scholarly journals 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

2006 ◽  
Vol 397 (1) ◽  
pp. 195-201 ◽  
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.

scholarly journals Identification of IRF8 As a Potent Tumor Suppressor in Murine Acute Promyelocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1518-1518
Author(s):  
Sangeetha Surianarayanan ◽  
Coline M Gaillard ◽  
Trevor Bentley ◽  
Matthew R. Warr ◽  
Briana Fitch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fusion Protein ◽  
Expression Pattern ◽  
Genetic Alterations ◽  
Chromosome 8 ◽  
Single Mutation ◽  
Wild Type ◽  
Leukemic Transformation ◽  
Protein Levels ◽  
The Impact

Abstract The classical paradigm suggests that PML/RARA fusion protein is the main driver of pathogenesis in APL. It is believed that the fusion oncogenic protein mediates this effect by potentially repressing key myeloid maturation genes involved in differentiation processes. However, the underlying mechanism is not completely understood. We recently challenged this model re-opening fundamental questions as to understand the precise contribution of the fusion protein to leukemic transformation. This knowledge on the mechanistic pathways can lead to better tailored combinatorial therapeutics. To understand the role of the PML/RARA fusion protein in leukemogenesis, we initially did a transcriptome analysis in our murine MRP8-PML/RARA APL model. Interestingly, we observed only moderate alterations in gene expression pattern of the key myeloid genes that we thought to be actively involved in differentiation processes. Of particular note, we found significant downregulation of the Irf8 in our promyelocyte compartments. IRF8 is a known regulator of hematopoiesis. The IRF8 myeloid transcription factor (TF) is expressed in several lineages of the hematopoietic tree and plays an important role in orchestrating specification and differentiation of B cells, dendritic cells and monocytes. Herein, we speculate lower levels of IRF8 could potentially impact tumorigenesis in the context of PML/RARA. In order to address this question, technically, we employed stringent staining and sorting strategy to distinctly differentiate early and late promyelocytes and looked at the expression pattern of Irf8 gene both at the transcript and protein levels. Results from qRT-PCR demonstrated 4.8 fold decrease in Irf8 expression compared to wildtype controls both in preleukemic promyelocytes and fully differentiated leukemic cells suggesting PML/RARA could be a target of IRF8 and this association could potentially be involved in the emergence and maintenance of leukemia. We next asked whether these changes are reflective at the protein levels and performed a Western blot analysis in our highly purified promyelocyte population and found a dramatic decrease in IRF8 levels in comparison to wild type controls again suggesting a possible protein-protein interaction under normal conditions that may provide an advantage for the cells from turning oncogenic. In order to study how low levels of IRF8 impact promyelocyte expansion, we generated double knock-outs of mice harboring both PML/RARA Irf8-/- mutations and compared their phenotype with mice harboring single mutations in either PML/RARA or Irf8 gene. As previously observed, young PML/RARA mice had a substantially increased number of marrow promyelocytes in comparison to wild-type mice. Fascinatingly, loss of Irf8 alone resulted in an essentially identical expansion of promyelocytes (as well as a loss of earlier myeloid progenitors in the bone marrow, not seen in PML/RARA mice) and a combination of PML/RARA expression and IRF8 loss did not result in a statistically significant further expansion of promyelocytes. These results suggest an epistatic relationship between PML/RARA and IRF8, compatible with downregulation of IRF8 by PML/RARA as being a key mechanism by which t(15;17) expands promyelocytes in the initiation of APL. Furthermore, in order to assess the impact of single/double genetic alterations on the overall and leukemia free survival we transplanted lethally irradiated mice with bone marrow cells derived from PML/RARA, Irf8-/- and PML/RARA Irf8-/-double knock outs and followed these mice over a period of one year. We observed there is no difference in their overall survival rate among the different groups of mice. However, looking specifically at the acute leukemic deaths, we observed a reduced latency in our PML/RARA Irf8-/- cohorts compared to mice carrying single mutation at PML/RARA loci. We also noticed that all the acute leukemias in the PML/RARA Irf8-/- cohort occurred prior to the first appearance of acute leukemia in the PML/RARA cohorts. Altogether, these data support a model of APL leukemogenesis in which the translocation of chromosomes 15 and 17 initiates leukemia development, in part by downregulating IRF8, and in which the resulting expansion of the promyelocyte compartment contributes to acquisition of additional cooperating events (e.g. trisomy of chromosome 8, mutation of FLT3) that complete leukemic transformation. Disclosures No relevant conflicts of interest to declare.

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

2007 ◽  
Vol 73 (22) ◽  
pp. 7291-7299 ◽  
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.

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

Archaea ◽  
2009 ◽  
Vol 2 (4) ◽  
pp. 233-239 ◽  
Author(s):  
Suzanne Wolterink-van Loo ◽  
Marco A. J. Siemerink ◽  
Georgios Perrakis ◽  
Thijs Kaper ◽  
Servé W. M. Kengen ◽  
...  
Keyword(s):  
Specific Activity ◽  
Aldol Condensation ◽  
Van Der Waals Interactions ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Optimal Activity ◽  
Threefold Increase ◽  
Aldol Condensation Reactions ◽  
Synthesis Reactions ◽  
Increased Activity

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.

scholarly journals Protein Engineering of Epoxide Hydrolase from Agrobacterium radiobacter AD1 for Enhanced Activity and Enantioselective Production of (R)-1-Phenylethane-1,2-Diol

2005 ◽  
Vol 71 (7) ◽  
pp. 3995-4003 ◽  
Author(s):  
Lingyun Rui ◽  
Li Cao ◽  
Wilfred Chen ◽  
Kenneth F. Reardon ◽  
Thomas K. Wood
Keyword(s):  
Active Site ◽  
Epoxide Hydrolase ◽  
Styrene Oxide ◽  
Saturation Mutagenesis ◽  
Dna Shuffling ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Agrobacterium Radiobacter ◽  
Enantiomeric Ratio ◽  
Enhanced Activity

ABSTRACT DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of Agrobacterium radiobacter AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (R)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (R)-1-phenylethane-1,2-diol (1.96 ± 0.09 versus 1.04 ± 0.07 μmol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (R)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (R) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 ± 2 versus 2.4 ± 0.3 μmol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 ± 0.5 versus 2.6 ± 0.0 μmol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 ± 0.3 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 ± 0.8 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.

scholarly journals New Efficient Recombinant Expression System To Engineer Candida antarctica Lipase B

2010 ◽  
Vol 76 (8) ◽  
pp. 2684-2687 ◽  
Author(s):  
St�phane Emond ◽  
C�dric Montanier ◽  
Jean-Marc Nicaud ◽  
Alain Marty ◽  
Pierre Monsan ◽  
...  
Keyword(s):  
Recombinant Expression ◽  
Expression System ◽  
Candida Antarctica ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Lipase B ◽  
Expression Host ◽  
Screening Protocol ◽  
Recombinant Expression System

ABSTRACT Here, we report the use of Yarrowia lipolytica as a versatile expression host for developing protein engineering approaches to modify the properties of Candida antarctica lipase B. A reliable screening protocol was defined and validated using a saturation mutagenesis library, yielding mutants displaying higher catalytic efficiencies than the wild-type enzyme.

 L-allo-Threonine aldolase with an H128Y/S292R mutation fromAeromonas jandaeiDK-39 reveals the structural basis of changes in substrate stereoselectivity

2014 ◽  
Vol 70 (6) ◽  
pp. 1695-1703 ◽  
Author(s):  
Hui-Min Qin ◽  
Fabiana Lica Imai ◽  
Takuya Miyakawa ◽  
Michihiko Kataoka ◽  
Nahoko Kitamura ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Active Site ◽  
Fold Increase ◽  
Saturation Mutagenesis ◽  
Structural Basis ◽  
Tyrosine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Threonine Aldolase

L-allo-Threonine aldolase (LATA), a pyridoxal-5′-phosphate-dependent enzyme fromAeromonas jandaeiDK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50 Å resolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123–Pro131, where His128 moved 4.2 Å outwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonineversusL-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.

scholarly journals Probing the Substrate Specificity of Aminopyrrolnitrin Oxygenase (PrnD) by Mutational Analysis

2006 ◽  
Vol 188 (17) ◽  
pp. 6179-6183 ◽  
Author(s):  
Jung-Kul Lee ◽  
Ee-Lui Ang ◽  
Huimin Zhao
Keyword(s):  
Substrate Specificity ◽  
Mutational Analysis ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Enzyme Substrate ◽  
Molecular Determinants

ABSTRACT Molecular modeling and mutational analysis (site-directed mutagenesis and saturation mutagenesis) were used to probe the molecular determinants of the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) from Pseudomonas fluorescens Pf-5. There are 17 putative substrate-contacting residues, and mutations at two of the positions, positions 312 and 277, could modulate the enzyme substrate specificity separately or in combination. Interestingly, several of the mutants obtained exhibited higher catalytic efficiency (approximately two- to sevenfold higher) with the physiological substrate aminopyrrolnitrin than the wild-type enzyme exhibited.

scholarly journals Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone

2004 ◽  
Vol 186 (14) ◽  
pp. 4705-4713 ◽  
Author(s):  
Ying Tao ◽  
Ayelet Fishman ◽  
William E. Bentley ◽  
Thomas K. Wood
Keyword(s):  
Burkholderia Cepacia ◽  
Saturation Mutagenesis ◽  
Hydroxyl Groups ◽  
Pseudomonas Mendocina ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Protein Variant ◽  
Catalytic Center ◽  
Hydrogen Bonding Interactions ◽  
Formed Structure

ABSTRACT Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent V max values of 6.6 ± 0.9 to 10.7 ± 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 ± 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184 :344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41 :3176-3188, 2002). By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 ± 0.2 versus 0.21 ± 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol. Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.

Sign in / Sign up

Export Citation Format

Share Document