Bioinformatics Analysis and Gene Cloning of L-Lactate Dehydrogenase from Lactobacillus rhamnosus
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摘要: 本研究以鼠李糖乳杆菌（Lactobacillus rhamnosus）L-乳酸脱氢酶（Lr-L-LDH）为研究对象，以研究较多的Lr-L-LDH1为对照，对基因组注释的两个Lr-L-LDH1和Lr-L-LDH2基因，进行异同分析。采用在线网站和专业软件对Lr-L-LDH1和Lr-L-LDH2的一级结构、基本特性、亲疏水性、二级结构进行分析和预测，对三级结构进行同源建模以及酶和底物的分子对接分析，并对酶的编码基因进行系统发育分析，克隆表达及酶活性检测。结果显示：相较于Lr-L-LDH1，Lr-L-LDH2有着相似的分子特性，二级和三级结构，但Lr-L-LDH2编码序列短，氨基酸序列同源性低（48.08%），有不同的进化地位；Lr-L-LDH2也含有乳酸脱氢酶催化活性位点序列和保守的NAD+结合位点序列（GXGXXG），能够形成典型的活性三维口袋域，是NAD+依赖型四聚体结构L-乳酸脱氢酶，在细胞中需要果糖1,6-二磷酸（FBP）来激活，催化丙酮酸还原为L-乳酸；体外克隆表达和酶学分析表明，Lr-L-LDH2酶活力极显著低于Lr-L-LDH1（P<0.01）。Lr-L-LDH1和Lr-L-LDH2都具有乳酸脱氢酶活性，推测二者在乳酸表达调控中相互作用，对鼠李糖乳杆菌L-乳酸脱氢酶的分子改造应同时考虑Lr-L-LDH1和Lr-L-LDH2的作用，研究对乳酸发酵工业的基因工程改造提供分子基础和科学依据。Abstract: In this study, L-lactate dehydrogenase (Lr-L-LDH) from Lactobacillus rhamnosus was investigated. The well-known Lr-L-LDH1 was taken as the control, differences between Lr-L-LDH1 and Lr-L-LDH2 annotated from the genome of L. rhamnosus were analyzed. The primary structure, basic properties, hydrophobicity, secondary structure of Lr-L-LDH1 and Lr-L-LDH2 were predicted and analyzed using online websites and professional software. Homology modeling of the tertiary structure, molecular docking of enzymes and substrates, phylogenetic analysis, in vitro cloning, expression, and enzyme activity assays were further studied. The results showed that, in comparison to Lr-L-LDH1, although Lr-L-LHD2 had similar molecular characteristics, secondary and tertiary structures, Lr-L-LHD2 exhibited differences: A shorter sequences, low amino acid identities (48.08%), and a different phylogenetic status. Lr-L-LHD2 also contained a catalytically active site, a highly conserved NAD+ binding site sequence (GXGXXG), a three-dimensional active pocket domain, was an NAD+ dependent tetrameric L-lactate dehydrogenase, and be activated by fructose 1,6-diphosphate in the cytoplasm for catalyzing the reduction of pyruvate to L-lactic acid. In vitro enzyme activity was significantly lower than Lr-L-LDH1 (P<0.01). In conclusion, both Lr-L-LDH1 and Lr-L-LHD2 showed enzyme activities of L-lactate dehydrogenase, indicating they may regulate the L-lactate metabolism pathway together. Considerations of both Lr-L-LDH1 and Lr-L-LHD2 should be taken for the future molecular modifications of L-LDH in L. rhamnosus, and the study provided the molecular and scientific basis for the gene engineering modifications of lactic acid fermentation industry.
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图 1 Lr-L-ldh1和Lr-L-ldh2的DNA序列系统进化树
Figure 1. Phylogenetic analysis of DNA sequences of Lr-L-ldh1 and Lr-L-ldh2
注：系统进化树为Cladogram的拓扑结构，无色部分为L-ldh1 Group；灰色部分为L-ldh2 Group。
图 2 Lr-L-LDH的亲疏水性分析
Figure 2. Hydrophobicity analysis of Lr-L-LDH
注：A：Lr-L-LDH1；B：Lr-L-LDH2。
图 3 Lr-L-LDH的跨膜区预测
Figure 3. Transmembrane domain prediction of Lr-L-LDH
注：A：Lr-L-LDH1；B：Lr-L-LDH2。
图 4 Lr-L-LDH1与Lr-L-LDH2的序列比对和分析
Figure 4. Alignment of multiple sequences and analysis of Lr-L-LDH1 and Lr-L-LDH2
图 5 Lr-L-LDH的二级结构预测
Figure 5. Secondary structure prediction of Lr-L-LDH
注：A：Lr-L-LDH1；B：Lr-L-LDH2；蓝色代表α-螺旋（Alpha helix）；红色代表延伸链（Extended strand），绿色代表β-转角（Beta turn）；紫色代表无规则卷曲（Random coil）。
图 6 Lr-L-LDH1 和Lr-L-LDH2的三维结构预测和分子对接分析
Figure 6. Three-dimensional structure prediction and molecular docking analysis of Lr-L-LDH1 and Lr-L-LDH2
注：A：Lr-L-LDH1；B：Lr-L-LDH2；A1：Lr-L-LDH1单体三维结构模拟；A2：Lr-L-LDH1四聚体三维结构模拟；A3、A4：Lr-L-LDH1酶-丙酮酸底物作用关系分析；B1：Lr-L-LDH2单体三维结构模拟；B2：Lr-L-LDH2四聚体三维结构模拟；B3、B4：Lr-L-LDH2酶-丙酮酸底物作用关系分析；橙黄色虚线表示盐桥（Salt bridge），绿色虚线表示氢键（Conventional hydrogen bond）。
图 7 PCR验证和酶切验证
Figure 7. The result of PCR confirmation and restriction endonuclease digestion
注：A：M：DNA Marker 2000；1：Lr-L-ldh1；2：Lr-L-ldh2；B：M：DNA Marker 15000；1；pET-28a-Lr-L-ldh1；2：pET-28a-Lr-L-ldh2。
图 8 Lr-L-LDH1和Lr-L-LDH2的诱导表达（A）和酶活检测（B）
Figure 8. Induced expression (A) and enzyme activity detection (B) of Lr-L-LDH1and Lr-L-LDH2
注：A：M：Marker；1：E. coli-BL21（DE3）-pET-28a-Lr-L-ldh1；2：E. coli-BL21（DE3）-pET-28a-Lr-L-ldh2；3：E. coli-BL21（DE3）；B：不同小写字母表示差异极显著（P<0.01）。
表 1 本研究使用的引物
Table 1. Primer used in this study
引物 引物序列（5'-3'） 用途 ldhL1-F CATGGATCCATGCACATAAGAAAGGATGAT 扩增Lr-L-ldh1基因 ldhL1-R GCACTCGAGTTACTGACGTGTTTCGATGTC 扩增Lr-L-ldh1基因 ldhL2-F CATGGATCCATGCAACATAGCGGAAATATT 扩增Lr-L-ldh2基因 ldhL2-R GCACTCGAGCTAGGCTTCCTGTGCCTTCTT 扩增Lr-L-ldh2基因 
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表 2 Lr-L-LDH基本性质分析
Table 2. Analysis of the basic characteristics of the Lr-L-LDH
基本性质 Lr-L-LDH1 Lr-L-LDH2 氨基酸残基数量 335 312 分子质量（Da） 36608.80 33557.10 等电点 5.34 5.12 带负电荷氨基酸（Asp+Glu） 45 39 带正电荷氨基酸（Arg+Lys） 36 28 分子式 C1639H2613N433O500S7 C1487H2377N407O461S7 原子总数 5192 4739 消光系数（mol/cm） 32890 26025 不稳定指数 30.10 37.35 脂肪族指数 99.88 104.17 亲水性平均系数 -0.096 -0.020
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表 3 Lr-L-LDH信号肽预测
Table 3. Signal peptide prediction of Lr-L-LDH
蛋白类型 可能性 Lr-L-LDH1 Lr-L-LDH2 信号肽（Sec/SPI） 0.0116 0.1173 TAT信号肽（Tat/SPI） 0.0019 0.01 脂蛋白信号肽（Sec/SPII） 0.0009 0.039 其他 0.9856 0.8337
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表 4 二级结构中组件的比率
Table 4. The proportion of components in a secondary structure
二级结构 比率（%） Lr-L-LDH1 Lr-L-LDH2 α-螺旋 36.72 41.03 延伸链 22.99 20.19 β-转角 8.96 9.29 无规则卷曲 31.34 29.49
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