Cited By ~ 22

Author(s):

Mirella Di Lorenzo ◽

Aurelio Hidalgo ◽

Rafael Molina ◽

Juan A. Hermoso ◽

Domenico Pirozzi ◽

...

Keyword(s):

Staining Method ◽

Rhizopus Oryzae ◽

Specific Activity ◽

Oxidation Products ◽

Saturation Mutagenesis ◽

Amino Acid Residues ◽

Wild Type ◽

Wild Type Enzyme ◽

Lipid Oxidation Products ◽

The Stability

ABSTRACT A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.

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Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase

Biochemical Journal ◽

10.1042/bj3260047 ◽

1997 ◽

Vol 326 (1) ◽

pp. 47-51 ◽

Cited By ~ 16

Author(s):

Shoshana KEYNAN ◽

Nigel M. HOOPER ◽

Anthony J. TURNER

Keyword(s):

Specific Activity ◽

Mammalian Species ◽

Competitive Inhibitor ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Renal Metabolism ◽

Leukotriene D4 ◽

Histidine Residues

Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin–Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

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

Biochemical Journal ◽

10.1042/bj20051847 ◽

2006 ◽

Vol 397 (2) ◽

pp. 305-312 ◽

Cited By ~ 46

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.

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

Biochemical Journal ◽

10.1042/bj2300569 ◽

1985 ◽

Vol 230 (3) ◽

pp. 569-578 ◽

Cited By ~ 26

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.

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