醇脱氢酶KpADH的127位点对催化活性和对映选择性的影响

doi:10.16085/j.issn.1000-6613.2019-0472

摘要/Abstract

摘要：

醇脱氢酶可用于合成手性化合物，在医药、材料等领域应用广泛。来源于多孢克鲁维酵母（Kluyveromyces polysporus）的醇脱氢酶KpADH同时具有还原(4-氯苯基)-(吡啶-2-基)-甲酮（CPMK）和氧化异丙醇、1,4-丁二醇的活性。通过分子对接和结构分析，发现Y127是紧靠底物的关键位点。本文通过对127位点定点饱和突变来提高催化活性和对映选择性，突变体Y127V和Y127I还原CPMK的比活力达到95.0U/mg和84.0U/mg，分别是野生型的6.5倍和5.8倍，突变体Y127L催化CPMK生成(R)-(4-氯苯基)-(吡啶-2-基)-甲醇[(R)-CPMA]的e.e.值由82％提高到99.2％。同时，127位点的突变提高了对醇类底物的氧化活性，突变体Y127I对异丙醇的比活力是野生型的1.46倍，而Y127C更青睐于对1,4-丁二醇的催化氧化，其比活力是野生型的3.00倍。分子间作用力分析表明，Y127L与底物CPMK间增加的氢键和π-π作用力稳定了底物构象，进一步提高了还原CPMK的对映选择性。本文为醇脱氢酶KpADH的分子改造和机制解析提供了指导，提高了该酶的工业应用潜力。

关键词: 醇脱氢酶, 定点饱和突变, 比活力, 对映选择性, 生物催化

Abstract:

Alcohol dehydrogenases can be used to synthesize chiral compounds, which are widely applied in pharmaceuticals, materials and other fields. Alcohol dehydrogenase from Kluyveromyces polysporus (KpADH) exhibited both reducing activity toward (4-chlorophenyl)-(pyridin-2-yl)-methanone (CPMK) and oxidizing activity toward isopropanol and 1,4-butanediol. Based on molecular docking and structural analysis, Y127 was identified as the key site close to the substrate. To improve the catalytic performance and enantioselectivity, site-saturation mutagenesis was applied at Y127. Specific activity toward CPMK of variants Y127V and Y127I increased to 95.0U/mg and 84.0U/mg, which were 6.5- and 5.8-fold of wild type KpADH (^WTKpADH), respectively. The enantioselectivity toward CPMK of variant Y127L increased from 82% to 99.2% e.e. (R). Mutations at 127 also enhanced the oxidizing activity toward alcohol substrates. Specific activity toward isopropanol of Y127I was 1.46-fold of ^WTKpADH. Variant Y127C displayed higher activity in the oxidation of 1,4-butanediol, whose specific activity was 3.00-fold of ^WTKpADH. Intermolecular forces analysis indicated that an increased hydrogen bond and extra π-π interactions stabilized the conformation, which resulted in enhanced enantioselectivity. This study provides guidance for molecular engineering and mechanism analysis of alcohol dehydrogenase KpADH, and extends its application potential in industry.

Key words: alcohol dehydrogenase, site-saturation mutagenesis, specific activity, enantioselectivity, biocatalysis

中图分类号:

Q554

朱诚,许国超,戴威,周婕妤,倪晔. 醇脱氢酶KpADH的127位点对催化活性和对映选择性的影响[J]. 化工进展, 2019, 38(12): 5504-5511.

Cheng ZHU,Guochao XU,Wei DAI,Jieyu ZHOU,Ye NI. Effect of position 127 on the activity and enantioselectivity of alcohol dehydrogenase KpADH[J]. Chemical Industry and Engineering Progress, 2019, 38(12): 5504-5511.

图/表 10

参考文献 19

1	FARINA V, REEVES J T, SENANAYAKE C H, et al. Asymmetric synthesis of active pharmaceutical ingredients[J]. Chemical Reviews, 2006, 106(7): 2734-2793.
2	SCHMIDT F, STEMMLER R T, RUDOLPH J, et al. Catalytic asymmetric approaches towards enantiomerically enriched diarylmethanols and diarylmethylamines[J]. Chemical Society Reviews, 2006, 35(5): 454-470.
3	SUI Y Z, ZHANG X C, WU J W, et al. Cu^II-catalyzed asymmetric hydrosilylation of diaryl- and aryl heteroaryl ketones: application in the enantioselective synthesis of orphenadrine and neobenodine[J]. Chemistry: A European Journal, 2012, 18(24): 7486-7492.
4	HOLLMANN F, ARENDS I W, HOLTMANN D. Enzymatic reduction for the chemist[J]. Green Chemistry, 2011, 13(9): 2285-2314.
5	谌容, 王秋岩, 殷晓浦, 等. 醇脱氢酶不对称还原制备手性醇的研究进展[J].化工进展, 2011, 30(7): 1562-1569.
	KAN R, WANG Q Y, YIN X P, et al. Progress of asymmetric reduction with alcohol dehydrogenase for preparation of chiral alcohols, [J].Chemical Industry and Engineering Progress, 2011, 30(7): 1562-1569.
6	TRUPPO M D, POLLARD D, DEVINE P. Enzyme-catalyzed enantioselective diaryl ketone reductions[J]. Organic Letters, 2007, 9(2): 335-338.
7	NI Y, ZHOU J Y, SUN Z H. Production of a key chiral intermediate of betahistine with a newly isolated Kluyveromyces sp. in an aqueous two-phase system[J]. Process Biochemistry, 2012, 47(7): 1042-1048.
8	ZHU D M, YANG Y, MAJKOWICZ S, et al. Inverting the enantioselectivity of a carbonyl reductase via substrate-enzyme docking guiding point mutation[J]. Organic Letters, 2008, 10(4): 525-528.
9	LI H M, ZHU D M, HUA L, et al. Enantioselective reduction of diaryl ketones catalyzed by a carbonyl reductase from Sporobolomyces salmonicolor and its mutant enzymes[J]. Advanced Synthesis & Catalysis, 2009, 351(4): 583-588.
10	唐铭烩, 许国超, 倪晔. 双芳基酮还原酶的基因挖掘及催化性质[J].食品与生物技术学报, 2018, 37(3): 240-249.
	TANG M H, XU G C, NI Y. Genime mining and characterization of diaryl ketone reductase from kluyveromyces polysporus[J]. Journal of Food Science and Biotechnology, 2018, 37(3): 240-249.
11	XU G C, WANG Y, TANG M H, et al. Hydroclassified combinatorial saturation mutagenesis: reshaping substrate binding pockets of KpADH for enantioselective reduction of bulky-bulky ketones[J]. ACS Catalysis, 2018, 8(9): 8336-8345.
12	ZHOU J Y, WANG Y, XU G C, et al. Structural insight into enantioselective inversion of an alcohol dehydrogenase reveals a “polar gate” in stereorecognition of diaryl ketones[J]. Journal of the American Chemical Society, 2018, 140(39): 12645-12654.
13	ARNOLD F H. Combinatorial and computational challenges for biocatalyst design[J]. Nature, 2001, 409(6817): 253-257.
14	BRANNIGAN J A, WILKINSON A J. Protein engineering 20 years on[J]. Nature Reviews Molecular Cell Biology, 2002, 3(12): 964-970.
15	姜恬, 冯旭东, 李岩, 等. 底物特异性的生物催化与酶设计改造[J].化工进展, 2019, 38(1): 606-614.
	JIANG T, FENG X D, LI Y, et al. The biocatalysis and enzyme modification of substrate specificity[J]. Chemical Industry and Engineering Progress, 2019, 38(1): 606-614.
16	冯旭东, 吕波, 李春, 等. 酶分子稳定性改造研究进展[J].化工学报, 2016, 67(1): 277-284.
	FENG X D, LÜ B, LI C,et al. Advances in enzyme stability modification[J]. CIESC Journal, 2016, 67(1): 277-284.
17	BETTS M J, RUSSELL R B. Bioinformatics for geneticists[M]. New York: Wiley, 2003: 291-315.
18	SUN Z T, LONSDALE R, KONG X D, et al. Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino acid alphabets in directed evolution[J]. Angewandte Chemie: International Edition, 2015, 54(42): 12410-12415.
19	KARA S, SPICKERMANN D, SCHEITTWIESER J H, et al. More efficient redox biocatalysis by utilising 1,4-butanediol as a ‘smart cosubstrate’[J]. Green Chemistry, 2013, 15(2): 330-335.

引物名称	引物序列（5’-3’）	引物序列（5’-3’）
Y127Q	PF: ATGACTGCTTCTCAGGCTTCA	PR: CATAATTGAAGCCTGAGAAGC
Y127C	PF: ATGACTGCTTCTTGCGCTTCA	PR: CATAATTGAAGCGCAAGAAGC
Y127I	PF: ATGACTGCTTCTATTGCTTCA	PR: CATAATTGAAGCAATAGAAGC
Y127N	PF: ATGACTGCTTCTAATGCTTCA	PR: CATAATTGAAGCATTAGAAGC
Y127K	PF: ATGACTGCTTCTAAAGCTTCA	PR: CATAATTGAAGCTTTAGAAGC
Y127M	PF: ATGACTGCTTCTATGGCTTCA	PR: CATAATTGAAGCCATAGAAGC
Y127V	PF: ATGACTGCTTCTGTGGCTTCA	PR: CATAATTGAAGCCACAGAAGC
Y127A	PF: ATGACTGCTTCTGCGGCTTCA	PR: CATAATTGAAGCCGCAGAAGC
Y127G	PF: ATGACTGCTTCTGGCGCTTCA	PR: CATAATTGAAGCGCCAGAAGC
Y127W	PF: ATGACTGCTTCTTGGGCTTCA	PR: CATAATTGAAGCCCAAGAAGC
Y127F	PF: ATGACTGCTTCTTTTGCTTCA	PR: CATAATTGAAGCAAAAGAAGC
Y127H	PF: ATGACTGCTTCTCATGCTTCA	PR: CATAATTGAAGCATGAGAAGC
Y127E	PF: ATGACTGCTTCTGAAGCTTCA	PR: CATAATTGAAGCTTCAGAAGC
Y127L	PF: ATGACTGCTTCTCTGGCTTCA	PR: CATAATTGAAGCCAGAGAAGC
Y127S	PF: ATGACTGCTTCTAGCGCTTCA	PR: CATAATTGAAGCGCTAGAAGC
Y127P	PF: ATGACTGCTTCTCCGGCTTCA	PR: CATAATTGAAGCCGGAGAAGC
Y127D	PF: ATGACTGCTTCTGATGCTTCA	PR: CATAATTGAAGCATCAGAAGC
Y127R	PF: ATGACTGCTTCTCGTGCTTCA	PR: CATAATTGAAGCACGAGAAGC
Y127T	PF: ATGACTGCTTCTACCGCTTCA	PR: CATAATTGAAGCGGTAGAAGC

引物名称	引物序列（5’-3’）	引物序列（5’-3’）
Y127Q	PF: ATGACTGCTTCTCAGGCTTCA	PR: CATAATTGAAGCCTGAGAAGC
Y127C	PF: ATGACTGCTTCTTGCGCTTCA	PR: CATAATTGAAGCGCAAGAAGC
Y127I	PF: ATGACTGCTTCTATTGCTTCA	PR: CATAATTGAAGCAATAGAAGC
Y127N	PF: ATGACTGCTTCTAATGCTTCA	PR: CATAATTGAAGCATTAGAAGC
Y127K	PF: ATGACTGCTTCTAAAGCTTCA	PR: CATAATTGAAGCTTTAGAAGC
Y127M	PF: ATGACTGCTTCTATGGCTTCA	PR: CATAATTGAAGCCATAGAAGC
Y127V	PF: ATGACTGCTTCTGTGGCTTCA	PR: CATAATTGAAGCCACAGAAGC
Y127A	PF: ATGACTGCTTCTGCGGCTTCA	PR: CATAATTGAAGCCGCAGAAGC
Y127G	PF: ATGACTGCTTCTGGCGCTTCA	PR: CATAATTGAAGCGCCAGAAGC
Y127W	PF: ATGACTGCTTCTTGGGCTTCA	PR: CATAATTGAAGCCCAAGAAGC
Y127F	PF: ATGACTGCTTCTTTTGCTTCA	PR: CATAATTGAAGCAAAAGAAGC
Y127H	PF: ATGACTGCTTCTCATGCTTCA	PR: CATAATTGAAGCATGAGAAGC
Y127E	PF: ATGACTGCTTCTGAAGCTTCA	PR: CATAATTGAAGCTTCAGAAGC
Y127L	PF: ATGACTGCTTCTCTGGCTTCA	PR: CATAATTGAAGCCAGAGAAGC
Y127S	PF: ATGACTGCTTCTAGCGCTTCA	PR: CATAATTGAAGCGCTAGAAGC
Y127P	PF: ATGACTGCTTCTCCGGCTTCA	PR: CATAATTGAAGCCGGAGAAGC
Y127D	PF: ATGACTGCTTCTGATGCTTCA	PR: CATAATTGAAGCATCAGAAGC
Y127R	PF: ATGACTGCTTCTCGTGCTTCA	PR: CATAATTGAAGCACGAGAAGC
Y127T	PF: ATGACTGCTTCTACCGCTTCA	PR: CATAATTGAAGCGGTAGAAGC

突变体	CPMK/U·mg^-1	疏水参数lgP
^WTKpADH	14.6±0.7	-1.3
Y127Q	4.77±0.2	-3.5
Y127C	15.7±0.5	2.5
Y127I	84.0±1.8	4.5
Y127N	0.834±0.3	-3.5
Y127K	15.6±0.4	-3.9
Y127M	15.4±0.5	1.9
Y127V	95.0±7.6	4.2
Y127A	2.82±0.2	1.8
Y127G	0.175±0.01	-0.4
Y127W	1.41±0.1	-0.9
Y127F	7.08±0.2	2.8
Y127H	2.74±0.1	-3.2
Y127E	5.14±0.1	-3.5
Y127L	5.27±1.0	3.8
Y127S	2.59±0.2	-0.8
Y127P	＜0.1	-1.6
Y127D	＜0.1	-3.5
Y127R	＜0.1	-4.5
Y127T	＜0.1	-0.7

突变体	CPMK/U·mg^-1	疏水参数lgP
^WTKpADH	14.6±0.7	-1.3
Y127Q	4.77±0.2	-3.5
Y127C	15.7±0.5	2.5
Y127I	84.0±1.8	4.5
Y127N	0.834±0.3	-3.5
Y127K	15.6±0.4	-3.9
Y127M	15.4±0.5	1.9
Y127V	95.0±7.6	4.2
Y127A	2.82±0.2	1.8
Y127G	0.175±0.01	-0.4
Y127W	1.41±0.1	-0.9
Y127F	7.08±0.2	2.8
Y127H	2.74±0.1	-3.2
Y127E	5.14±0.1	-3.5
Y127L	5.27±1.0	3.8
Y127S	2.59±0.2	-0.8
Y127P	＜0.1	-1.6
Y127D	＜0.1	-3.5
Y127R	＜0.1	-4.5
Y127T	＜0.1	-0.7

突变体	1,4-丁二醇/U·g^-1	异丙醇/U·mg^-1
^WTKpADH	57.8±0.5	14.9±1.5
Y127Q	29.2±1.6	2.06±0.1
Y127C	173±8.0	1.58±0.1
Y127I	37.1±1.1	21.8±0.3
Y127N	30.2±0.8	0.987±0.1
Y127K	143±11.0	12.0±0.5
Y127M	147±5.0	11.5±0.1
Y127V	124±5.0	15.7±0.4
Y127A	93.0±1.9	5.64±0.2
Y127G	8.89±1.4	0.202±0.01
Y127W	30.7±0.1	1.83±0.1
Y127F	157±3.0	18.8±0.7
Y127H	48.1±1.2	4.19±0.4
Y127E	6.15±0.1	0.221±0.01
Y127L	91.4±5.3	14.9±0.6
Y127S	31.5±2.3	5.76±0.1
Y127P	0.495±0.1	＜0.1
Y127D	0.913±0.1	＜0.1
Y127R	2.75±0.5	0.233±0.01
Y127T	＜0.1	＜0.1