scholarly journals Synthesis of Glucosyltrehaloses by a Thermostable .ALPHA.-Glucosidase with Broad Substrate Specificity and High Transglucosylation Activity: Assessment Using Wild-type Enzyme and Mutants with Amino Acid Substitution near the Active Site.

2002 ◽  
Vol 55 (1) ◽  
pp. 19-25
Author(s):  
Maki Okada ◽  
Toru Nakayama ◽  
Akio Noguchi ◽  
Tokuzo Nishino ◽  
Yukiko Kan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Substitution ◽  
Active Site ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Broad Substrate Specificity ◽  
Activity Assessment ◽  
Alpha Glucosidase

scholarly journals Impact of the E540V Amino Acid Substitution in GyrB ofMycobacterium tuberculosison Quinolone Resistance

2011 ◽  
Vol 55 (8) ◽  
pp. 3661-3667 ◽  
Author(s):  
Hyun Kim ◽  
Chie Nakajima ◽  
Kazumasa Yokoyama ◽  
Zeaur Rahim ◽  
Youn Uck Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Dna Cleavage ◽  
Recombinant Dna ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Quinolone Resistance ◽  
Wild Type ◽  
Wild Type Enzyme

ABSTRACTAmino acid substitutions conferring resistance to quinolones inMycobacterium tuberculosishave generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance inM. tuberculosis, we expressed recombinant DNA gyrases inEscherichia coliand characterized themin vitro. Wild-type and GyrB-E540V DNA gyrases were reconstitutedin vitroby mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance inM. tuberculosis.

scholarly journals Alteration of Chain Length Substrate Specificity of Aeromonas caviae R-Enantiomer-Specific Enoyl-Coenzyme A Hydratase through Site-Directed Mutagenesis

2003 ◽  
Vol 69 (8) ◽  
pp. 4830-4836 ◽  
Author(s):  
Takeharu Tsuge ◽  
Tamao Hisano ◽  
Seiichi Taguchi ◽  
Yoshiharu Doi
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Chain Length ◽  
Acyl Chain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Acyl Chain Length ◽  
Aeromonas Caviae

ABSTRACT Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJAc) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid β-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJAc by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJAc revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C8) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C12) was examined by using the mutated PhaJAc as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1 Ps). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C8) and 3-hydroxydecanoate (C10) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.

scholarly journals New Insights into the Fructosyltransferase Activity of Schwanniomyces occidentalis β-Fructofuranosidase, Emerging from Nonconventional Codon Usage and Directed Mutation

2010 ◽  
Vol 76 (22) ◽  
pp. 7491-7499 ◽  
Author(s):  
Miguel Álvaro-Benito ◽  
Miguel de Abreu ◽  
Francisco Portillo ◽  
Julia Sanz-Aparicio ◽  
María Fernández-Lobato
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Transferase Activity ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Schwanniomyces Occidentalis ◽  
Directed Mutation

ABSTRACT Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k cat/Km ) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.

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.

scholarly journals LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity

2006 ◽  
Vol 398 (3) ◽  
pp. 531-538 ◽  
Author(s):  
Yukiko Mizutani ◽  
Akio Kihara ◽  
Yasuyuki Igarashi
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Family Members ◽  
Fatty Acyl ◽  
Ceramide Synthase ◽  
Broad Substrate Specificity ◽  
Acid Protein ◽  
Amino Acid Protein

The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

Prediction of amino acid residues participated in substrate recognition by cytochrome P450 subfamilies with broad substrate specificity

2013 ◽  
Vol 26 (2) ◽  
pp. 86-91 ◽  
Author(s):  
Maria S. Zharkova ◽  
Boris N. Sobolev ◽  
Nina Yu. Oparina ◽  
Alexander V. Veselovsky ◽  
Alexander I. Archakov
Keyword(s):  
Cytochrome P450 ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Substrate Recognition ◽  
Amino Acid Residues ◽  
Broad Substrate Specificity

scholarly journals A Single Amino Acid Substitution in the West Nile Virus Nonstructural Protein NS2A Disables Its Ability To Inhibit Alpha/Beta Interferon Induction and Attenuates Virus Virulence in Mice

2006 ◽  
Vol 80 (5) ◽  
pp. 2396-2404 ◽  
Author(s):  
Wen Jun Liu ◽  
Xiang Ju Wang ◽  
David C. Clark ◽  
Mario Lobigs ◽  
Roy A. Hall ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Nonstructural Protein ◽  
Mutant Virus ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Wild Type ◽  
Alpha Beta ◽  
A30p Mutation

ABSTRACT Alpha/beta interferons (IFN-α/β) are key mediators of the innate immune response against viral infection. The ability of viruses to circumvent IFN-α/β responses plays a crucial role in determining the outcome of infection. In a previous study using subgenomic replicons of the Kunjin subtype of West Nile virus (WNVKUN), we demonstrated that the nonstructural protein NS2A is a major inhibitor of IFN-β promoter-driven transcription and that a single amino acid substitution in NS2A (Ala30 to Pro [A30P]) dramatically reduced its inhibitory effect (W. J. Liu, H. B. Chen, X. J. Wang, H. Huang, and A. A. Khromykh, J. Virol. 78:12225-12235). Here we show that incorporation of the A30P mutation into the WNVKUN genome results in a mutant virus which elicits more rapid induction and higher levels of synthesis of IFN-α/β in infected human A549 cells than that detected following wild-type WNVKUN infection. Consequently, replication of the WNVKUNNS2A/A30P mutant virus in these cells known to be high producers of IFN-α/β was abortive. In contrast, both the mutant and the wild-type WNVKUN produced similar-size plaques and replicated with similar efficiency in BHK cells which are known to be deficient in IFN-α/β production. The mutant virus was highly attenuated in neuroinvasiveness and also attenuated in neurovirulence in 3-week-old mice. Surprisingly, the mutant virus was also partially attenuated in IFN-α/βγ receptor knockout mice, suggesting that the A30P mutation may also play a role in more efficient activation of other antiviral pathways in addition to the IFN response. Immunization of wild-type mice with the mutant virus resulted in induction of an antibody response of similar magnitude to that observed in mice immunized with wild-type WNVKUN and gave complete protection against challenge with a lethal dose of the highly virulent New York 99 strain of WNV. The results confirm and extend our previous original findings on the role of the flavivirus NS2A protein in inhibition of a host antiviral response and demonstrate that the targeted disabling of a viral mechanism for evading the IFN response can be applied to the development of live attenuated flavivirus vaccine candidates.

R to Q Amino Acid Substitution in the GFFKR Sequence of the Cytoplasmic Domain of the Integrin IIb Subunit in a Patient With a Glanzmann’s Thrombasthenia-Like Syndrome

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4178-4187 ◽  
Author(s):  
O. Peyruchaud ◽  
A.T. Nurden ◽  
S. Milet ◽  
L. Macchi ◽  
A. Pannochia ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Chinese Hamster ◽  
Cytoplasmic Domain ◽  
Site Directed Mutagenesis ◽  
Heterozygous Mutation ◽  
Polymorphism Analysis ◽  
Wild Type ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

Abstract The integrin IIbβ3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of IIb and β3. We now report the molecular analysis of IIbβ3 from a patient with a Glanzmann’s thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of IIbβ3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of IIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the IIb gene and a single allele of the β3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated IIb together with wild-type β3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type IIb and β3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary–transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the IIb cytoplasmic domain.

scholarly journals Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

2019 ◽  
Vol 20 (18) ◽  
pp. 4412
Author(s):  
Denis L. Atroshenko ◽  
Mikhail D. Shelomov ◽  
Sophia A. Zarubina ◽  
Nikita Y. Negru ◽  
Igor V. Golubev ◽  
...  
Keyword(s):  
Amino Acid ◽  
Thermal Denaturation ◽  
Catalytic Properties ◽  
Acid Oxidase ◽  
Cephalosporin C ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Amino Acid Oxidase ◽  
Catalytic Constant ◽  
Biotechnological Processes

d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin C oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin C reported by others.

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