基于同源模型的比较和分析,发现羰基还原酶SCR1辅酶结合域P124和W125位点对辅酶NADPH的结合形成了一定的空间位阻效应.通过对该位点进行小侧基氨基酸的取代突变,该酶的底物专一性和立体选择性均发生了不同程度的改变,表明该位点是酶与辅酶有效结合的关键位点,而且它与辅酶结合的空间效应进一步影响了底物结合域活性中心对不同构型的底物及其对映体产物的亲和作用.在底物专一性方面,野生型酶对2-羟基苯乙酮和2-溴苯乙酮及其衍生物等底物表现出较高的催化活性,而突变株W125A,W125G,P124A/W125A和P124G/W125G对苯乙酮及其部分衍生物和2-辛酮等底物的催化活性均有所提高.对于酶的立体选择性,部分突变株发生了转化产物对映体构型反转的现象,突变株P124A/W125A和P124G/W125G催化还原2-羟基苯乙酮和4-氯乙酰乙酸乙酯均生成了(R)-型产物.
参考文献
| [1] | Patel R N .[J].Enzyme and Microbial Technology,2002,31:804. |
| [2] | Goldberg K;Schroer K;Lutz S;Liese A .Biocatalytic ketone reduction - a powerful tool for the production of chiral alcohols - part I: processes with isolated enzymes[J].Applied Microbiology and Biotechnology,2007(2):237-248. |
| [3] | Schroer K;Mackfeld U;Tan IA;Wandrey C;Heuser F;Bringer-Meyer S;Weckbecker A;Hummel W;Daussmann T;Pfaller R;Liese A;Lutz S .Continuous asymmetric ketone reduction processes with recombinant Escherichia coli[J].Journal of Biotechnology,2007(4):438-444. |
| [4] | Ema T;Yagasaki H;Okita N;Nishikawa K;Korenaga T;Sakai T .Asymmetric reduction of a variety of ketones with a recombinant carbonyl reductase: identification of the gene encoding a versatile biocatalyst[J].Tetrahedron, Asymmetry: The International Journal for Repid Publication on all Aspects of Asymmetry in Orgainc, Inorganic, Organometallic, Physical and Bio-Organic Chemistry,2005(6):1075-1078. |
| [5] | Xie, Y;Xu, JH;Lu, WY;Lin, GQ .Adzuki bean: A new resource of biocatalyst for asymmetric reduction of aromatic ketones with high stereoselectivity and substrate tolerance[J].Bioresource Technology,2009(9):2463-2468. |
| [6] | Bougioukou DJ;Stewart JD .Opposite stereochemical courses for enzyme-mediated alkene reductions of an enantiomeric substrate pair[J].Journal of the American Chemical Society,2008(24):7655-7658. |
| [7] | Gamenara D;Dominguez de Maria P .Candida spp. redox machineries: An ample biocatalytic platform for practical applications and academic insights[J].Biotechnology Advances: An International Review Journal,2009(3):278-285. |
| [8] | Inoue K;Makino Y;Itoh N .Production of (R)-chiral alcohols by a hydrogen-transfer bioreduction with NADH-dependent Leifsonia alcohol dehydrogenase (LSADH)[J].Tetrahedron, Asymmetry: The International Journal for Repid Publication on all Aspects of Asymmetry in Orgainc, Inorganic, Organometallic, Physical and Bio-Organic Chemistry,2005(15):2539-2549. |
| [9] | Strohmeier G A;Pichler H;May O;Gruber-Khadjawi M .[J].Chemical Reviews,2011,111:441. |
| [10] | 朱敦明.中国化学会第27届学术年会第06分会场摘要集[M].厦门:厦门大学出版社,2010:06-O-002. |
| [11] | Burton SG;Cowan DA;Woodley JM .The search for the ideal biocatalyst [Review][J].Nature biotechnology,2002(1):37-45. |
| [12] | 朱敦明,吴洽庆.生物催化剂立体选择性的基因工程改造[J].生物工程学报,2009(12):1770-1778. |
| [13] | Heiss C;Laivenieks M;Zeikus J G;Phillips R S .[J].Bioorganic and Medicinal Chemistry Letters,2001,9:1659. |
| [14] | Baerga-Ortiz A;Popovic B;Siskos AP;O'Hare HM;Spiteller D;Williams MG;Campillo N;Spencer JB;Leadlay PF .Directed mutagenesis alters the stereochemistry of catalysis by isolated ketoreductase domains from the erythromycin polyketide synthase[J].Chemistry & biology,2006(3):277-285. |
| [15] | Qi X H;Chen Y L;Jiang K;Zuo W P Luo Z F Wei Y T Du L Q Wei H Huang R B Du Q S .[J].Journal of Biotechnology,2009,144:43. |
| [16] | Stewart J D .[J].Current Opinion in Chemical Biology,2001,5:120. |
| [17] | Persson B;Kallberg Y;Bray JE;Bruford E;Dellaporta SL;Favia AD;Duarte RG;Jornvall H;Kavanagh KL;Kedishvili N;Kisiela M;Maser E;Mindnich R;Orchard S;Penning TM;Thornton JM;Adamski J;Oppermann U .The SDR (short-chain dehydrogenase/reductase and related enzymes) nomenclature initiative.[J].Chemico-biological interactions,2009(1/3):94-98. |
| [18] | Oppermann U;Filling C;Hult M;Shafqat N;Wu X;Lindh M;Shafqat J;Nordling E;Kallberg Y;Persson B;Jornvall H .Short-chain dehydrogenases/reductases (SDR): the 2002 update.[J].Chemico-biological interactions,2003(0):247-253. |
| [19] | Kavanagh K L;J(c)rnvall H;Persson B;Oppermanna U .[J].Cellular and Molecular Life Sciences,65:3895. |
| [20] | Nie, Y.;Xiao, R.;Xu, Y.;Montelione, G.T. .Novel anti-Prelog stereospecific carbonyl reductases from Candida parapsilosis for asymmetric reduction of prochiral ketones[J].Organic & biomolecular chemistry,2011(11):4070-4078. |
| [21] | Yan Z;Nie Y;Xu Y;Liu X Xiao R .[J].Tetrahedron Letters,2011,52:999. |
| [22] | Reetz M T;Bocola M;Carballeira J D;Zha D X Vogel A .[J].Angewandte Chemie International Edition,2005,44:4192. |
| [23] | Carrigan JB;Engel PC .Probing the determinants of coenzyme specificity in Peptostreptococcus asaccharolyticus glutamate dehydrogenase by site-directed mutagenesis[J].The FEBS journal,2007(19):5167-5174. |
| [24] | 闫真 .[D].无锡:江南大学,2010. |
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