近平滑假丝酵母立体选择性还原酶表达、结构解析与改造
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摘要
氧化还原酶是一种具有高度立体选择性的生物催化剂,常用于生物转化光学活性的手性化合物。在众多的微生物氧化还原酶源中,来源于近平滑假丝酵母(Candida parapsilosis)CCTCC M203011的(R)-和(S)-羰基还原酶催化2-羟基苯乙酮产生不同立体化学性质的苯基乙二醇,其中(R)-羰基还原酶催化2-羟基苯乙酮到(R)-苯基乙二醇的可逆反应,而(S)-羰基还原酶催化2-羟基苯乙酮的还原反应,产生(S)-苯基乙二醇。为了更好地提高上述两种生物催化剂手性转化的适用性能,本研究通过改良(R)-羰基还原酶的编码基因,优化了目标蛋白的表达量;同时用X-衍射和分子置换的方法解析了(S)-羰基还原酶的蛋白空间结构,基于精细的三维结构及催化功能域的研究,利用蛋白质工程技术,理性设计了辅酶特异性和界面特性变化等一系列功能突变体,为酶催化功能的认识和改造提供了新的研究视点。
依据大肠杆菌(Escherichia coli)对密码子的偏好性,用细胞偏爱的密码子同源置换出(R)-羰基还原酶编码基因中的9个稀有密码子,同时切除5’末端的无序结构序列4-27 bp。研究结果表明密码子的优化较好地提高了蛋白表达量,酶活力比优化前提高了35.7%。原子力显微镜观察结果表明高浓度的酶单体分子多聚为网状结构,但是当加入少量的辅酶(NAD+,NADH)或底物时,蛋白由网状结构转为椭圆形低聚态,与低浓度酶形成的聚合态相同。动力学研究表明酶催化2-羟基苯乙酮的KM和kcat分别是催化(R)-苯基乙二醇的0.5和1.8倍,即它们的kcat/KM比值约等于4.0。同时酶与辅酶NAD+的结合常数Kd是酶与NADH的近3倍。说明酶更易催化NADH链接的2-羟基苯乙酮的还原反应,较难催化NAD+链接的(R)-苯基乙二醇的氧化反应。
采用SOE-PCR法克隆出增强型荧光蛋白标记的(R)-和(S)-羰基还原酶蛋白融合基因,构建到真核pYX212表达载体中,以荧光蛋白为筛选标志,发现两种羰基还原酶在酿酒酵母(Saccharomyces cerevisiae W303-1A)细胞中多定位于细胞膜(如高尔基体等内膜系等),少数成点状分布于胞内。根据荧光强度分析可知(S)-羰基还原酶的表达水平明显高于(R)-羰基还原酶。荧光蛋白融合型(R)-羰基还原酶催化2-羟基苯乙酮获得产物(R)-苯基乙二醇的光学纯度和产率分别为86.6%和70.4%。荧光蛋白融合型(S)-羰基还原酶催化2-羟基苯乙酮生成产物(S)-型对映体的光学纯度和产率分别92.3%和81.8%。说明两种羰基还原酶与荧光蛋白的融合不影响酶蛋白的正确折叠和生理催化功能。
利用分子重组技术,将6×Histidine的标签分别与(R)-和(S)-羰基还原酶进行融合,克隆其融合基因,置于AOX1的甲醇诱导型启动子下游,构建重组毕赤氏酵母菌株GS115,实现了两种羰基还原酶的高效异源表达。经培养条件的优化,它们的最佳表达条件基本相同:pH7.0,OD600为2.0–2.5,甲醇的日诱导添加量为1%,甲醇诱导后菌体培养的时间为3.5–4天。在优化表达条件下,(R)-和(S)-羰基还原酶外泌蛋白的表达量分别为185 mg/L和270 mg/L,酶比活分别为1.35 U/mg和3.41U/mg。Western杂交结果显示6×Histidine标签已特异性地结合于两种酶蛋白上,经过Ni2+柱一步法亲和层析后的靶蛋白纯度均高于90%。生物转化实验表明纯化后的(R)-和(S)-羰基还原酶的最适反应的pH分别为8.0–9.0和7.0,其中(R)-羰基还原酶催化产生(R)-苯基乙二醇的光学纯度和产率分别为89.5%和78.4%。(S)-羰基还原酶催化合成产物(S)-苯基乙二醇的光学纯度和产率分别为96.9%和88.7%。低浓度(5 mmol/L)的Zn2+对(R)-羰基还原酶催化2-羟基苯乙酮的还原反应具有明显的促进作用,当反应体系中添加5 mM Zn2+时,产物(R)-苯基乙二醇的光学纯度和产率分别可以达到94.3%和86.5%。
将近平滑假丝酵母(C. parapsilosis)的(R)-和(S)-羰基还原酶基因构建到含双克隆位点的表达载体pETDuetTM-1中,构建重组质粒pETDuet-RCR-SCR。同时将大肠杆菌(E. coli)的嘧啶核苷酸转氢酶A和B亚基基因克隆到表达载体pACYCDuet-1上,构建重组质粒pCACYD-PNTA-PNTB。将两种重组质粒分别转化或共转化表达宿主E. coli BL21 (DE3),构建同时带有两个或四个目的基因的重组菌株。生物转化实验表明共转化重组菌具有高效生物催化(R)-苯基乙二醇,一步法转化(S)-苯基乙二醇的功能,其产物光学纯度为96.3%e.e.,产率为91.9%,与只含有(R)-和(S)-羰基还原酶基因共表达体系相比较,产物(S)-苯基乙二醇的光学纯度和产率分别提高了32%和39.2%。
采用悬滴法对纯化的重组(S)-羰基还原酶进行结晶条件筛选及条件优化,获知该蛋白晶体的最佳生长条件为:18% (w/v) polyethylene glycol 2000 monomethyl ether (PEG2K MME)和8% (v/v)异丙醇。X-衍射最好的晶体大约在5天后获得,其大小为0.4×0.3×0.3 mm。以Agaricus bisporus甘露醇脱氢酶(MtDH,1H5Q)为同源模型,采用分子置换法解析(S)-羰基还原酶的结构,其空间群属于P212121,晶胞参数为:a=104.7 ?,b=142.8 ?和c=151.8 ?。其最终结构模型的分辨率修正到2.69-?,Rfree和Rwork分别26.4%和20.5%,具有很好的立体化学特性。一个不对称单位中有八个单体分子,组成两个四聚体,或者说一个不对称单位有四个二聚体,每个二聚体关于所谓的Q轴对称。从已解析出来的结构发现该酶与典型短链脱氢酶不同,主要表现在三个方面:(1)N端有一个约含30个氨基酸的肽段尾巴,起着平衡Q轴的作用;(2)二元对称轴不规范,有大约15°的偏角。因而形成非标准的2-2-2对称的四聚体;(3)在四聚体中,四个之中的二个NADPH可能的结合区域被周围的肽段所占据。分子筛排阻实验结果表明辅酶NADPH与(S)-羰基还原酶的结合能够诱导一个无活性的四聚体向有活性四聚体的转化。
采用定点突变的方法,构建了包括N末端的肽段,保守的催化三元区域以及位于界面与界面之间的关键氨基酸位点的突变。实验结果表明(S)-羰基还原酶的N-末端肽段在很大程度上起着平衡四聚体的Q轴作用,对酶活几乎没有贡献。与其它短链脱氢酶一样,(S)-羰基还原酶也使用相同的保守催化三元区域Ser-Tyr-Lys。定点突变的研究结果表明保守的Ser-Tyr-Lys催化三元区域对催化功能是必需的。在(S)-羰基还原酶二元对称轴的疏水界面上,βG折叠中的一个紧密折叠的缬氨酸被一个带负电的氨基酸天冬氨酸即V270D,打破了原始的四聚态。超速分析的结果证实突变体V270D在溶液中表现为同源二聚体,动力学研究表明二聚体酶仍旧保持原始的催化功能。圆二色谱温度扫描结果显示所有突变体蛋白的稳定性均低于野生型酶,它们的熔化温度比野生型酶低4-7℃。
为迎合工业应用需求,使(S)-羰基还原酶的辅酶依赖性由昂贵的NADPH转变为较便宜的NADH将产生非常重要的意义。因而根据(S)-羰基还原酶蛋白结构,理性设计了位于辅酶结合口袋或其附近的βB和αC之间所谓磷酸结合区域中关键氨基酸的定点突变,包括Ser67Asp、His68Asp和Pro69Asp点突变的不同组合。生物转化实验研究表明所有的突变体均催化2-羟基苯乙酮产生(R)-苯基乙二醇,但产物的光学活性和产率各不相同。其中在辅酶NADH链接反应中,突变体酶S67D/H68D催化产生(R)-对映体,其光学活性和产率分别为95.4%和83.1%。与野生型酶相比较,当NADH和NADPH作辅酶时,突变体S67D/H68D的kcat/KM值分别增加10倍和降低20倍,而kcat值保持不变。NADPH链接反应中,突变体S67D/H68D和野生型酶的Kd值分别是NADH链接反应中的0.28和1.9倍,表明突变体S67D/H68D酶具有更强NADPH的亲和性和更低的NADH依赖性。圆二色谱分析结果表明突变体S67D/H68D与野生型表现出相似的蛋白二级结构和熔化温度。温度变性和尿素变性实验结果表明NAD(H)对突变体S67D/H68D具有很好的保护作用,而以此相对应的是NADP(H)对野生型酶具有很好的保护作用。由此可见双位点S67D/H68D的突变在没有改变酶的稳定性的基础上,不仅成功改变了辅酶的特异性,而且改变了产物的空间选择性。该研究为采用蛋白质工程技术改变短链脱氢酶的辅酶特异性和产物的空间选择性提供了一个崭新的例证,同时也为手性醇的工业制备提供了有价值的研究。
Oxidoreductase, a kind of biocatalyst with high stereoselectivity, is always used in bioconversion of optically active chiral compounds. Among various of stereospecific oxidoreductases from microorganisms, (R)- and (S)-specific carbonyl reductases from Candida parapsilosis CCTCC M203011 catalyze 2-hydroxyacetophenone to stereospecific 1-phenyl-1,2-ethanediol (PED). (R)-carbonyl reductase (RCR) catalyzes the reversible reaction between 2-hydroxyacetophenone and (R)-PED, and (S)-carbonyl reductase (SCR) catalyzes the reduction of 2-hydroxyacetophenone to (S)-PED. In order to further facilitate the two biocatalysts for stereoselective conversion and modify their biocatalytic functions, the coding sequences of RCR were optimized to improve its protein expression using genetic engineering methods. The crystal structure of SCR was also determined. Based on its refined three dimensional structure and catalytic functional domain, a series of mutants were rationally designed to change the cofactor specificity and interface characteristics using protein engineering technique. This work will supply the new research viewpoint for further understanding and modifying the catalytic functions of the enzyme.
According to the preferred codons in Escherichia coli, nine rare codons were synonymously replaced with those used at higher frequency and the disorder sequence of 4-27 bp at 5’-terminus was truncaed in the RCR coding gene. The results showed that the total enzyme activity of the codon variant was increased 35.7% than before optimization and its protein production was also considerably improved. Atomic force microscopy images showed that the apo-enzyme adopted a netlike aggregate morphology at a high concentration. When the protein was complexed with NAD+, NADH or the ligand of 2-hydroxyacetophenone, most molecules of the protein resembled flat elliptical cylinders, which is similar to that of the enzyme at the low concentrations. By kinetic analysis the ratios of KM and kcat between RCR catalyzing 2-hydroxyacetophenone and (R)-PED were 0.5 and 1.8, respectively, and their ratio value of kcat/KM was about 4.0. The results showed that RCR catalyzed the NADH-linked reduction of 2-hydroxyacetophenone more easily than the NAD+-linked oxidation of (R)-PED.
The RCR or SCR genes fused with enhanced green fluorescence protein (EGFP) were cloned and constructed on the eukaryotic expression vector pYX212. Using EGFP as specific biomarkers, two carbonyl reductases were mostly found to locate on the cell membrane, i.e. golgi complex endomembrane system, few dotted in the cells. According to the intensity of EGFP the level of SCR expression was found higher than RCR in Saccharomyces cerevisiae W303-1A cells. The fusion enzyme RCR-EGFP catalyzed the reduction of 2-hydroxyacetophenone to (R)-PED with the optical purity of 86.6% and yield of 70.4%, while the enzyme SCR-EGFP transformed the (S)-isomer with the optical purity of 92.3% in a yield of 81.8%. The results showed that the fusion of RCR or SCR with EGFP showed no effect in correct protein folding and physical catalytic functions.
The 6×Histidine tag was inserted N terminus of RCR or SCR, and their fused genes were cloned using molecular recombinant technique and located downstream of the AOX1 promoter. The recombinant Pichia pastoris were successfully constructed. The highly heterogenous expressions of two carbonyl reductases were obtained. By analysis of the pre-expression and optimized expression process, the optimal culturing conditions were achieved as follows: pH 7.0, the initial OD600 2.0–2.5, methanol daily addition concentration of 1.0% (v/v) and the optimal induction time points at about 3.5–4.0 d for the strain GS115. Under these conditions, the productions of recombinant RCR and SCR were 185 mg/L and 270 mg/L in P. pastoris, the specific activity of both enzymes were 1.35 U/mg and 3.41 U/mg. The western blotting result with only one band appeared on polyvinylidene fluoride (PVDF) membrane revealed that Ab1 bound specifically to the 6×Histidine tag of RCR and SCR. The purity of both proteins was over 90% after a single Ni2+ -cherating purification step. The experiments showed that the optimal pH values for biotransfomation were 8–9 and 7.0 for RCR and SCR respectively. The purified RCR catalyzed the product (R)-PED with the optical purity of 83.7% and yield of 67.2%, while the enzyme SCR transformed (S)-isomer with the optical purity of 96.9% in a yield of 88.7%. The results also revealed that the addition of the“functional elements”Zn2+ of 5 mM considerably enhanced the biotransformation efficiency of (R)-PED, and the optical purity and yield of the product reached 94.3% and 86.5%.
The RCR and SCR genes from C. parapsilosis were constructed an expression vector pETDuetTM-1 contain double cloning sites simultaneously and the recombinant plasmid pETDuet-RCR-SCR was obtained. Pyrimidine nucleotide transhydrogenase A and B genes from E. coli were inserted into the expression vector pACYCDuet-1 and the recombinant plasmid pACYD-PNTA-PNTB was constructed. The plasmids pETDuet-RCR-SCR and/or pACYD-PNTA-PNTB were then transformed the competent cells of E. coli BL21 (DE3), and the recombinant strains containing two or four target genes. The biotransformation experiments showed that strains catalyzed (R)-PED to (S)-isomer in one-step efficiently. The product was afforded with high optical purity of 96.3%e.e. and yield of 91.9%. When compared to the co-expression system containing RCR and SCR, the optical purity and yield was increased with 32% and 39.2%.
The recombinant SCR was purified and crystallized, the crystal form was obtained using the hanging-drop vapor-diffusion method and reservoir solution of 18% (w/v) polyethylene glycol 2000 monomethyl ether and 8% (v/v) isopropyl as the precipitant. The crystals obtained after 5 d of growth were good enough for data collection and were rhomboid in shape with average dimensions of 0.3×0.3×0.4 mm. The crystal structure of mannitol 2-dehydrogenase (MtDH, PDB file 1H5Q) from Agaricus bisporus was used as a search model for molecular replacement. The crystal belongs to the space groups P212121 with cell dimensions of a= 104.7 ?; b= 142.8 ?; and c= 151.8 ?. The crystal structure of the apo-form was solved to a 2.69-? resolution to final Rwork of 20.5% (Rfree of 26.4%), with good stereochemical properties. Eight SCR molecules were identified per asymmetric unit and were organized into two tetramers, or eight SCR molecules in an asu form four dimmers, each is related by the so-called Q-axis dyad symmetry. Three novel features were found between the crystal structure of SCR and the other short-chain dehydrogenase/reductase members: (1) an extended N-terminal peptide tail which stabilizes the Q-axis related dimerization; (2) an unusual tetramerization which is devoid of 2-2-2 point-group symmetry, particularly in the R-axis related dimer interface; and (3) two of the four potential NADPH binding sites in the tetramer are occupied by surrounding peptides in the apo-enzyme form. Gel filtration experiments further suggests that a conformational change in the SCR subunits induced by NADPH binding converts the tetramer into an active form.
A number of mutagenesis-enzymatic analyses were carried out based on the new structural information. The mutations of N-terminal peptide, the putative catalytic Ser-Tyr-Lys triad and key amino acids between interfaces were carried out. The results showed that the functional role of N-terminal peptide stabilizes the Q-axis related dimerization and has no any contribution to enzyme activity. SCR uses the same conserved catalytic triad as other SDR proteins. To investigate a possible functional role of the A-B interface, a dimer-breaking mutation in the dyad symmetrical hydrophobic interface was introduced. In particular, a tightly packed Val residue at position 270 in theβG strand was replaced with a negatively charged residue, Asp, which was expected to disrupt the A-B type dimerization without affecting the A-C interface. Consistent with our prediction, analytic ultracentrifugation measurements confirmed that the V270D mutant of SCR exists as a homo-dimer in solution. Kinetic studies indicate that SCR may maintain its enzymatic activity in a dimer form. Thermal stability of all the variants were analyzed using a circular dichroism temperature scan, resulting in a 45–48℃melting temperature, 4–7℃lower than that of the wild type SCR.
For industrial applications, converting the cofactor specificity of an enzyme from NADPH to NADH would be of great significance as NADH is substantially cheaper than NADPH. Based on the determined quaternary structure of SCR, the mutations were rationally designed in order to explore the possibility of converting SCR from a NADPH-dependent enzyme into an NADH-dependent one. Using site-directed mutagenesis, the mutants were designed with different combinations of Ser67Asp, His68Asp and Pro69Asp substitutions inside or adjacent to the coenzyme binding pocket of so-called phosphate-binding loop betweenβB andαC. All mutations caused a significant shift of enantioselectivity toward the (R)-configuration during 2-hydroxyacetophenone reduction with different optical purity and yield. The mutant S67D/H68D produced (R)-PED with high opitical purity of 95.4% and yield of 83.1% in the NADH-linked reaction. By kinetic analysis, the mutant S67D/H68D resulted in a nearly 10-fold increase and a 20-fold decrease in the kcat/KM value when NADH and NADPH were used as the cofactors respectively, but maintaining kcat essentially the same with respect to wild-type SCR. The ratio of Kd values between NADH and NADPH for the S67D/H68D mutant and SCR were 0.28 and 1.9 respectively, which indicates the S67D/H68D mutant has a stronger preference for NADH and weaker binding for NADPH. Moreover, the S67D/H68D enzyme exhibited the similar secondary structure and melting temperature to the wild-type from circular dichroism analysis. It was also found that NADH provided maximal protection against thermal and urea denaturation for S67D/H68D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the double point mutation S67D/H68D converted the coenzyme specificity of SCR from NADP(H) to NAD(H) as well as the product enantioselectivity successfully without disturbing enzyme stability. The work provides a protein engineering approach to modify the co-enzyme specificity and enantioselectivity of ketone reduction for short-chain reductases and will likely have valuable industrial applications.
依据大肠杆菌(Escherichia coli)对密码子的偏好性,用细胞偏爱的密码子同源置换出(R)-羰基还原酶编码基因中的9个稀有密码子,同时切除5’末端的无序结构序列4-27 bp。研究结果表明密码子的优化较好地提高了蛋白表达量,酶活力比优化前提高了35.7%。原子力显微镜观察结果表明高浓度的酶单体分子多聚为网状结构,但是当加入少量的辅酶(NAD+,NADH)或底物时,蛋白由网状结构转为椭圆形低聚态,与低浓度酶形成的聚合态相同。动力学研究表明酶催化2-羟基苯乙酮的KM和kcat分别是催化(R)-苯基乙二醇的0.5和1.8倍,即它们的kcat/KM比值约等于4.0。同时酶与辅酶NAD+的结合常数Kd是酶与NADH的近3倍。说明酶更易催化NADH链接的2-羟基苯乙酮的还原反应,较难催化NAD+链接的(R)-苯基乙二醇的氧化反应。
采用SOE-PCR法克隆出增强型荧光蛋白标记的(R)-和(S)-羰基还原酶蛋白融合基因,构建到真核pYX212表达载体中,以荧光蛋白为筛选标志,发现两种羰基还原酶在酿酒酵母(Saccharomyces cerevisiae W303-1A)细胞中多定位于细胞膜(如高尔基体等内膜系等),少数成点状分布于胞内。根据荧光强度分析可知(S)-羰基还原酶的表达水平明显高于(R)-羰基还原酶。荧光蛋白融合型(R)-羰基还原酶催化2-羟基苯乙酮获得产物(R)-苯基乙二醇的光学纯度和产率分别为86.6%和70.4%。荧光蛋白融合型(S)-羰基还原酶催化2-羟基苯乙酮生成产物(S)-型对映体的光学纯度和产率分别92.3%和81.8%。说明两种羰基还原酶与荧光蛋白的融合不影响酶蛋白的正确折叠和生理催化功能。
利用分子重组技术,将6×Histidine的标签分别与(R)-和(S)-羰基还原酶进行融合,克隆其融合基因,置于AOX1的甲醇诱导型启动子下游,构建重组毕赤氏酵母菌株GS115,实现了两种羰基还原酶的高效异源表达。经培养条件的优化,它们的最佳表达条件基本相同:pH7.0,OD600为2.0–2.5,甲醇的日诱导添加量为1%,甲醇诱导后菌体培养的时间为3.5–4天。在优化表达条件下,(R)-和(S)-羰基还原酶外泌蛋白的表达量分别为185 mg/L和270 mg/L,酶比活分别为1.35 U/mg和3.41U/mg。Western杂交结果显示6×Histidine标签已特异性地结合于两种酶蛋白上,经过Ni2+柱一步法亲和层析后的靶蛋白纯度均高于90%。生物转化实验表明纯化后的(R)-和(S)-羰基还原酶的最适反应的pH分别为8.0–9.0和7.0,其中(R)-羰基还原酶催化产生(R)-苯基乙二醇的光学纯度和产率分别为89.5%和78.4%。(S)-羰基还原酶催化合成产物(S)-苯基乙二醇的光学纯度和产率分别为96.9%和88.7%。低浓度(5 mmol/L)的Zn2+对(R)-羰基还原酶催化2-羟基苯乙酮的还原反应具有明显的促进作用,当反应体系中添加5 mM Zn2+时,产物(R)-苯基乙二醇的光学纯度和产率分别可以达到94.3%和86.5%。
将近平滑假丝酵母(C. parapsilosis)的(R)-和(S)-羰基还原酶基因构建到含双克隆位点的表达载体pETDuetTM-1中,构建重组质粒pETDuet-RCR-SCR。同时将大肠杆菌(E. coli)的嘧啶核苷酸转氢酶A和B亚基基因克隆到表达载体pACYCDuet-1上,构建重组质粒pCACYD-PNTA-PNTB。将两种重组质粒分别转化或共转化表达宿主E. coli BL21 (DE3),构建同时带有两个或四个目的基因的重组菌株。生物转化实验表明共转化重组菌具有高效生物催化(R)-苯基乙二醇,一步法转化(S)-苯基乙二醇的功能,其产物光学纯度为96.3%e.e.,产率为91.9%,与只含有(R)-和(S)-羰基还原酶基因共表达体系相比较,产物(S)-苯基乙二醇的光学纯度和产率分别提高了32%和39.2%。
采用悬滴法对纯化的重组(S)-羰基还原酶进行结晶条件筛选及条件优化,获知该蛋白晶体的最佳生长条件为:18% (w/v) polyethylene glycol 2000 monomethyl ether (PEG2K MME)和8% (v/v)异丙醇。X-衍射最好的晶体大约在5天后获得,其大小为0.4×0.3×0.3 mm。以Agaricus bisporus甘露醇脱氢酶(MtDH,1H5Q)为同源模型,采用分子置换法解析(S)-羰基还原酶的结构,其空间群属于P212121,晶胞参数为:a=104.7 ?,b=142.8 ?和c=151.8 ?。其最终结构模型的分辨率修正到2.69-?,Rfree和Rwork分别26.4%和20.5%,具有很好的立体化学特性。一个不对称单位中有八个单体分子,组成两个四聚体,或者说一个不对称单位有四个二聚体,每个二聚体关于所谓的Q轴对称。从已解析出来的结构发现该酶与典型短链脱氢酶不同,主要表现在三个方面:(1)N端有一个约含30个氨基酸的肽段尾巴,起着平衡Q轴的作用;(2)二元对称轴不规范,有大约15°的偏角。因而形成非标准的2-2-2对称的四聚体;(3)在四聚体中,四个之中的二个NADPH可能的结合区域被周围的肽段所占据。分子筛排阻实验结果表明辅酶NADPH与(S)-羰基还原酶的结合能够诱导一个无活性的四聚体向有活性四聚体的转化。
采用定点突变的方法,构建了包括N末端的肽段,保守的催化三元区域以及位于界面与界面之间的关键氨基酸位点的突变。实验结果表明(S)-羰基还原酶的N-末端肽段在很大程度上起着平衡四聚体的Q轴作用,对酶活几乎没有贡献。与其它短链脱氢酶一样,(S)-羰基还原酶也使用相同的保守催化三元区域Ser-Tyr-Lys。定点突变的研究结果表明保守的Ser-Tyr-Lys催化三元区域对催化功能是必需的。在(S)-羰基还原酶二元对称轴的疏水界面上,βG折叠中的一个紧密折叠的缬氨酸被一个带负电的氨基酸天冬氨酸即V270D,打破了原始的四聚态。超速分析的结果证实突变体V270D在溶液中表现为同源二聚体,动力学研究表明二聚体酶仍旧保持原始的催化功能。圆二色谱温度扫描结果显示所有突变体蛋白的稳定性均低于野生型酶,它们的熔化温度比野生型酶低4-7℃。
为迎合工业应用需求,使(S)-羰基还原酶的辅酶依赖性由昂贵的NADPH转变为较便宜的NADH将产生非常重要的意义。因而根据(S)-羰基还原酶蛋白结构,理性设计了位于辅酶结合口袋或其附近的βB和αC之间所谓磷酸结合区域中关键氨基酸的定点突变,包括Ser67Asp、His68Asp和Pro69Asp点突变的不同组合。生物转化实验研究表明所有的突变体均催化2-羟基苯乙酮产生(R)-苯基乙二醇,但产物的光学活性和产率各不相同。其中在辅酶NADH链接反应中,突变体酶S67D/H68D催化产生(R)-对映体,其光学活性和产率分别为95.4%和83.1%。与野生型酶相比较,当NADH和NADPH作辅酶时,突变体S67D/H68D的kcat/KM值分别增加10倍和降低20倍,而kcat值保持不变。NADPH链接反应中,突变体S67D/H68D和野生型酶的Kd值分别是NADH链接反应中的0.28和1.9倍,表明突变体S67D/H68D酶具有更强NADPH的亲和性和更低的NADH依赖性。圆二色谱分析结果表明突变体S67D/H68D与野生型表现出相似的蛋白二级结构和熔化温度。温度变性和尿素变性实验结果表明NAD(H)对突变体S67D/H68D具有很好的保护作用,而以此相对应的是NADP(H)对野生型酶具有很好的保护作用。由此可见双位点S67D/H68D的突变在没有改变酶的稳定性的基础上,不仅成功改变了辅酶的特异性,而且改变了产物的空间选择性。该研究为采用蛋白质工程技术改变短链脱氢酶的辅酶特异性和产物的空间选择性提供了一个崭新的例证,同时也为手性醇的工业制备提供了有价值的研究。
Oxidoreductase, a kind of biocatalyst with high stereoselectivity, is always used in bioconversion of optically active chiral compounds. Among various of stereospecific oxidoreductases from microorganisms, (R)- and (S)-specific carbonyl reductases from Candida parapsilosis CCTCC M203011 catalyze 2-hydroxyacetophenone to stereospecific 1-phenyl-1,2-ethanediol (PED). (R)-carbonyl reductase (RCR) catalyzes the reversible reaction between 2-hydroxyacetophenone and (R)-PED, and (S)-carbonyl reductase (SCR) catalyzes the reduction of 2-hydroxyacetophenone to (S)-PED. In order to further facilitate the two biocatalysts for stereoselective conversion and modify their biocatalytic functions, the coding sequences of RCR were optimized to improve its protein expression using genetic engineering methods. The crystal structure of SCR was also determined. Based on its refined three dimensional structure and catalytic functional domain, a series of mutants were rationally designed to change the cofactor specificity and interface characteristics using protein engineering technique. This work will supply the new research viewpoint for further understanding and modifying the catalytic functions of the enzyme.
According to the preferred codons in Escherichia coli, nine rare codons were synonymously replaced with those used at higher frequency and the disorder sequence of 4-27 bp at 5’-terminus was truncaed in the RCR coding gene. The results showed that the total enzyme activity of the codon variant was increased 35.7% than before optimization and its protein production was also considerably improved. Atomic force microscopy images showed that the apo-enzyme adopted a netlike aggregate morphology at a high concentration. When the protein was complexed with NAD+, NADH or the ligand of 2-hydroxyacetophenone, most molecules of the protein resembled flat elliptical cylinders, which is similar to that of the enzyme at the low concentrations. By kinetic analysis the ratios of KM and kcat between RCR catalyzing 2-hydroxyacetophenone and (R)-PED were 0.5 and 1.8, respectively, and their ratio value of kcat/KM was about 4.0. The results showed that RCR catalyzed the NADH-linked reduction of 2-hydroxyacetophenone more easily than the NAD+-linked oxidation of (R)-PED.
The RCR or SCR genes fused with enhanced green fluorescence protein (EGFP) were cloned and constructed on the eukaryotic expression vector pYX212. Using EGFP as specific biomarkers, two carbonyl reductases were mostly found to locate on the cell membrane, i.e. golgi complex endomembrane system, few dotted in the cells. According to the intensity of EGFP the level of SCR expression was found higher than RCR in Saccharomyces cerevisiae W303-1A cells. The fusion enzyme RCR-EGFP catalyzed the reduction of 2-hydroxyacetophenone to (R)-PED with the optical purity of 86.6% and yield of 70.4%, while the enzyme SCR-EGFP transformed the (S)-isomer with the optical purity of 92.3% in a yield of 81.8%. The results showed that the fusion of RCR or SCR with EGFP showed no effect in correct protein folding and physical catalytic functions.
The 6×Histidine tag was inserted N terminus of RCR or SCR, and their fused genes were cloned using molecular recombinant technique and located downstream of the AOX1 promoter. The recombinant Pichia pastoris were successfully constructed. The highly heterogenous expressions of two carbonyl reductases were obtained. By analysis of the pre-expression and optimized expression process, the optimal culturing conditions were achieved as follows: pH 7.0, the initial OD600 2.0–2.5, methanol daily addition concentration of 1.0% (v/v) and the optimal induction time points at about 3.5–4.0 d for the strain GS115. Under these conditions, the productions of recombinant RCR and SCR were 185 mg/L and 270 mg/L in P. pastoris, the specific activity of both enzymes were 1.35 U/mg and 3.41 U/mg. The western blotting result with only one band appeared on polyvinylidene fluoride (PVDF) membrane revealed that Ab1 bound specifically to the 6×Histidine tag of RCR and SCR. The purity of both proteins was over 90% after a single Ni2+ -cherating purification step. The experiments showed that the optimal pH values for biotransfomation were 8–9 and 7.0 for RCR and SCR respectively. The purified RCR catalyzed the product (R)-PED with the optical purity of 83.7% and yield of 67.2%, while the enzyme SCR transformed (S)-isomer with the optical purity of 96.9% in a yield of 88.7%. The results also revealed that the addition of the“functional elements”Zn2+ of 5 mM considerably enhanced the biotransformation efficiency of (R)-PED, and the optical purity and yield of the product reached 94.3% and 86.5%.
The RCR and SCR genes from C. parapsilosis were constructed an expression vector pETDuetTM-1 contain double cloning sites simultaneously and the recombinant plasmid pETDuet-RCR-SCR was obtained. Pyrimidine nucleotide transhydrogenase A and B genes from E. coli were inserted into the expression vector pACYCDuet-1 and the recombinant plasmid pACYD-PNTA-PNTB was constructed. The plasmids pETDuet-RCR-SCR and/or pACYD-PNTA-PNTB were then transformed the competent cells of E. coli BL21 (DE3), and the recombinant strains containing two or four target genes. The biotransformation experiments showed that strains catalyzed (R)-PED to (S)-isomer in one-step efficiently. The product was afforded with high optical purity of 96.3%e.e. and yield of 91.9%. When compared to the co-expression system containing RCR and SCR, the optical purity and yield was increased with 32% and 39.2%.
The recombinant SCR was purified and crystallized, the crystal form was obtained using the hanging-drop vapor-diffusion method and reservoir solution of 18% (w/v) polyethylene glycol 2000 monomethyl ether and 8% (v/v) isopropyl as the precipitant. The crystals obtained after 5 d of growth were good enough for data collection and were rhomboid in shape with average dimensions of 0.3×0.3×0.4 mm. The crystal structure of mannitol 2-dehydrogenase (MtDH, PDB file 1H5Q) from Agaricus bisporus was used as a search model for molecular replacement. The crystal belongs to the space groups P212121 with cell dimensions of a= 104.7 ?; b= 142.8 ?; and c= 151.8 ?. The crystal structure of the apo-form was solved to a 2.69-? resolution to final Rwork of 20.5% (Rfree of 26.4%), with good stereochemical properties. Eight SCR molecules were identified per asymmetric unit and were organized into two tetramers, or eight SCR molecules in an asu form four dimmers, each is related by the so-called Q-axis dyad symmetry. Three novel features were found between the crystal structure of SCR and the other short-chain dehydrogenase/reductase members: (1) an extended N-terminal peptide tail which stabilizes the Q-axis related dimerization; (2) an unusual tetramerization which is devoid of 2-2-2 point-group symmetry, particularly in the R-axis related dimer interface; and (3) two of the four potential NADPH binding sites in the tetramer are occupied by surrounding peptides in the apo-enzyme form. Gel filtration experiments further suggests that a conformational change in the SCR subunits induced by NADPH binding converts the tetramer into an active form.
A number of mutagenesis-enzymatic analyses were carried out based on the new structural information. The mutations of N-terminal peptide, the putative catalytic Ser-Tyr-Lys triad and key amino acids between interfaces were carried out. The results showed that the functional role of N-terminal peptide stabilizes the Q-axis related dimerization and has no any contribution to enzyme activity. SCR uses the same conserved catalytic triad as other SDR proteins. To investigate a possible functional role of the A-B interface, a dimer-breaking mutation in the dyad symmetrical hydrophobic interface was introduced. In particular, a tightly packed Val residue at position 270 in theβG strand was replaced with a negatively charged residue, Asp, which was expected to disrupt the A-B type dimerization without affecting the A-C interface. Consistent with our prediction, analytic ultracentrifugation measurements confirmed that the V270D mutant of SCR exists as a homo-dimer in solution. Kinetic studies indicate that SCR may maintain its enzymatic activity in a dimer form. Thermal stability of all the variants were analyzed using a circular dichroism temperature scan, resulting in a 45–48℃melting temperature, 4–7℃lower than that of the wild type SCR.
For industrial applications, converting the cofactor specificity of an enzyme from NADPH to NADH would be of great significance as NADH is substantially cheaper than NADPH. Based on the determined quaternary structure of SCR, the mutations were rationally designed in order to explore the possibility of converting SCR from a NADPH-dependent enzyme into an NADH-dependent one. Using site-directed mutagenesis, the mutants were designed with different combinations of Ser67Asp, His68Asp and Pro69Asp substitutions inside or adjacent to the coenzyme binding pocket of so-called phosphate-binding loop betweenβB andαC. All mutations caused a significant shift of enantioselectivity toward the (R)-configuration during 2-hydroxyacetophenone reduction with different optical purity and yield. The mutant S67D/H68D produced (R)-PED with high opitical purity of 95.4% and yield of 83.1% in the NADH-linked reaction. By kinetic analysis, the mutant S67D/H68D resulted in a nearly 10-fold increase and a 20-fold decrease in the kcat/KM value when NADH and NADPH were used as the cofactors respectively, but maintaining kcat essentially the same with respect to wild-type SCR. The ratio of Kd values between NADH and NADPH for the S67D/H68D mutant and SCR were 0.28 and 1.9 respectively, which indicates the S67D/H68D mutant has a stronger preference for NADH and weaker binding for NADPH. Moreover, the S67D/H68D enzyme exhibited the similar secondary structure and melting temperature to the wild-type from circular dichroism analysis. It was also found that NADH provided maximal protection against thermal and urea denaturation for S67D/H68D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the double point mutation S67D/H68D converted the coenzyme specificity of SCR from NADP(H) to NAD(H) as well as the product enantioselectivity successfully without disturbing enzyme stability. The work provides a protein engineering approach to modify the co-enzyme specificity and enantioselectivity of ketone reduction for short-chain reductases and will likely have valuable industrial applications.
引文
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