scholarly journals Ser67Asp and His68Asp Substitutions in Candida parapsilosis Carbonyl Reductase Alter the Coenzyme Specificity and Enantioselectivity of Ketone Reduction

2009 ◽  
Vol 75 (7) ◽  
pp. 2176-2183 ◽  
Author(s):  
Rongzhen Zhang ◽  
Yan Xu ◽  
Ying Sun ◽  
Wenchi Zhang ◽  
Rong Xiao
Keyword(s):  
Candida Parapsilosis ◽  
Enzyme Stability ◽  
Optical Purity ◽  
Carbonyl Reductase ◽  
Site Directed Mutagenesis ◽  
Short Chain ◽  
Significant Shift ◽  
Wild Type ◽  
Ketone Reduction ◽  
Coenzyme Specificity

ABSTRACT A short-chain carbonyl reductase (SCR) from Candida parapsilosis catalyzes an anti-Prelog reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol (PED) and exhibits coenzyme specificity for NADPH over NADH. By using site-directed mutagenesis, the mutants were designed with different combinations of Ser67Asp, His68Asp, and Pro69Asp substitutions inside or adjacent to the coenzyme binding pocket. All mutations caused a significant shift of enantioselectivity toward the (R)-configuration during 2-hydroxyacetophenone reduction. The S67D/H68D mutant produced (R)-PED with high optical purity and yield in the NADH-linked reaction. By kinetic analysis, the S67D/H68D mutant resulted in a nearly 10-fold increase and a 20-fold decrease in the k cat/Km value when NADH and NADPH were used as the cofactors, respectively, but maintaining a k cat value essentially the same with respect to wild-type SCR. The ratio of Kd (dissociation constant) values between NADH and NADPH for the S67D/H68D mutant and SCR were 0.28 and 1.9 respectively, which indicates that the S67D/H68D mutant has a stronger preference for NADH and weaker binding for NADPH. Moreover, the S67D/H68D enzyme exhibited a secondary structure and melting temperature similar to the wild-type form. It was also found that NADH provided maximal protection against thermal and urea denaturation for S67D/H68D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the double point mutation S67D/H68D successfully converted the coenzyme specificity of SCR from NADP(H) to NAD(H) as well as the product enantioselectivity without disturbing enzyme stability. This work provides a protein engineering approach to modify the coenzyme specificity and enantioselectivity of ketone reduction for short-chain reductases.

scholarly journals ML-12 MECHANISMS OF CELL DEATH INHIBITION THROUGH CHANGE IN SUBCELLULAR LOCALIZATION OF PIM-1 BY PIM1 GENE MUTATION RECURRENTLY FOUND IN PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA

2019 ◽  
Vol 1 (Supplement_2) ◽  
pp. ii34-ii34
Author(s):  
Nobuyoshi Sasaki ◽  
Koichi Ichimura ◽  
Motoo Nagane ◽  
Arata Tomiyama
Keyword(s):  
Central Nervous System ◽  
Cell Death ◽  
Nervous System ◽  
Subcellular Localization ◽  
Human Cancer ◽  
Central Nervous System Lymphoma ◽  
B Cell Lymphoma ◽  
Site Directed Mutagenesis ◽  
Significant Shift ◽  
Wild Type

Abstract BACKGROUNDS In a study of Next Generation Sequencing in primary central nervous system lymphoma (PCNSL), we have previously reported several mutations of high frequency, in comparison with systemic diffuse large B cell lymphoma (DLBCL)s. Consequences of these specific mutations in PCNSL are unknown. In this study, we have analyzed the functional consequence of mutations in the PIM1 gene, observed in 100% of PCNSL patients, which encodes a serine/threonine kinase and is known to drive tumorigenesis in several malignancies. METHODS Four most frequent mutations of PIM1 in PCNSL, S77N, K115N, P216S, L275F, were chosen from our previous study, and each mutant was generated by site directed mutagenesis in PIM1 cDNA cloned in an expression vector. Resulting vectors were transiently transfected into human cancer cell lines. Functional studies were carried out using various biochemical methods. RESULTS Among the four mutants, increased phosphorylation of BCL-2 associated death promoter (BAD) at Ser112, which is a phosphorylation target of Pim-1, was observed by expression of K115N mutant compared with wild type PIM1 in Hela and Nagai cells expressing endogeneous BAD. Decreased cell death under campthothecin treatment was also observed in K115N mutant expressing Nagai cells compared with wild type PIM1-expressed cells. We also observed a significant shift in subcellular localization of Pim-1 carrying K115N mutant; from the nucleus, main sublocalization for wild type Pim-1, into the cytosol determined by immunocytochemistry and immunoblotting of nuclear and cytosolic fraction of the cells. Augmented cytosolic localization of Pim-1 carrying K115N mutant was suppressed by inhibition of glycosylation. DISCUSSION It is suggested that PIM1 K115N mutant may drive chemoresistance through increased BAD phosphorylation that suppresses cell death compared with wild-type PIM1 through modification of its subcellular localization, which might be regulated by its glycosylation status.

scholarly journals The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography

2004 ◽  
Vol 385 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Barbara PETSCHACHER ◽  
Stefan LEITGEB ◽  
Kathryn L. KAVANAGH ◽  
David K. WILSON ◽  
Bernd NIDETZKY
Keyword(s):  
Double Mutant ◽  
Profile Analysis ◽  
Xylose Reductase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binary Complex ◽  
Wild Type ◽  
Coenzyme Specificity ◽  
X Ray ◽  
X Ray Crystallography

CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2′-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme–NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274→Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R–N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 °C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 Å resolution.

scholarly journals Site-directed mutagenesis of the 1,3-β glucan synthase catalytic subunit ofPneumocystis jiroveciiand susceptibility assays suggest its sensitivity to caspofungin

2018 ◽  
Author(s):  
A. Luraschi ◽  
S. Richard ◽  
P. M. Hauser
Keyword(s):  
Candida Parapsilosis ◽  
Pneumocystis Carinii ◽  
Culture Method ◽  
Catalytic Subunit ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Resistant Species ◽  
Rats And Mice

AbstractThe echinocandin caspofungin inhibits the catalytic subunit Gsc1 of the enzymatic complex synthetizing 1,3-β glucan, an essential compound of the fungal wall. Studies in rodents showed that caspofungin can treatPneumocystisinfections. However, its efficacy againstPneumocystis jirovecii, the species infecting exclusively humans, remains controversial. The aim of this study was to assess the sensitivity to caspofungin of theP. jiroveciiGsc1 subunit, as well as of those ofPneumocystis cariniiandPneumocystis murinainfecting respectively rats and mice. In absence of an establishedin vitroculture method forPneumocystisspecies, we used functional complementation of theSaccharomyces cerevisiae gsc1deletant. In the fungal pathogenCandida albicans, mutations leading to amino acid substitutions in Gsc1 confer resistance to caspofungin. We introduced the corresponding mutations into thePneumocystis gsc1genes using site-directed mutagenesis. In spot dilution tests, the sensitivity to caspofungin of the complemented strains decreased with the number of mutations introduced, suggesting that the wild-type enzymes are sensitive. The minimum inhibitory concentrations of caspofungin determined by E-test®and Yeastone®for strains complemented withPneumocystisenzymes (respectively 0.125 and 0.12 microg/ml) were identical to those upon complementation with the enzyme ofC. albicansthat is sensitive to caspofungin. However, they were lower than the MICs upon complementation with the enzyme of the resistant speciesCandida parapsilosis(0.19 and 0.25). Sensitivity levels of Gsc1 enzymes of the threePneumocystisspecies were similar. Our results suggest thatP. jiroveciiis sensitive to caspofungin during infections, asP. cariniiandP. murina.

scholarly journals Mutation of tyrosine-194 and lysine-198 in the catalytic site of pig 3α/β,20β-hydroxysteroid dehydrogenase

1998 ◽  
Vol 334 (3) ◽  
pp. 553-557 ◽  
Author(s):  
Shizuo NAKAJIN ◽  
Noriko TAKASE ◽  
Shuji OHNO ◽  
Satoshi TOYOSHIMA ◽  
Michael E. BAKER
Keyword(s):  
Catalytic Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Carbonyl Reductase ◽  
Lysine Residue ◽  
Short Chain ◽  
Tyrosine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Aldehydes And Ketones ◽  
Reduction Of Ketones

Pig 3α/β,20β-hydroxysteroid dehydrogenase is an NADPH-dependent enzyme that catalyses the reduction of ketones on steroids and aldehydes and ketones on various xenobiotics, like its homologue carbonyl reductase. 3α/β,20β-Hydroxysteroid dehydrogenase and carbonyl reductase are members of the short-chain dehydrogenases/reductase family, in which a tyrosine residue and a lysine residue have been identified as catalytically important. In pig 20β-hydroxysteroid dehydrogenase these residues are tyrosine-194 and lysine-198. Here we report the effect on the reduction of two ketone and two aldehyde substrates by pig 3α/β,20β-hydroxysteroid dehydrogenase in which tyrosine-194 has been mutated to phenylalanine and cysteine, and lysine-198 has been mutated to isoleucine and arginine. Mutants with phenylalanine-194 or isoleucine-198 are inactive. Depending on the substrate, the mutant with cysteine-194 has a catalytic efficiency of 0.4–1% and the mutant with arginine-198 has a catalytic efficiency of 4–23% of the wild-type enzyme. We also mutated tyrosine-81 and tyrosine-253 to phenylalanine. Although both tyrosines are conserved in 3α/β,20β-hydroxysteroid dehydrogenase and carbonyl reductase, depending on the substrate, the mutant enzymes are as active as, or more active than, wild-type enzyme.

Expression and Characterization of Clustered Charge-to-Alanine Mutants of Low Mr Single-Chain Urokinase-Type Plasminogen Activator

1994 ◽  
Vol 71 (01) ◽  
pp. 134-140 ◽  
Author(s):  
S Ueshima ◽  
P Holvoet ◽  
H R Lijnen ◽  
L Nelles ◽  
V Seghers ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Solvent Accessibility ◽  
Site Directed Mutagenesis ◽  
Clot Lysis ◽  
Wild Type ◽  
Single Chain ◽  
Charged Amino Acids ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Pharmacokinetic Properties

SummaryIn an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M r single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala).The rscu-PA 32k moictic3 were expressed in High Five Ttichoplasiani cells, and purified to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/1. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion In lwo chain moieties by plasmin were comparable for mutant and wild-type rscu PA 32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 pi 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant lysis with 16 pg/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min). In hamsters with pulmonary embolism, continuous intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type rscu-PA-32k.These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed to eliminate charged regions with the highest solvent accessibility, do not have significantly improved functional, fibrinolytic or pharmacokinetic properties.

scholarly journals Suberoylanilide hydroxamic acid enhances the radiosensitivity of lung cancer cells through acetylated wild-type and mutant p53-dependent modulation of mitochondrial apoptosis

2021 ◽  
Vol 49 (2) ◽  
pp. 030006052098154
Author(s):  
Kan Wu ◽  
Xueqin Chen ◽  
Xufeng Chen ◽  
Shirong Zhang ◽  
Yasi Xu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Cells ◽  
Hydroxamic Acid ◽  
Site Directed Mutagenesis ◽  
Lung Cancer Cells ◽  
Mutant P53 ◽  
Suberoylanilide Hydroxamic Acid ◽  
Mitochondrial Apoptosis ◽  
Wild Type ◽  
Clonogenic Cell

Objective Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has shown potential as a candidate radiosensitizer for many types of cancers. This study aimed to explore the radiosensitization mechanism of SAHA in lung cancer cells. Methods Mutations in p53 were generated by site-directed mutagenesis using polymerase chain reaction. Transfection was performed to generate H1299 cells carrying wild-type or mutant p53. The radiosensitizing enhancement ratio was determined by clonogenic assays. Mitochondrial apoptosis was detected using JC-1 staining and flow cytometry analysis. Results Our results showed that SAHA induced radiosensitization in H1299 cells expressing wild-type p53, p53R175H or p53P223L, but this enhanced clonogenic cell death was not observed in parental H1299 (p53-null) cells or H1299 cells expressing p53 with K120R, A161T and V274R mutations. In SAHA-sensitized cells, mitochondrial apoptosis was induced following exposure to irradiation. Additionally, we observed that a secondary mutation at K120 (K120R) could eliminate p53-mediated radiosensitization and mitochondrial apoptosis. Conclusions The results of this study suggest that wild-type and specific mutant forms of p53 mediate SAHA-induced radiosensitization by regulating mitochondrial apoptosis, and the stabilization of K120 acetylation by SAHA is the molecular basis contributing to radiosensitization in lung cancer cells.

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 N-terminal fatty acylation of the α-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation

1994 ◽  
Vol 303 (3) ◽  
pp. 697-700 ◽  
Author(s):  
F Galbiati ◽  
F Guzzi ◽  
A I Magee ◽  
G Milligan ◽  
M Parenti
Keyword(s):  
Palmitic Acid ◽  
G Protein ◽  
Fatty Acyl ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Α Subunit ◽  
Metabolic Labelling ◽  
Fatty Acylation ◽  
Absolute Requirement ◽  
Cdna Construct

The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.

scholarly journals Mutations in the Escherichia coli UvrB ATPase motif compromise excision repair capacity

1989 ◽  
Vol 86 (17) ◽  
pp. 6577-6581 ◽  
Author(s):  
T W Seeley ◽  
L Grossman
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Excision Repair ◽  
Site Directed Mutagenesis ◽  
Sequence Motif ◽  
Amino Acid Side Chain ◽  
Uv Resistance ◽  
Wild Type ◽  
General Applicability ◽  
Repair Capacity

The Escherichia coli UvrB protein possesses an amino acid sequence motif common to many ATPases. The role of this motif in UvrB has been investigated by site-directed mutagenesis. Three UvrB mutants, with amino acid replacements at lysine-45, failed to confer UV resistance when tested in the UV-sensitive strain N364 (delta uvrB), while five other mutants constructed near this region of UvrB confer wild-type levels of UV resistance. Because even the conservative substitution of arginine for lysine-45 in UvrB results in failure to confer UV resistance, we believe we have identified an amino acid side chain in UvrB essential to nucleotide excision repair in E. coli. The properties of two purified mutant UvrB proteins, lysine-45 to alanine (K45A) and asparagine-51 to alanine (N51A), were analyzed in vitro. While the K45A mutant is fully defective in incision of UV-irradiated DNA, K45A is capable of interaction with UvrA in forming an ATP-dependent nucleoprotein complex. The K45A mutant, however, fails to activate the characteristic increase in ATPase activity observed with the wild-type UvrB in the presence of UvrA and DNA. From these results we conclude that there is a second nucleotide-dependent step in incision following initial complex formation, which is defective in the K45A mutant. This experimental approach may prove of general applicability in the study of function and mechanism of other ATPase motif proteins.

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