Structural analysis of aspartate kinase enzyme in Corynebacterium species to find the best enzyme structure for high industrial production of lysine amino acid

پژوهشهای علوم دامی ایران

Article 8, Volume 11, Issue 4 - Serial Number 40, December 2020, Pages 501-512   

Document Type: Genetics & breeding

DOI: 10.22067/ijasr.v11i4.79758

Authors

Morteza Hashemi َAttar¹; Mohammad Reza Nassiry^{* 2}; Ali Javadmanesh¹; Bahar Nayeri Fasaei³

¹Department Animal Science, Faculty of agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.

²Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

³Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Iran

Abstract

Introduction Lysine is one of the essential amino acids not synthesized biologically in the human body and mammals so should be supplied through diets. Among the industrially important amino acids, L-lysine is in 1st position, which is used in pharmaceuticals, animals, human feeds and precursors for the production of peptides or agrochemicals. As L-lysine has large applications, the demand for it is increasing constantly year by year. To minimize the gap between increasing demand and production of L-lysine, it has to be produced in large scale. Corynebacterium species especially Corynebacterium glutamicum is widely used for the industrial production of amino acids especially L-glutamate and L- lysine. The C. glutamicum from long period has been used for the industrial production of various amino acids, primary metabolites and nucleotides. This organism is an aerobic gram positive, rod shaped and non-sporulating bacteria which used for the industrial production of amino acids of L-lysine and L-glutamate. This bacterium uses a variety of carbohydrates, alcohols and organic acids as single sources of carbon and energy for growth and also for the amino acid production. The quantity of lysine production by wild (natural) type of Corynebacterium glutamycum is very low, and its cultivation and propagation cannot provide the amino acid required by markets, therefor the wild type of this bacterium is not suitable and cost-effective for industrial purposes. To minimize the gap between increasing demand and production of L-lysine, it has to be produced in large scale. Aspartate kinase (AK) is one of the key enzymes in the biosynthesis of aspartate-derived amino acids such as lysine. This enzyme catalyzes the transfer of the C-phosphate group of ATP to aspartic acid. In most bacteria, the reaction is the first step of branched biosynthetic pathway for lysine, threonine, isoleucine and methionine and is known to be regulated by the end metabolites through feedback inhibition. For example, aspartate kinase from Corynebacterium glutamicum is concertedly inhibited by lysine and threonine, while aspartate kinase I and III from Escherichia coli is inhibited by threonine and lysine, respectively. Due to industrial production of lysine amino acid by using Corynebacterium species, a lot of researches have been done to improve the genetic modification of these microorganisms. Today, bioinformatics tools are available as online access through web-based databases and software, which can be used to study best structures of aspartate kinase enzyme with the least cost and time.  Also due to high laboratory costs, the use of bioinformatics methods will be important in obtaining the final result. The aim of this study was to investigate the bioinformatics structure of aspartate kinase enzyme in different species of Corynebacterium by authenticated bioinformatics databases to suggest best bioinformatics structure of the aspartate kinase enzyme for applying in laboratory cloning and production of lysine amino acid for industrial purposes.
Material and methods The amino acid sequences of the aspartic kinase enzyme from 33 species of the Corynebacterium was obtained from the NCBI (https://www.ncbi.nlm.nih.gov/protein) and stored as FASTA. In order to study the genetic distances and similarities in 33 species of Corynebacterium a phylogenetic tree was constructed using the Neighbor Joining method using the Mega software (MEGA 6). (A bootstrap check with 1000 replications was also conducted to obtain a confidence level for the branches) ClC main work bench5 software was used to investigate genetic similarities using protein sequences. The Evolutionary properties, physiological and physicochemical properties of aspartate kinase enzyme were studied and investigated in 33 different species of Corynebacterium through valid databases and software of NCBI, MEGA, ProtScale and ProtParam. In order to predict the second structure, two proteins selected from the psipred server were used (http://bioinf.cs.ucl.ac.uk/psipred). For this purpose, the protein sequences of aspartate kinase enzyme in Corynebacterium glutamicum with access numbers of CAO00530.1 and SJM57548.1 introduced into the psipred and their second structure was mapped. Afterward, three-dimensional structure of mentioned protein was modeled using Swiss-model server (https://swissmodel.expasy.org) Then the quality of the two predicted models evaluated by the Rampage server (http://mordred.bioc.cam.ac.uk/ ~ rapper / rampage2.php), and in the next step its ramachandran plot mapped.
Results and discussions The results of evolutionary tree analysis in Corynebacterium species showed that derivation time of aspartate kinase protein in these 33 species is very close. The results of the ProtScale and ProtParam databases showed that the aspartatekinase enzyme of Corynebacterium glutamicum with the access number of CAO00530.1 and SJM57548.1 have the best physicochemical and maximum stability among 33 different species of study. Afterward, with further in silico investigation by the Swiss-Model server and Rampage tool, it was found that the two access numbers of CAO00530.1 and SJM57548.1 had the best three-dimensional structure. From the results of in silico analyses, it can be inferred that the aspartate kinase enzyme with the two access numbers of CAO00530.1 and SJM57548.1 have the best physicochemical properties, the most stable and also the best three-dimensional structure and therefore could be offered for laboratory cloning and production of lysine amino acid for industrial purposes.
Conclusion Due to wide applications and importance of lysine production in our country and also the necessity of selecting appropriate strain of Corynebacterium for genetic engineering and industrial production, this bioinformatics study was done to predict best structure of aspartate kinase enzyme and best strain of Corynebacterium. Based on the results of our in silico analysis, it is suggested that corynebacterium glutamicum has the best protein structure of aspartate kinase enzyme and may be beneficial to increase Industrial lysine amino acid production.

Keywords

Aspartate kinase; Bioinformatics; Corynebacterium; Lysine amino acid production

References

-Becker, J., C. Klopprogge, O. Zelder, E. Heinzle, and C Wittmann. 2005. Amplified Expression of Fructose 1,6-Bisphosphatase in Corynebacterium glutamicum Increases In Vivo Flux through the Pentose Phosphate Pathway and Lysine Production on Different Carbon Sources. Applied and Environmental Microbiology, 71(12): 8587–8596.

2-Chou P. Y., and G. D. Fasman. 1974. Conformational parameters for amino acids in helical, β-sheet, and random coil regions calculated from proteins. Biochemistry, 13: 211-222.

3-Fallahzadeh Mamaghani V. 2016. Characterization of Conserved Hypothetical Proteins from Proteome of Xanthomonas citri subsp. citri, with Ethylene Induction Activity on Arabidopsis thaliana. Genetic Engineering and Biosafety, 5(1): 31-39.

4-Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39: 783-791.

5-Gasteiger, E., C. Hoogland, A.Gattiker, S. Duvaud, M. R. Wilkins, R. D. Appel, and A. Bairoch. 2005. Protein identification and analysis tools on the ExPASy server. The Proteomics Protocols Handbook. Humana Press, pp. 571–607.

6-Hermann, T. 2003. Industrial production of amino acids by Coryneform bacteria. Journal of Biotechnology, 104: 155-172.

7-Ikai A. 1980. Thermostability and aliphatic index of globular proteins. Journal of Biochemistry, 88: 1895-1898.

8-Kent, J. T., Mardia, K. V., and C. C. Taylor. 2010. Matching Unlabelled Configurations and Protein Bioinformatics. Research Report STAT10-01, University of Leeds, Leeds, UK.

9-Lovell S. C., I. W. Davis, W. B. Arendall, P. I. W. de Bakker, J. M. Word, M. G. Prisant, J. S. Richardson and D. C. Richardson. 2002. Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins, 50: 437-450.

10-Plachys, J., and S. Ulbert. 1985. Preparation of lysine by mutants of Corynebacterium. Kvasny Prumysl, 31: 159-160.

11-Pramanik, K., P. Rajbhar. 2017. Functional and Phylogenetic Analyses of Corynebacterium Aspartokinase: An Enzyme of Aspartate Family of Amino Acids. Microbiology Laboratory, Department of Botany, The University of Burdwan Burdwan, PIN-713104, West Bengal, India.

12-Rafalski J. A., and S. C. Falco. 2004. Structure of the yeast HOM3 gene which encodes aspartokinase. Journal of Biological Chemistry, 263(5):2146-2151.

13-Rastegari H., M. Chiani, A. Akbarzadeh, S. Cheraghi, Z. Saffari, M. R. Mehrabi. 2013. Improvement in the Production of L-Lysine by Over expression of Aspartokinase (ASK) in C. glutamicum ATCC21799. Tropical Journal of Pharmaceutical Research, 12 (1): 51-56.

14-Saitou N. and M. Nei. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4: 406-425.

15-Seibold, G., M. Auchter, S. Berens, J. Kalinowski, B. J. Eikmanns. 2006. Utilization of soluble starch by a recombinant Corynebacterium glutamicum strain: Growth and lysine production. Journal of Biotechnology, 124(2): 381-391.

16-Tamura K., G. Stecher, D.Peterson, A. Filipski, and S. Kumar. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30: 2725-2729.

17-Tryfona, T., and M. T. Bustard. 2005. Fermentative production of lysine by Corynebacterium glutamicum: transmembrane transport and metabolic flux analysis. Process Biochemistry, 40(2): 499-508.

18-Yoshida, A., and T. Tomita. 2010. Mechanism of Concerted Inhibition of 2-type Hetero-oligomeric Aspartate Kinase from Corynebacterium glutamicum. The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

19-Zhu, Z. Y., and T. L. Blundell. 1996. The use of amino acid patterns of classified helices and strands in secondary structure prediction. Journal of Molecular Biology, 260: 261-276.