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Enhancing the Thermostability and Activity of Uronate Dehydrogenase from Agrobacterium Tumefaciens LBA4404 by Semi-Rational Engi
Su et al. Bioresour. Bioprocess. (2019) 6:36 https://doi.org/10.1186/s40643-019-0267-3 RESEARCH Open Access Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering Hui‑Hui Su1†, Fei Peng1†, Pei Xu1, Xiao‑Ling Wu1, Min‑Hua Zong1, Ji‑Guo Yang2 and Wen‑Yong Lou1* Abstract Background: Glucaric acid, one of the aldaric acids, has been declared a “top value‑added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efcient and economical than chemical synthesis. Uronate dehydro‑ genases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi‑rational design, is a major research goal. Results: In the present work, three UDHs were obtained from diferent Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tume- 1 1 faciens (At) LBA4404 had a better catalytic efciency than the other two UDHs (800 vs 600 and 530 s− mM− ). To fur‑ ther boost the catalytic performance of the UDH from AtLBA4404, site‑directed mutagenesis based on semi‑rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400‑fold improvement in half‑life at 59 °C, a 10 5 °C improvement in T50 value and a 2.5‑fold improvement in specifc activity at 30 °C compared to wild‑type UDH. -
Global Regulation of Differential Gene Expression by C-Abl/Arg Oncogenic
LAB/IN VITRO RESEARCH e-ISSN 1643-3750 © Med Sci Monit, 2017; 23: 2625-2635 DOI: 10.12659/MSM.904888 Received: 2017.04.16 Accepted: 2017.05.08 Global Regulation of Differential Gene Published: 2017.05.30 Expression by c-Abl/Arg Oncogenic Kinases Authors’ Contribution: ABCE 1 Qincai Dong* 1 Laboratory of Genetic Engineering, Beijing Institute of Biotechnology, Beijing, Study Design A BC 2 Chenggong Li* P.R. China Data Collection B 2 Institute of Health Sciences, Anhui University, Hefei, Anhui, P.R. China Statistical Analysis C BC 3 Xiuhua Qu 3 Center of Basic Medical Sciences, Navy General Hospital of PLA, Beijing, Data Interpretation D AFG 1 Cheng Cao P.R. China Manuscript Preparation E AEG 1 Xuan Liu Literature Search F Funds Collection G * These 2 authors contributed equally to this work Corresponding Authors: Xuan Liu, e-mail: [email protected], Cheng Cao, e-mail: [email protected] Source of support: This work was supported by the National Natural Science Foundation of China [31070674] Background: Studies have found that c-Abl oncogenic kinases may regulate gene transcription by RNA polymerase II phos- phorylation or by direct regulation of specific transcription factors or coactivators. However, the global regu- lation of differential gene expression by c-Abl/Arg is largely unknown. In this study, differentially expressed genes (DEGs) regulated by c-Abl/Arg were identified, and related cellular functions and associated pathways were investigated. Material/Methods: RNA obtained from wild-type and c-Abl/Arg gene-silenced MCF-7 cells was analyzed by RNA-Seq. DEGs were identified using edgeR software and partially validated by qRT-PCR. -
Functional and Physiological Discovery in the Mannonate Dehydratase Subgroup of the Enolase Superfamily
FUNCTIONAL AND PHYSIOLOGICAL DISCOVERY IN THE MANNONATE DEHYDRATASE SUBGROUP OF THE ENOLASE SUPERFAMILY BY DANIEL JOSEPH WICHELECKI DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2014 Urbana, Illinois Doctoral Committee: Professor John Gerlt, Chair Professor John Cronan Professor Scott Silverman Professor Wilfred van der Donk ABSTRACT In the current post-genomic world, the exponential amassing of protein sequences is overwhelming the scientific community’s ability to experimentally assign each protein’s function. The use of automated, homology-based annotations has allowed a reprieve from this efflux of data, but has led to widespread misannotation and nonannotation in protein sequence databases. This dissertation details the functional and physiological characterization of the mannonate dehydratase subgroup (ManD) of the enolase superfamily (ENS). The outcome affirms the dangers of homology-based annotations while discovering novel metabolic pathways. Furthermore, the experimental verification of these pathways ( in vitro and in vivo ) has provided a platform to test the general strategies for improved functional and metabolic characterization being developed by the Enzyme Function Initiative (EFI). Prior to this study, one member of the ManD subgroup had been characterized and was shown to dehydrate D-mannonate to 2-keto-3-deoxy-D-gluconate. Forty-two additional members of the ManD, selected from across the sequence space of the subgroup, were screened for activity and kinetic constants were determined. The members of the once isofunctional subgroup were found to differ in both catalytic efficiency and substrate specificity: 1) high 3 4 -1 -1 efficiency (k cat /K M = 10 to 10 M s ) dehydration of D-mannonate, 2) low efficiency (k cat /K M = 10 1 to 10 2 M-1s-1) dehydration of D-mannonate and/or D-gluconate, and 3) no-activity with either D-mannonate or D-gluconate (or any other acid sugar tested). -
The Regulation of Human Carbonyl Reductase 3 (CBR3) in Epithelial Cell Lines
Charles university in Prague Faculty of pharmacy in Hradec Kralov´ e´ Department of biochemical sciences Diploma thesis The regulation of human carbonyl reductase 3 (CBR3) in epithelial cell lines Regulace lidske´ karbonylreduktasy 3 v buneˇcnˇ ych´ lini´ıch epithelu Petra Malatkov´ a´ 2009 Supervisors: Prof. Dr. Edmund Maser Dr. Bettina Ebert Christian Albrechts University in Kiel Faculty of Medicine Department of Toxicology and Pharmacology for Natural Scientists Prof. Ing. Vladim´ır Wsol,´ Ph.D. Charles University in Prague Faculty of Pharmacy in Hradec Kralov´ e´ Department of Biochemical Sciences Declaration I declare that this thesis is my original author’s work which I have worked out independently. All literature and other sources, from which I gather during elaboration, are listed in the bibli- ography and properly cited in the work. 4th May 2009 2 Acknowledgements First, I would like to express sincere thanks to Dr. Bettina Ebert for teaching me new techniques and performing cell culture experiments. I thank you for lending your time and providing de- tailed comments. Also, I would like to thank to Prof. Dr. Edmund Maser and Prof. Ing. Vladim´ır Wsol,´ Ph.D. for offering useful advice and, in particular, giving me chance to do the diploma thesis at Department of Toxicology and Pharmacology in Kiel. Next, I would like to thank to Yasser El-Hawari, who helps me with LaTex and contributes to improving my German skills. I would also like to thank to Michael Kisiela for his helpful assistance with any occurred problems. Finally, I would like to extend a big thank to my parents for their love and support. -
Directed Mutagenesis Sites of Agrobacterium Tumefaciens Uronate Dehydrogenase
Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 22, No. (1) : 2020 : 140-148 © Global Science Publications ISSN-0972-3005 EVOLUTION-GUIDED OPTIMIZATION FOR SELECTING SITE- DIRECTED MUTAGENESIS SITES OF AGROBACTERIUM TUMEFACIENS URONATE DEHYDROGENASE MURUGAN R., PRATHIVIRAJ R., DIPTI MOTHAY AND CHELLAPANDI P.* Molecular Systems Engineering Lab, Department of Bioinformatics, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India (Received 19 October, 2019; accepted 26 November, 2019) Key words : Uronate dehydrogenase, Agrobacterium tumefaciens, Site-directed mutagenesis, Coevolution, Mutant stability, Substrate specificity, Molecular evolution Abstract – Uronate dehydrogenase (EC:1.1.1.203) belongs to the NAD-dependent epimerase/dehydratase (NDE/D) subfamily, which converts D-galacturonic acid and D-glucuronic acid into D-galactaric acid and D-glucaric acid, respectively. Uronate dehydrogenase-catalyzed reaction is reversible and no substrate- specific activity in nature. Therefore, evolution-guided optimization approach was employed for screening, selection, and evaluation of its mutants to increase the substrate specificity with NAD+ and D-glucuronic acid. The phylogenetic analysis described that uronate dehydrogenase from A. tumefaciens evolved from the UDP-glucose-4-epimerase subfamily members and not related to closely related soil bacteria. Molecular conservation of its sequence-structure-function integrity was retained in this organism by imposing purifying selection and amino acid substitution patterns. A single amino acid substitution in its proton relay system or substrate-binding site found to bring several changes in the local structural environments. It hasenforced to optimize the substrate-binding site that recognizes the D-galacturonic acid or D-glucuronic acid. Hence, site-directed mutagenesis targets detectedinthis study would be useful for engineering uronate dehydrogenase subjected to be used in the biotransformation process of D-glucaric acid production. -
V13a192-Bando Pgmkr
Molecular Vision 2007; 13:1722-9 <http://www.molvis.org/molvis/v13/a192/> ©2007 Molecular Vision Received 14 February 2007 | Accepted 12 September 2007 | Published 18 September 2007 NADH photo-oxidation is enhanced by a partially purified λ-crystallin fraction from rabbit lens Masayasu Bando,1 Mikako Oka,2 Kenji Kawai,1 Hajime Obazawa,3 Makoto Takehana2 1Department of Ophthalmology, Tokai University School of Medicine, Isehara, Japan; 2Department of Molecular Function and Physiology, Kyoritsu University of Pharmacy, Tokyo, Japan; 3Eye Research Institute of Cataract Foundation, Tokyo, Japan Purpose: In the rabbit lens, high levels of reduced nicotinamide adenine dinucleotide (NADH) can function as a near- ultraviolet light (near-UV) filter, an effect apparently achieved by specific nucleotide binding to λ-crystallin. The present investigation asks whether λ-crystallin enhances NADH photo-oxidation by superoxide radicals produced via a photosen- sitization reaction of near-UV with NADH. Methods: λ-Crystallin was partially purified from rabbit lens soluble fraction by a two-step gel filtration and affinity column chromatography procedure. NADH solutions with or without partially purified λ-crystallin were subjected to near-UV irradiation or exposed to superoxide generated enzymatically by the xanthine/xanthine oxidase system. NADH oxidation was determined by assaying the decrease of absorbance at 340 nm. Results: When irradiated with near-UV, free NADH was oxidized very little in the absence of λ-crystallin. In contrast, NADH photo-oxidation was rapidly initiated in the presence of partially purified λ-crystallin. This λ-crystallin-enhanced NADH photo-oxidation was totally inhibited by adding superoxide dismutase. We also found that λ-crystallin largely increased NADH oxidation by a superoxide that is generated enzymatically. -
All Enzymes in BRENDA™ the Comprehensive Enzyme Information System
All enzymes in BRENDA™ The Comprehensive Enzyme Information System http://www.brenda-enzymes.org/index.php4?page=information/all_enzymes.php4 1.1.1.1 alcohol dehydrogenase 1.1.1.B1 D-arabitol-phosphate dehydrogenase 1.1.1.2 alcohol dehydrogenase (NADP+) 1.1.1.B3 (S)-specific secondary alcohol dehydrogenase 1.1.1.3 homoserine dehydrogenase 1.1.1.B4 (R)-specific secondary alcohol dehydrogenase 1.1.1.4 (R,R)-butanediol dehydrogenase 1.1.1.5 acetoin dehydrogenase 1.1.1.B5 NADP-retinol dehydrogenase 1.1.1.6 glycerol dehydrogenase 1.1.1.7 propanediol-phosphate dehydrogenase 1.1.1.8 glycerol-3-phosphate dehydrogenase (NAD+) 1.1.1.9 D-xylulose reductase 1.1.1.10 L-xylulose reductase 1.1.1.11 D-arabinitol 4-dehydrogenase 1.1.1.12 L-arabinitol 4-dehydrogenase 1.1.1.13 L-arabinitol 2-dehydrogenase 1.1.1.14 L-iditol 2-dehydrogenase 1.1.1.15 D-iditol 2-dehydrogenase 1.1.1.16 galactitol 2-dehydrogenase 1.1.1.17 mannitol-1-phosphate 5-dehydrogenase 1.1.1.18 inositol 2-dehydrogenase 1.1.1.19 glucuronate reductase 1.1.1.20 glucuronolactone reductase 1.1.1.21 aldehyde reductase 1.1.1.22 UDP-glucose 6-dehydrogenase 1.1.1.23 histidinol dehydrogenase 1.1.1.24 quinate dehydrogenase 1.1.1.25 shikimate dehydrogenase 1.1.1.26 glyoxylate reductase 1.1.1.27 L-lactate dehydrogenase 1.1.1.28 D-lactate dehydrogenase 1.1.1.29 glycerate dehydrogenase 1.1.1.30 3-hydroxybutyrate dehydrogenase 1.1.1.31 3-hydroxyisobutyrate dehydrogenase 1.1.1.32 mevaldate reductase 1.1.1.33 mevaldate reductase (NADPH) 1.1.1.34 hydroxymethylglutaryl-CoA reductase (NADPH) 1.1.1.35 3-hydroxyacyl-CoA -
Cloning and Characterization of Uronate Dehydrogenases from Two
JOURNAL OF BACTERIOLOGY, Mar. 2009, p. 1565–1573 Vol. 191, No. 5 0021-9193/09/$08.00ϩ0 doi:10.1128/JB.00586-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved. Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58ᰔ‡ Sang-Hwal Yoon, Tae Seok Moon, Pooya Iranpour,† Amanda M. Lanza, and Kristala Jones Prather* Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139 Received 28 April 2008/Accepted 30 November 2008 Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel comple- mentation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (kcat) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (Km) for galacturonate was lower than that for glucuronate. -
Synthetic Scaffold Systems for Increasing the Efficiency Of
biology Review Synthetic Scaffold Systems for Increasing the Efficiency of Metabolic Pathways in Microorganisms Almando Geraldi 1,2,* , Fatiha Khairunnisa 2,3, Nadya Farah 4, Le Minh Bui 5 and Ziaur Rahman 6 1 Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya 60115, Indonesia 2 Research Center for Bio-Molecule Engineering, Universitas Airlangga, Surabaya 60115, Indonesia; [email protected] 3 Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Surabaya 60115, Indonesia 4 Department of Biology, Faculty of Mathematics and Life Sciences, Indonesia Defense University, Bogor 16810, Indonesia; [email protected] 5 NTT Hi-Tech Institute, Nguyen Tat Thanh University (NTTU), Ho Chi Minh City 700000, Vietnam; [email protected] 6 Department of Microbiology, Abdul Wali Khan University Mardan, Mardan, Khyber Pakhtunkhwa 23200, Pakistan; [email protected] * Correspondence: [email protected]; Tel.: +62-31-5936501 Simple Summary: Biotechnology involves the use of living organisms to create high-value products. Bacteria and yeast, in particular, are widely applied for such processes, but they may not naturally produce certain products, such as amino acids, organic acids, and alcohols in large amounts, if at all. Hence, the field of metabolic engineering has emerged for “tweaking” the biosynthetic pathways of these cells to encourage the high production of desired products. However, the complexity of the many metabolic pathways in natural cells makes it difficult to ensure that only the molecular components and pathways related to the desired product are enhanced. Very often, competing Citation: Geraldi, A.; Khairunnisa, F.; metabolic pathways and toxic intermediates will lower the production efficiency. -
Uncorrected Proof
Process Biochemistry xxx (xxxx) xxx-xxx Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com Chromohalobacter salixigens uronate dehydrogenase: Directed evolution for improved thermal stability and mutant CsUDH-inc X-ray crystal structure Kurt Wagschal a , ⁎, Victor J. Chan a , Jose H. Pereira b , Peter H. Zwart c , Banumathi Sankaran d , ⁎ a USDA Agricultural Research Service, Western Regional Research Center, Albany, CA 94710, USA b Molecular Biophysics and Integrated Bioimaging, Joint BioEnergy Institute, Emeryville, CA, 94608, USA c Molecular Biophysics and Integrated Bioimaging & Center for Advanced Mathematics for Energy Research Applications, Lawrence Berkeley National Laboratories,1 Cyclotron Road, Berkeley, CA, 94703, USA d Molecular Biophysics and Integrated Bioimaging, Berkeley Center for Structural Biology, Lawrence Berkeley National Laboratory, Berkeley,PROOFCalifornia, 94720, USA ARTICLE INFO ABSTRACT Keywords: Chromohalobacter salixigens contains a uronate dehydrogenase termed CsUDH that can convert uronic acids to Uronate dehydrogenase their corresponding C1,C6-dicarboxy aldaric acids, an important enzyme reaction applicable for biotechnological Directed evolution use of sugar acids. To increase the thermal stability of this enzyme for biotechnological processes, directed evolu- Pectin tion using gene family shuffling was applied, and the hits selected from 2-tier screening of a shuffled gene family Alginate library contained in total 16 mutations, only some of which when examined individually appreciably increased Gene shuffling thermal stability. Most mutations, while having minimal or no effect on thermal stability when tested in isola- Protein thermal stability 0.5 tion, were found to exhibit synergy when combined; CsUDH-inc containing all 16 mutations had ΔK t +18 °C, such that kcat was unaffected by incubation for 1 h at ∼70 °C. -
Bioassay Technology
BIOASSAY TECHNOLOGY - CHINA SR#: CAT#: PRODUCT NAME SIZE 1 E0000Hu Human transthyretin,TTR ELISA Kit 96 Tests 2 E0001Hu Human True insulin,TI ELISA Kit 96 Tests 3 E0002Hu Human Islet amyloid polypeptide,IAPP ELISA Kit 96 Tests 4 E0003Hu Human advanced glycation end products,AGEs ELISA Kit 96 Tests 5 E0004Hu Human Pentosidine ELISA Kit 96 Tests 6 E0005Hu Human ReverseTri-iodothyronine,rT3 ELISA Kit 96 Tests 7 E0006Hu Human C-Polypeptide,CP ELISA Kit 96 Tests 8 E0007Hu Human beta cellulin,BTC ELISA Kit 96 Tests 9 E0008Hu Human Thyroglobulin,TG ELISA Kit 96 Tests 10 E0009Hu Human C-Peptide ELISA Kit 96 Tests 11 E0010Hu Human Insulin,INS ELISA Kit 96 Tests 12 E0011Hu Human Neonatal Thyroxine,NN-T4 ELISA Kit 96 Tests 13 E0012Hu Human Free Tri-iodothyronine Indes,Free-T3 ELISA Kit 96 Tests 14 E0013Hu Human Free Thyroxine,FT4 ELISA Kit 96 Tests 15 E0014Hu Human trypsinogen activation peptide,TAP ELISA Kit 96 Tests 16 E0015Hu Human Ultrasensitivity Tri-iodothyronine,u-T3 ELISA Kit 96 Tests 17 E0016Hu Human Amylin ELISA Kit 96 Tests Human Glucose dependent insulin releasing polypeptide,GIP 18 E0017Hu 96 Tests ELISA Kit 19 E0018Hu Human Proinsulin,PI ELISA Kit 96 Tests 20 E0019Hu Human beta,beta-carotene 9',10'-oxygenase,BCO2 ELISA Kit 96 Tests 21 E0020Hu Human Tri-iodothyronine,T3 ELISA Kit 96 Tests 22 E0021Hu Human thyroxine,T4 ELISA Kit 96 Tests 23 E0022Hu Human Glucagon-like peptide 1,GLP-1 ELISA Kit 96 Tests 24 E0023Hu Human Glycated hemoglobin A1c,GHbA1c ELISA Kit 96 Tests 25 E0024Hu Human Insulin Receptor β,ISR-β ELISA KIT 96 Tests 26 E0025Hu -
Thermostabilization of the Uronate Dehydrogenase From
Roth et al. AMB Expr (2017) 7:103 DOI 10.1186/s13568-017-0405-2 ORIGINAL ARTICLE Open Access Thermostabilization of the uronate dehydrogenase from Agrobacterium tumefaciens by semi‑rational design Teresa Roth1,2, Barbara Beer1, André Pick1 and Volker Sieber1,3,4* Abstract Aldaric acids represent biobased ‘top value-added chemicals’ that have the potential to substitute petroleum-derived chemicals. Until today they are mostly produced from corresponding aldoses using strong chemical oxidizing agents. An environmentally friendly and more selective process could be achieved by using natural resources such as sea- weed or pectin as raw material. These contain large amounts of uronic acids as major constituents such as glucuronic acid and galacturonic acid which can be converted into the corresponding aldaric acids via an enzyme-based oxida- tion using uronate dehydrogenase (Udh). The Udh from Agrobacterium tumefaciens (UdhAt) features the highest cata- 1 1 lytic efciency of all characterized Udhs using glucuronic acid as substrate (829 s− mM− ). Unfortunately, it sufers from poor thermostability. To overcome this limitation, we created more thermostable variants using semi-rational design. The amino acids for substitution were chosen according to the B factor in combination with four additional knowledge-based criteria. The triple variant A41P/H101Y/H236K showed higher kinetic and thermodynamic stability 15 with a T50 value of 62.2 °C (3.2 °C improvement) and a ∆∆GU of 2.3 kJ/mol compared to wild type. Interestingly, it was only obtained when including a neutral mutation in the combination. Keywords: Uronate dehydrogenase, Glucuronic acid, Agrobacterium tumefaciens, Thermostability, B factor, Neutral drift Introduction not selective.