Characterization of the Enzymatic properties of a single mutant of homoserine dehydrogenase from Corynebacterium glutamicum

Abstract

Abstract: Homoserine dehydrogenase (HSD) is a key enzyme in the synthesis of aspartic acid in Corynebacterium glutamicum, which plays an important role in the synthesis of methionine, threonine, and isoleucine. Through site-directed mutagenesis, the catalytic activity of homoserine dehydrogenase is improved, and the feedback inhibition and repression of metabolites in the pathway are reduced.According to the docking result of HSD with the substrate homoserine molecule andanalysis of their spatial structuretwo key sites, Gly25 and Asp61, were selected for site-directed saturation mutation. Through enzyme activity screening, it was found that the mutants A61L and G25G were stronger than the wild type (WT). The enzyme activity was significantly increased, so the kinetics and enzymatic properties of these two mutants were studied.、The kinetic results showed that compared with WT, the Km value of G25G and A61L decreased, the substrate affinity increased, and the enzyme activity increased by 1.21 times and 1.35 times, respectively; the n value decreased, and the positive synergy increased.The optimum temperature of A61L and G25G was 40°C, the same as WT; the optimum pH of A61L was 8.0, the optimum pH of G25G was 8.5, which was higher than that of WT; the enzymatic half-lives of A61L and G25G were 1h and 0.5h longer than WT, respectively; Different concentrations of K+, Mg2+, Ca2+ can activate the mutant and the wild type;different concentrations of methanol, ethanol, acetonitrile and dimethyl sulfoxide had significant inhibitory effects on the mutant and wild type; under the concentration of 1mmol/L~25mmol/L inhibitor , the inhibitory effect of the mutant was significantly weaker than that of the wild type. In this study, the mutants G25G and A61L with improved enzyme activity and weakened allosteric inhibition were obtained, which provided a reference for optimizing the biosynthetic pathway of high serine dehydrogenase and constructing high-yield methionine, threonine and isoleucine strains.

Key words: Corynebacterium glutamicum, homoserine dehydrogenase, enzymatic properties

CLC Number:

Q517

泽沅江 Yu-Zhe LIU Xin GAO Min WeiHong. Characterization of the Enzymatic properties of a single mutant of homoserine dehydrogenase from Corynebacterium glutamicum[J]. FOOD SCIENCE, 0, (): 0-0.

References

[1] Navik U, Sheth V G, Khurana A, et al. Methionine as a double-edged sword in health and disease: Current perspective and future challenges[J]. Ageing Research Reviews, 2021, 72: 101500. DOI:10.1016/j.arr.2021.101500.
[2] Lee H S, Hwang B J. Methionine biosynthesis and its regulation in Corynebacterium glutamicum : parallel pathways of transsulfuration and direct sulfhydrylation[J]. Applied Microbiology and Biotechnology, 2003, 62(5-6): 459-467. DOI:10.1007/s00253-003-1306-7.
[3] 李莹.基于代谢工程选育谷氨酸棒杆菌L-蛋氨酸高产菌[D]. 哈尔滨工业大学, 2016: 3-5.
[4] 秦天宇.代谢工程改造谷氨酸棒杆菌生产L-甲硫氨酸[D]. 江南大学, 2014: 1-4.
[5] Kumar D, Gomes J. Methionine production by fermentation[J]. Biotechnology Advances, 2005, 23(1): 41-61. DOI:10.1016/j.biotechadv.2004.08.005.
[6] Thomas D, Barbey R, Surdin-Kerja Y. Evolutionary relationships between yeast and bacterial homoserine dehydrogenases[J]. Febs Letters, 1993: 289-293. DOI:10.1016/0014-5793(93)81359-8.
[7] Akai S, Ikushiro H, Sawai T, et al. The crystal structure of homoserine dehydrogenase complexed with L-homoserine and NADPH in a closed form[J]. Journal of biochemistry, 2018. DOI:10.1093/jb/mvy094.
[8] Berghuis A M, DeLaBarre B, Thompson P R, et al. Crystal structures of homoserine dehydrogenase suggest a novel catalytic mechanism for oxidoreductases[J]. Nature structural & molecular biology, 2000, 7(3): 238-244. DOI:10.1038/73359.
[9] Hayashi J, Inoue S, Kim K, et al. Crystal Structures of a Hyperthermophilic Archaeal Homoserine Dehydrogenase Suggest a Novel Cofactor Binding Mode for Oxidoreductases[J]. Scientific Reports, 2015, 5(1). DOI:10.1038/srep11674.
[10] Tomonaga Y, Kaneko R, Goto M, et al. Structural insight into activation of homoserine dehydrogenase from the archaeon Sulfolobus tokodaii via reduction[J]. Biochemistry and Biophysics Reports, 2015, 3: 14-17. DOI:10.1016/j.bbrep.2015.07.006.
[11] Navratna V, Reddy G, Gopal B. Structural basis for the catalytic mechanism of homoserine dehydrogenase[J]. Acta Crystallographica Section D Biological Crystallography, 2015, 71(5): 1216-1225. DOI:10.1107/S1399004715004617.
[12] Kim D H, Nguyen Q T, Ko G S, et al. Molecular and Enzymatic Features of Homoserine Dehydrogenase fromBacillus subtilis[J]. Journal of Microbiology and Biotechnology, 2020, 30(12): 1905-1911. DOI:10.4014/jmb.2004.04060.
[13] Tang W, Guo M, Jiang X, et al. Expression, purification, and biochemical characterization of an NAD-dependent homoserine dehydrogenase from the symbiotic Polynucleobacter necessarius subsp. necessarius.[J]. Protein expression and purification, 2021, 188: 105977. DOI:10.1016/j.pep.2021.105977.
[14] Tang W, Dong X, Meng J, et al. Biochemical characterization and redesign of the coenzyme specificity of a novel monofunctional NAD+-dependent homoserine dehydrogenase from the human pathogen Neisseria gonorrhoeae[J]. Protein Expression and Purification, 2021, 186: 105909. DOI:10.1016/j.pep.2021.105909.
[15] Ogata K, Yajima Y, Nakamura S, et al. Inhibition of homoserine dehydrogenase by formation of a cysteine-NAD covalent complex[J]. Scientific Reports, 2018, 8(1). DOI:10.1038/s41598-018-24063-1.
[16] Kubota T, Kurihara E, Watanabe K, et al. Conformational changes in the catalytic region are responsible for heat-induced activation of hyperthermophilic homoserine dehydrogenase[J]. Communications Biology, 2022, 5(1). DOI:10.1038/s42003-022-03656-7.
[17] Li N, Zeng W, Zhou J, et al. O-Acetyl-L-homoserine production enhanced by pathway strengthening and acetate supplementation in Corynebacterium glutamicum[J]. Biotechnology for Biofuels and Bioproducts, 2022, 15(1). DOI:10.1186/s13068-022-02114-0.
[18] 申术霞, 朱运明, 闵伟红, 等. 北京棒杆菌AS1.299高丝氨酸脱氢酶突变体L200F/D215K的异源表达及酶学性质[J]. 微生物学报, 2014, 54(10): 1178-1184. DOI:10.13343/j.cnki.wsxb.2014.10.010.
[19] 樊占青. 北京棒杆菌（Corynebacterium pekinense）天冬氨酸激酶催化位点空间改造及别构调控机制研究[D]. 长春: 吉林农业大学,2021: 8-9.
[20] 任军. 天冬氨酸激酶定点突变及酶学性质表征[D]. 长春: 吉林农业大学,2013: 44-45.
[21] 王亚南.北京棒杆菌（Corynebacterium pekinense）天冬氨酸激酶催化活性位点的空间改造及工程菌构建[D]. 吉林农业大学, 2021: 13-16.
[22] 申术霞.北京棒杆菌高丝氨酸脱氢酶突变体的异源表达及酶学性质表征[D]. 吉林农业大学, 2014: 15-16.
[23] 许金坤.北京棒杆菌AS1.299高丝氨酸脱氢酶定点突变及酶学性质表征[D]. 吉林农业大学, 2013: 23-26.
[24] Bailey S F, Alonso Morales L A, Kassen R. Effects of Synonymous Mutations beyond Codon Bias: The Evidence for Adaptive Synonymous Substitutions from Microbial Evolution Experiments[J]. Genome Biology and Evolution, 2021, 13(9). DOI:10.1093/gbe/evab141.
[25] Brule C E, Grayhack E J. Synonymous Codons: Choose Wisely for Expression[J]. Trends in Genetics, 2017, 33(4): 283-297. DOI:10.1016/j.tig.2017.02.001.
[26] Supek F. The Code of Silence: Widespread Associations Between Synonymous Codon Biases and Gene Function[J]. Journal of Molecular Evolution, 2016, 82(1): 65-73. DOI:10.1007/s00239-015-9714-8.
[27] 郑志强.耐热性细菌漆酶的高效表达及其在纸浆生物漂白中的应用[D]. 江南大学, 2015: 35-36.
[28] 董聪, 高庆华, 王玥, 等. 基于密码子优化的FAD依赖葡萄糖脱氢酶在毕赤酵母中的高效表达及酶学性质[J]. 生物技术通报, 2019, 35(07): 114-120. DOI:10.13560/j.cnki.biotech.bull.1985.2018-0941.