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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Research Article

Heterologous Expression and Characterization of Flavinadenine Dinucleotide Synthetase from Candida famata for Flavin Adenine Dinucleotide Production

Author(s): Guoqiang Zhou, Qiaoqiao Pan, Zeyu Hu, Juanping Qiu and Zhiliang Yu*

Volume 28, Issue 2, 2021

Published on: 08 July, 2020

Page: [229 - 239] Pages: 11

DOI: 10.2174/0929866527666200708151327

Price: $65

Abstract

Background: Flavin adenine dinucleotide (FAD) is a redox-active coenzyme that regulates several important enzymatic reactions during metabolism. FAD is used in the medicinal and food industries and FAD supplements have been used to treat some inheritable diseases. FAD can be biosynthesized from flavin mononucleotide (FMN) and adenosine triphosphate (ATP), catalyzed by FAD synthetase (FADS).

Objective: The aim of this study was to heterologously express the gene encoding FADS from the flavinogenic yeast Candida famata (FADSCf) for biosynthesis of FAD.

Methods: The sequence encoding FADSCf was retrieved and heterologously expressed in Escherichia coli. The structure and enzymatic properties of recombinant FADSCf were characterized.

Results: FADSCf (279 amino acids) was successfully expressed in E. coli BL21 (DE3), with a theoretical molecular weight of 32299.79 Da and an isoelectric point of 6.09. Secondary structural analysis showed that the number of α-helices was 2-fold higher than the number of β-sheets, indicating that the protein was highly hydrophilic. Under fixed ATP concentration, FADSCf had a Km of 0.04737±0.03158 mM and a Vmax of 3.271±0.79 μM/min/mg. Under fixed FMN concentration, FADSCf had a Km of 0.1214±0.07464 mM and a Vmax of 2.6695±0.3715 μM/min/mg. Enzymatic reactions in vitro showed that expressed FADSCf could form 80 mM of FAD per mg of enzyme after 21 hours under the following conditions: 0.5 mM FMN, 5 mM ATP and 10 mM Mg2+.

Conclusion: Under optimized conditions (0.5 mM FMN, 5 mM ATP and 10 mM Mg2+), the production of FAD reached 80 mM per mg of FADSCf after a 21-hour reaction. Our results indicate that purified recombinant FADSCf can be used for the biosynthesis of FAD.

Keywords: Candida famata, FAD biosynthesis, flavin adenine dinucleotide synthetase, heterologous expression, sequence and structure analysis, E. coli.

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[1]
Huang, Y.F.; Liu, S.Y.; Yen, C.L.; Yang, P.W.; Shieh, C.C. Thapsigargin and flavin adenine dinucleotide ex vivo treatment rescues trafficking-defective gp91phox in chronic granulomatous disease leukocytes. Free Radic. Biol. Med., 2009, 47(7), 932-940.
[http://dx.doi.org/10.1016/j.freeradbiomed.2009.06.037] [PMID: 19631269]
[2]
Sebastián, M.; Anoz-Carbonell, E.; Gracia, B.; Cossio, P.; Aínsa, J.A.; Lans, I.; Medina, M. Discovery of antimicrobial compounds targeting bacterial type FAD synthetases. J. Enzyme Inhib. Med. Chem., 2018, 33(1), 241-254.
[http://dx.doi.org/10.1080/14756366.2017.1411910] [PMID: 29258359]
[3]
Vandamme, E.J. Production of vitamins, coenzymes and related biochemicals by biotechnological processes. J. Chem. Technol. Biotechnol., 1992, 53(4), 313-327.
[http://dx.doi.org/10.1002/jctb.280530402] [PMID: 1368195]
[4]
Yruela, I.; Arilla-Luna, S.; Medina, M.; Contreras-Moreira, B. Evolutionary divergence of chloroplast FAD synthetase proteins. BMC Evol. Biol., 2010, 10(18), 311-323.
[http://dx.doi.org/10.1186/1471-2148-10-311] [PMID: 20955574]
[5]
Barile, M.; Brizio, C.; Valenti, D.; De Virgilio, C.; Passarella, S. The riboflavin/FAD cycle in rat liver mitochondria. Eur. J. Biochem., 2000, 267(15), 4888-4900.
[http://dx.doi.org/10.1046/j.1432-1327.2000.01552.x] [PMID: 10903524]
[6]
Giancaspero, T.A.; Locato, V.; de Pinto, M.C.D.; De Gara, L.; Barile, M. The occurrence of riboflavin kinase and FAD synthetase ensures FAD synthesis in tobacco mitochondria and maintenance of cellular redox status. FEBS J., 2009, 276(1), 219-231.
[http://dx.doi.org/10.1111/j.1742-4658.2008.06775.x] [PMID: 19049514]
[7]
Herguedas, B.; Lans, I.; Sebastián, M.; Hermoso, J.A.; Martínez-Júlvez, M.; Medina, M. Structural insights into the synthesis of FMN in prokaryotic organisms. Acta Crystallogr. D Biol. Crystallogr., 2015, 71(Pt 12), 2526-2542.
[http://dx.doi.org/10.1107/S1399004715019641] [PMID: 26627660]
[8]
Mashhadi, Z.; Xu, H.; Grochowski, L.L.; White, R.H. Archaeal RibL: a new FAD synthetase that is air sensitive. Biochemistry, 2010, 49(40), 8748-8755.
[http://dx.doi.org/10.1021/bi100817q] [PMID: 20822113]
[9]
Leulliot, N.; Blondeau, K.; Keller, J.; Ulryck, N.; Quevillon-Cheruel, S.; van Tilbeurgh, H. Crystal structure of yeast FAD synthetase (Fad1) in complex with FAD. J. Mol. Biol., 2010, 398(5), 641-646.
[http://dx.doi.org/10.1016/j.jmb.2010.03.040] [PMID: 20359485]
[10]
de Montigny, J.; Spehner, C.; Souciet, J.; Tekaia, F.; Dujon, B.; Wincker, P.; Artiguenave, F.; Potier, S. Genomic exploration of the hemiascomycetous yeasts: 15. Pichia sorbitophila. FEBS Lett., 2000, 487(1), 87-90.
[http://dx.doi.org/10.1016/S0014-5793(00)02286-9] [PMID: 11152890]
[11]
Yatsyshyn, V.Y.; Fedorovych, D.V.; Sibirny, A.A. Metabolic and bioprocess engineering of the yeast Candida famata for FAD production. J. Ind. Microbiol. Biotechnol., 2014, 41(5), 823-835.
[http://dx.doi.org/10.1007/s10295-014-1422-7] [PMID: 24595668]
[12]
Abbas, C.A.; Sibirny, A.A. Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers. Microbiol. Mol. Biol. Rev., 2011, 75(2), 321-360.
[http://dx.doi.org/10.1128/MMBR.00030-10] [PMID: 21646432]
[13]
de Carvalho, C.C. Enzymatic and whole cell catalysis: finding new strategies for old processes. Biotechnol. Adv., 2011, 29(1), 75-83.
[http://dx.doi.org/10.1016/j.biotechadv.2010.09.001] [PMID: 20837129]
[14]
Yu, Z.; Ding, Y.; Yin, J.; Yu, D.; Zhang, J.; Zhang, M.; Ding, M.; Zhong, W.; Qiu, J.; Li, J. Dissemination of genetic acquisition/loss provides a variety of quorum sensing regulatory properties in Pseudoalteromonas. Int. J. Mol. Sci., 2018, 19(11), 3636-3654.
[http://dx.doi.org/10.3390/ijms19113636] [PMID: 30453700]
[15]
Chang, K.; Wang, Q.; Shi, X.; Wang, S.; Wu, H.; Nie, L.; Li, H. Stepwise partially overlapping primer-based PCR for genome walking. AMB Express, 2018, 8(1), 77-83.
[http://dx.doi.org/10.1186/s13568-018-0610-7] [PMID: 29744607]
[16]
See-Too, W.S.; Convey, P.; Pearce, D.A.; Chan, K.G. Characterization of a novel N-acylhomoserine lactonase, AidP, from Antarctic Planococcus sp. Microb. Cell Fact., 2018, 17(1), 179-192.
[http://dx.doi.org/10.1186/s12934-018-1024-6] [PMID: 30445965]
[17]
Ni, H.; Zeng, S.; Qin, X.; Sun, X.; Zhang, S.; Zhao, X.; Yu, Z.; Li, L. Molecular docking and site-directed mutagenesis of a Bacillus thuringiensis chitinase to improve chitinolytic, synergistic lepidopteran-larvicidal and nematicidal activities. Int. J. Biol. Sci., 2015, 11(3), 304-315.
[http://dx.doi.org/10.7150/ijbs.10632] [PMID: 25678849]
[18]
Yu, Z.; Wang, J.; Lin, J.; Zhao, M.; Qiu, J. Exploring regulation genes involved in the expression of L-amino acid oxidase in Pseudoalteromonas sp. Rf-1. PLoS One, 2015, 10(3), e0122741.
[http://dx.doi.org/10.1371/journal.pone.0122741] [PMID: 25815733]
[19]
Yu, Z.; Zhou, N.; Qiao, H.; Qiu, J. Identification, cloning, and expression of L-amino acid oxidase from marine Pseudoalteromonas sp. B3. SWJ, 2014, 2014, 979858.
[PMID: 24526926]
[20]
Idrees, D.; Kumar, S.; Rehman, SAA.; Gourinath, S.; Islam, A.; Ahmad, F.; Imtaiyaz Hassan, M. Cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase VA 3 Biotech., 2016, 6(1), 16-23.
[21]
Ding, GB.; Wu, G.; Li, B.; Yang, P.; Li, Z. High-yield expression in Escherichia coli, biophysical characterization, and biological evaluation of plant toxin gelonin 3 Biotech., 2019, 9(1), 19-26.
[22]
Asante, I.; Pei, H.; Zhou, E.; Liu, S.; Chui, D.; Yoo, E.; Louie, S.G. Simultaneous quantitation of folates, flavins and B6 metabolites in human plasma by LC-MS/MS assay: Applications in colorectal cancer. J. Pharm. Biomed. Anal., 2018, 158, 66-73.
[http://dx.doi.org/10.1016/j.jpba.2018.05.030] [PMID: 29860180]
[23]
Serrano, A.; Frago, S.; Velázquez-Campoy, A.; Medina, M. Role of key residues at the flavin mononucleotide (FMN):adenylyltransferase catalytic site of the bifunctional riboflavin kinase/flavin adenine dinucleotide (FAD) Synthetase from Corynebacterium ammoniagenes. Int. J. Mol. Sci., 2012, 13(11), 14492-14517.
[http://dx.doi.org/10.3390/ijms131114492] [PMID: 23203077]
[24]
Huerta, C.; Borek, D.; Machius, M.; Grishin, N.V.; Zhang, H. Structure and mechanism of a eukaryotic FMN adenylyltransferase. J. Mol. Biol., 2009, 389(2), 388-400.
[http://dx.doi.org/10.1016/j.jmb.2009.04.022] [PMID: 19375431]
[25]
Frago, S.; Martínez-Júlvez, M.; Serrano, A.; Medina, M. Structural analysis of FAD synthetase from Corynebacterium ammoniagenes. BMC Microbiol., 2008, 8(8), 160-171.
[http://dx.doi.org/10.1186/1471-2180-8-160] [PMID: 18811972]
[26]
Dym, O.; Eisenberg, D. Sequence-structure analysis of FAD-containing proteins. Protein Sci., 2001, 10(9), 1712-1728.
[http://dx.doi.org/10.1110/ps.12801] [PMID: 11514662]
[27]
Masuda, T.; Sawa, Y.; Asai, M. Application of chromatography. XXVII. On the formation of FAD in the culture of Eremothecium ashbyii. Pharm. Bull., 1955, 3(5), 375-378.
[http://dx.doi.org/10.1248/cpb1953.3.375] [PMID: 13289297]
[28]
Watanabe, T.; Uchida, T.; Kato, J.; Chibata, I. Production of flavine-adenine dinucleotide from riboflavine by a mutant of Sarcina lutea. Appl. Microbiol., 1974, 27(3), 531-536.
[http://dx.doi.org/10.1128/AEM.27.3.531-536.1974] [PMID: 4824882]
[29]
Shimizu, S.; Yamane, K.; Tani, Y.; Yamada, H. Enzymatic synthesis of flavin adenine dinucleotide. Appl. Biochem. Biotechnol., 1983, 8(3), 237-247.
[http://dx.doi.org/10.1007/BF02778261] [PMID: 6089661]
[30]
Hagihara, T.; Fujio, T.; Aisaka, K. Cloning of FAD synthetase gene from Corynebacterium ammoniagenes and its application to FAD and FMN production. Appl. Microbiol. Biotechnol., 1995, 42(5), 724-729.
[http://dx.doi.org/10.1007/BF00171952] [PMID: 7765913]

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