利用脯氨酸效应提高短乳杆菌谷氨酸脱羧酶的热稳定性
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摘要
以磷酸吡哆醛为辅酶的谷氨酸脱羧酶(Glutamate decarboxylase,GAD),能专一、不可逆地催化L-谷氨酸脱去α-羧基生成γ-氨基丁酸。为了提高GAD热稳定性为目标,本研究通过与嗜热古细菌Thermococcus kodakarensis中GAD氨基酸序列的比对及引入脯氨酸策略,最终在短乳杆菌Lactobacillus brevis CGMCC No.1306的GAD突变体中筛选得到热稳定性提高的突变酶G364P。结果显示,突变酶G364P在55℃的半衰期以及半失活温度分别比野生酶提高19.4 min和5.3℃,并且突变酶G364P的催化效率与野生酶相比没有明显变化。此外,利用分子动力学模拟来验证突变对蛋白质热稳定性的影响,突变酶G364P的均方根偏差(Rootmeansquare deviation,RMSD)以及含G364的loop区域均方根涨落(Root mean square fluctuation,RMSF)均比野生酶低,引入脯氨酸增加了364位氨基酸与相邻氨基酸的疏水相互作用。文中通过引入脯氨酸成功提高了L. brevis中GAD的热稳定性,同时也为其他酶热稳定性的理性设计提供了方法学指导。
Glutamate decarboxylase, a unique pyridoxal 5′-phosphate-dependent enzyme, catalyzes α-decarboxylation of L-glutamate to γ-aminobutyrate. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, proline was introduced at 13 different positions in glutamate decarboxylase by using the design strategy of homologous sequence alignment between Thermococcus kodakarensis and Lactobacillus brevis CGMCC No.1306. A mutant enzyme G364 P with higher thermostability was obtained. Compared to the wild type, thermostability of the mutant G364 P was significantly improved, the half-life time(t1/2) at 55 °C and the semi-inactivation temperature(T5015) of the mutant G364 P increased 19.4 min and 5.3 °C, respectively, while kcat/Km of the mutant enzyme remained nearly unchanged. Further analysis of their thermostability by molecular dynamics simulations were performed. The root mean square deviation of G364 P and root mean square fluctuation in the loop region including G364 were lower than the wild type at 313 K for 10 ns, and G364 P increased one hydrophobic interaction in the loop region. It proves that mutation of flexible 364-Gly to rigid proline endows glutamate decarboxylase with enhanced thermostability.
Glutamate decarboxylase, a unique pyridoxal 5′-phosphate-dependent enzyme, catalyzes α-decarboxylation of L-glutamate to γ-aminobutyrate. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, proline was introduced at 13 different positions in glutamate decarboxylase by using the design strategy of homologous sequence alignment between Thermococcus kodakarensis and Lactobacillus brevis CGMCC No.1306. A mutant enzyme G364 P with higher thermostability was obtained. Compared to the wild type, thermostability of the mutant G364 P was significantly improved, the half-life time(t1/2) at 55 °C and the semi-inactivation temperature(T5015) of the mutant G364 P increased 19.4 min and 5.3 °C, respectively, while kcat/Km of the mutant enzyme remained nearly unchanged. Further analysis of their thermostability by molecular dynamics simulations were performed. The root mean square deviation of G364 P and root mean square fluctuation in the loop region including G364 were lower than the wild type at 313 K for 10 ns, and G364 P increased one hydrophobic interaction in the loop region. It proves that mutation of flexible 364-Gly to rigid proline endows glutamate decarboxylase with enhanced thermostability.
引文
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[26]Ueno Y,Hayakawa K,Takahashi S,et al.Purification and characterization of glutamate decarboxylase from Lactobadllus brevis IFO 12005.Biosci Biotechnol Biochem,1997,61(7):1168-1171.
[27]Tomita H,Yokooji Y,Ishibashi T,et al.An archaeal glutamate decarboxylase homolog functions as an aspartate decarboxylase and is involved inβ-alanine and coenzyme a biosynthesis.J Bacteriol,2014196(6):1222-1230.
[28]Yu K,Hu S,Huang J,et al.A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase.Enzyme Microb Technol2011,49(3):272-276.
[29]Gromiha MM,Pathak MC,Saraboji K,et al Hydrophobic environment is a key factor for the stability of thermophilic proteins.Proteins,2013,81(4):715-721.
[30]Shirley BA,Stanssens P,Hahn U,et al.Contribution of hydrogen bonding to the conformational stability of ribonuclease T1.Biochemistry,1992,31(3):725-732.
[31]Burley SK,Petsko GA.Aromatic-aromatic interaction:a mechanism of protein structure stabilization.Science,1985,229(4708):23-28.
[32]Deckert G,Warren PV,Gaasterland T,et al.The complete genome of the hyperthermophilic bacterium Aquifex aeolicus.Nature,1998,392(6674):353-358.
[33]Tina KG,Bhadra R,Srinivasan N.PIC:protein interactions calculator.Nucleic Acids Res,2007,35(S2):W473-W476.
[34]Pace CN,Fu HL,Fryar KL,et al.Contribution of hydrophobic interactions to protein stability.J Mol Biol,2011,408(3):514-528.
[2]Inoue K,Shirai T,Ochiai H,et al.Blood-pressure-lowering effect of a novel fermented milk containingγ-aminobutyric acid(GABA)in mild hypertensives.Eur J Clin Nutr,2003,57(3):490-495.
[3]Kai KL,Ruusuvuori E,Seja P,et al.GABA actions and ionic plasticity in epilepsy.Curr Opin Neurobiol,2014,26:34-41.
[4]Diana M,Quílez J,Rafecas M.Gamma-aminobutyric acid as a bioactive compound in foods:a review.JFunct Foods,2014,10:407-420.
[5]Lammens TM,Franssen MCR,Scott EL,et al.Synthesis of biobased N-methylpyrrolidone by one-pot cyclization and methylation ofγ-aminobutyric acid.Green Chem,2010,12(8):1430-1436.
[6]Ueno H.Enzymatic and structural aspects on glutamate decarboxylase.J Mol Catal B:Enzym,2000,10(1/3):67-79.
[7]Huang J,Fang H,Gai ZC,et al.Lactobacillus brevis CGMCC 1306 glutamate decarboxylase:crystal structure and functional analysis.Biochem Biophys Res Commun,2018,503(3):1703-1709.
[8]Lyu CJ,Zhao WR,Hu S,et al.Physiology-oriented engineering strategy to improve gamma-aminobutyrate production in Lactobacillus brevis.J Agric Food Chem,2017,65(4):858-866.
[9]Foster JW.Escherichia coli acid resistance:tales of an amateur acidophile.Nat Rev Microbiol,20042(11):898-907.
[10]Li H,Cao Y.Lactic acid bacterial cell factories for gamma-aminobutyric acid.Amino Acids,2010,39(5):1107-1116.
[11]Dhakal R,Bajpai VK,Baek KH.Production of gaba(γ-Aminobutyric acid)by microorganisms:a review.Braz J Microbiol,2012,43(4):1230-1241.
[12]Huang J,Mei LH,Sheng Q,et al.Purification and characterization of glutamate decarboxylase of Lactobacillus brevis CGMCC 1306 isolated from fresh milk.Chin J Chem Eng,2007,15(2):157-161.
[13]Xia J.Breeding ofγ-aminobutyric acid-producing Lactobacillus and optimization of fermentation conditions[D].Hangzhou:Zhejiang University,2016(in Chinese).夏江.产γ-氨基丁酸的乳酸菌菌株选育及其发酵条件优化[D].杭州:浙江大学,2006.
[14]Fan EY,Huang J,Hu S,et al.Cloning,sequencing and expression of a glutamate decarboxylase gene from the GABA-producing strain Lactobacillus brevis CGMCC 1306.Ann Microbiol,2012,62(2):689-698.
[15]Yu K,Lin L,Hu S,et al.C-terminal truncation of glutamate decarboxylase from Lactobacillus brevis CGMCC 1306 extends its activity toward near-neutral pH.Enzyme Microb Technol,2012,50(4/5):263-269.
[16]Yang HQ,Liu L,Li JH,et al.Rational design to improve protein thermostability:recent advances and prospects.ChemBioEng Rev,2015,2(2):87-94.
[17]Durrenberger M,Ward TR.Recent achievments in the design and engineering of artificial metalloenzymes.Curr Opin Chem Biol,2014,19:99-106.
[18]Jones BJ,Lim HY,Huang J,et al.Comparison of five protein engineering strategies for stabilizing anα/β-hydrolase.Biochemistry,2017,56(50):6521-6532.
[19]Max KEA,Wunderlich M,Roske Y,et al.Optimized variants of the cold shock protein from in vitro selection:structural basis of their high thermostability.J Mol Biol,2007,369(4):1087-1097.
[20]Benedix A,Becker CM,de Groot BL,et al.Predicting free energy changes using structural ensembles.Nat Methods,2009,6(1):3-4.
[21]Reetz MT,Carballeira JD,Vogel A.Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability.Angew Chem Int Ed,2006,118(46):7909-7915.
[22]Xie DF,Fang H,Mei JQ,et al.Improving thermostability of(R)-selective amine transaminase from Aspergillus terreus through introduction of disulfide bonds.Biotechnol Appl Biochem,2018,65(2):255-262.
[23]Strickler SS,Gribenko AV,Gribenko AV,et al.Protein stability and surface electrostatics:a charged relationship.Biochemistry,2006,45(9):2761-2766.
[24]Huang J,Jones BJ,Kazlauskas RJ.Stabilization of anα/β-hydrolase by introducing proline residues:salicylic acid binding protein 2 from tobacco Biochemistry,2015,54(28):4330-4341.
[25]Chen XX,Li D,LüJX,et al.Determination ofγ-aminobutyric acid and glutamic acid in human cerebrospinal fluid by high performance liquid chromatography.Chin J Chromatogr,1997,15(3):237-239(in Chinese).陈希贤,李东,吕建新,等.高效液相色谱法测定人脑脊液中γ-氨基丁酸和谷氨酸.色谱,1997,15(3):237-239.
[26]Ueno Y,Hayakawa K,Takahashi S,et al.Purification and characterization of glutamate decarboxylase from Lactobadllus brevis IFO 12005.Biosci Biotechnol Biochem,1997,61(7):1168-1171.
[27]Tomita H,Yokooji Y,Ishibashi T,et al.An archaeal glutamate decarboxylase homolog functions as an aspartate decarboxylase and is involved inβ-alanine and coenzyme a biosynthesis.J Bacteriol,2014196(6):1222-1230.
[28]Yu K,Hu S,Huang J,et al.A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase.Enzyme Microb Technol2011,49(3):272-276.
[29]Gromiha MM,Pathak MC,Saraboji K,et al Hydrophobic environment is a key factor for the stability of thermophilic proteins.Proteins,2013,81(4):715-721.
[30]Shirley BA,Stanssens P,Hahn U,et al.Contribution of hydrogen bonding to the conformational stability of ribonuclease T1.Biochemistry,1992,31(3):725-732.
[31]Burley SK,Petsko GA.Aromatic-aromatic interaction:a mechanism of protein structure stabilization.Science,1985,229(4708):23-28.
[32]Deckert G,Warren PV,Gaasterland T,et al.The complete genome of the hyperthermophilic bacterium Aquifex aeolicus.Nature,1998,392(6674):353-358.
[33]Tina KG,Bhadra R,Srinivasan N.PIC:protein interactions calculator.Nucleic Acids Res,2007,35(S2):W473-W476.
[34]Pace CN,Fu HL,Fryar KL,et al.Contribution of hydrophobic interactions to protein stability.J Mol Biol,2011,408(3):514-528.