定义底物特异性的脂肪酶和磷脂酶考生
Summary
Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.
Abstract
微生物产生的那些，以使可用于生物体外部基板分泌（磷）的脂酶广泛光谱。另外，其他（磷酸）脂肪酶可以在物理上与生产菌引起的内在脂质的营业额，并经常引起细胞膜的重塑有关。虽然势（磷）的脂肪酶可以用数目的算法时该基因/蛋白质序列可用来预测，经常没有获得的酶的活性，底物特异性，和潜在的生理功能的实验证据。这个手稿描述的测定条件与未知底物特异性，以及如何利用这些优化条件下在搜索用于相应（磷）的脂肪酶的天然底物准（磷）的脂肪酶的最优化。采用人工发色底物，如对硝基苯衍生物，可有助于检测小为在标准条件下的预测（磷）的脂肪酶的酶活性。具有遇到这样的次要的酶活性，酶测定法的不同参数可以在为了获得人造基底的更有效的水解而变化。已确定在其下的酶工作良好的条件之后，各种潜在天然底物应测定其降解，其可以采用不同的层析方法进行随后的处理。底物特异性为新酶的定义，通常提供假设这些酶，然后可通过实验测试的潜在生理作用。遵循这些指导原则，我们能够确定降解磷脂酰胆碱磷酸胆碱到甘油二酯和，在膜中生长时的磷限制条件细菌根瘤菌重塑的关键一步磷脂酶C（SMc00171）。两个预测patatin-就像有机体一样的磷脂酶（SMc00930和SMc01003），我们可以重新确定自己的底物特异性和澄清SMc01003是甘油二酯脂肪酶。
Introduction
基于甘油脂质，如三酰甘油和（甘油）磷脂构成了重要的可能是最知名的类脂质1。三酰基甘油（变量）的脂肪或油，其通常用作存储脂类，因此作为潜在的能量和碳的来源。标签可以通过脂肪酶，这是经常被生产菌分泌的消化外部变量并使它们可作为碳源退化。另外，脂肪酶已经被广泛地研究，多年来，由于其重要的生物技术应用2。
由于它们的两亲性质及其近圆筒状的形状，（甘油）磷脂展览成膜性，通常构成了双层膜3的主要类脂组分。在简单的微生物，如细菌大肠杆菌中，只有三个主要头组的变体，磷脂酰甘油（PG），心磷脂（CL），和phosphatidylethanolamine（PE）遇到，尽管人们应该知道，他们中的每一个都可以用在sn相当数量的不同脂肪酰基链的-1或SN -2位置引起大量不同分子种类4的被取代。其他细菌可能具有除了或代替其他磷脂。例如， 苜蓿中华根瘤菌 ，土壤细菌，其能够形成与豆科苜蓿（ 紫花苜蓿 ）一个固氮根瘤共生，包含除了PE的第二两性离子磷脂，磷脂酰胆碱（PC），5。另外，不含有脂质磷或甘油可能是细胞膜的两亲性并形成部分。例如，在磷限制生长条件下，在S根瘤菌 ，（甘油）磷脂主要由膜脂质不包含磷， 即，硫脂，鸟氨酸脂质和diacylglyceryl trimethylho取代moserine（DGTS）6。在细菌中，DGTS从二酰甘油（DAG）在一个两步通路7形成，但是对于DAG生成源不明确。脉冲追踪实验表明个人电脑可能是DGTS 8的前体，并使用在这个手稿，我们可以识别磷脂酶C（PLCP，SMc00171）中描述的方法，该方法是磷限制性的条件下形成并且可PC转换为DAG和磷酸8。
在另一项研究中，我们发现，酰基-CoA合成酶（FADD）S的缺陷型突变体进入生长9的固定相时的根瘤菌或大肠杆菌积累游离脂肪酸。尽管这些脂肪酸似乎是从膜脂质衍生自游离脂肪酸或解放出来的酶的确切来源没有已知的。再次，采用的策略在这个手稿概述，二patatin启动类10（磷）的脂肪酶（SMc00930和SMc01003），该对S中形成的游离脂肪酸贡献根瘤菌 11进行了预测。令人惊讶地，SMc01003使用的DAG作为底物将其转换为单酰甘油和最后甘油和游离脂肪酸11。因此，SMc01003是DAG脂肪酶（DGLA）。
虽然用于预测潜在（磷）存在一些算法脂酶12,13，它们的精确的功能和生理作用通常是不知道的。在这里，我们勾勒出一个协议，克隆和过度预测的或潜在的（磷）的脂肪酶。这份手稿解释酶检测如何开发和利用人工色底物的过度表达（磷）脂肪酶进行了优化。我们提供的例子，这些研究结果可能如何与优化的酶测定可遇到的实际（磷酸），酶底物，以及如何丰富我们的微生物生理学的理解。
Protocol
Representative Results
Discussion
在过去的20年中，许多生物基因组研究已经被测序，虽然丰富的基因组序列数据已经产生，功能解释相对滞后，因此阻碍了我们的基因组功能的认识。在基因组中的基因功能的基础上相似已知功能或保守的基序的发生基因经常被分配。然而，给定基因的精确功能往往不是公知的。尤其，预计酶的结构基因不能由组学技术容易地探索由于这样的事实，大部分酶催化涉及两个基板和两个产品的复杂的?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由（在InvestigaciónCientíficaBASICA在Investigación连接国界德拉西恩西亚82614，153998，253549和178359以及118），并从提高妇女地位总署德从理事会全国国立科学城ŸTECNOLOGIA – 墨西哥（国家科学技术委员会和墨西哥）资助项目Asuntos去个人Académico-国立自治大学墨西哥（DGAPA，墨西哥国立自治大学; PAPIIT IN202616，IN203612）。
Materials
| Chloroform | JT Baker | 9180-03 | TLC analysis & Lipid extraction |
| Methanol | JT Baker | 9070-03 | TLC analysis & Lipid extraction |
| Acetic Acid | JT Baker | 9507-05 | TLC analysis & Lipid extraction |
| Hexanes | JT Baker | 9309-02 | TLC analysis & Lipid extraction |
| Diethylether | Sigma | 32203 | Enzymatic assays |
| bidistilled water | ANY | NA | Enzymatic assays |
| Tris Base | Sigma | T-1503 | Enzymatic assays |
| HCl | Baker | 9535-02 | Enzymatic assays |
| NaCl | Baker | 3624-01 | Enzymatic assays |
| Triton X-100 | Sigma | X-100 | Enzymatic assays |
| LB broth | ANY | NA | Bacterial growth, 10g tryptone + 5g yeast extract + 10g NaCl per liter of bidistilled water |
| tryptone | Becton Dickinson and Company | 211705 | Bacterial growth |
| yeast extract | Becton Dickinson and Company | 212750 | Bacterial growth |
| TY broth | ANY | NA | Bacterial growth, 8g tryptone + 3g yeast extract + 66mg CaCl2 2 H20 per liter of bidistilled water |
| CaCl2 2 H2O | Baker | 1332-01 | Enzymatic assays |
| isopropyl-β-D-thiogalactoside (IPTG) | Invitrogen | 15529-019 | Bacterial growth |
| Diethanolamine | Sigma | D-8885 | Enzymatic assays |
| MnCl2 | Sigma | 221279 | Enzymatic assays |
| Phospholipase A2 snake venom | Sigma | P0790 | Enzymatic assays |
| Phospholipase C Clostridium perfingrens | Sigma | P7633 | Enzymatic assays |
| Bis-p-nitrophenyl phosphate | Sigma | 07422AH | Enzymatic assays |
| p-nitrophenyl stearate | Sigma | N3627 | Enzymatic assays |
| p-nitrophenyl dodecanoate | Sigma | 61716 | Enzymatic assays |
| p-nitrophenyl decanoate | Sigma | N0252 | Enzymatic assays |
| p-nitrophenyl palmitate | Sigma | N2752 | Enzymatic assays |
| p-nitrophenyl butyrate | Sigma | N9876 | Enzymatic assays |
| p-nitrophenyl octanoate | Sigma | 21742 | Enzymatic assays |
| Acetic Acid, sodium salt [1-14C] | Perkin Elmer | NEC084 | Bacterial growth |
| dimethylsulfoxide (DMSO) | JT Baker | 9224-01 | Enzymatic assays |
| Aluminium HPTLC silica gel 60 plates. Silica gel HPTLC plates size 20 x 20 cm, 25 sheets. | Merck | 105547 | TLC analysis & Lipid extraction |
| Spectrometer UV/VIS Lambda 35 | Perkin Elmer | NA | Enzymatic assays |
| Storm 820 Phosphorimager | Molecular Dynamics | NA | Photostimulable Luminescence scanner |
| Multipurpose Scintillation Counter | Beckman Coulter | NA | Radioactivity Quantification |
| French Pressure Cell | ThermoSpectronic | NA | Breakage of cells |
| chromatography paper 3MM Chr | Whatman | 3030917 | TLC analysis |
| Sinorhizobium meliloti 1021our | reference 34 | studied strain | |
| Escherichia coli BL21 (DE3) pLysS Competent cells | Novagen | 69451 | protein expression strain |
| pET9a vector | Novagen | 69431 | protein expression vector |
| pET17b vector | Novagen | 69663 | protein expression vector |
| sterile polystyrene round-bottom tube (14 ml) Falcon | Becton Dickinson | 352057 | radiolabeling of bacterial cultures |
| polypropylene microcentrifuge tubes (1.5 ml) | Eppendorf | 30125.15 | Enzymatic assays |
| 1,2-dipalmitoyl-sn-glycerol | Sigma | D9135 | lipid standard |
| L-α-phosphatidylcholine, dipalmitoyl | Sigma | P6267 | lipid standard |
| DL-α-monopalmitin | Sigma | M1640 | lipid standard |
| palmitic acid | Sigma | P0500 | lipid standard |
Referências
- Nelson, D. L., Cox, M. M. . Lehninger, Principles of Biochemistry. , (2013).
- Jaeger, K. E., Eggert, T. Lipases for biotechnology. Curr. Opin. Biotechnol. 13 (4), 390-397 (2002).
- Dowhan, W., Bogdanov, M., Mileykovskaya, E. Functional roles of lipids in membranes. Biochemistry of Lipids, Lipoproteins and Membranes. , 1-37 (2008).
- Dowhan, W. Molecular basis for membrane phospholipid diversity: why are there so many lipids?. Annu. Rev. Biochem. 66, 199-232 (1997).
- De Rudder, K. E. E., Thomas-Oates, J. E., Geiger, O. Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine. J. Bacteriol. 179, 6921-6928 (1997).
- Geiger, O., Röhrs, V., Weissenmayer, B., Finan, T. M., Thomas-Oates, J. E. The regulator gene phoB mediates phosphate stress-controlled synthesis of the membrane lipid diacylglyceryl-N,N,N-trimethylhomoserine in Rhizobium (Sinorhizobium) meliloti. Mol. Microbiol. 32 (1), 63-73 (1999).
- Klug, R. M., Benning, C. Two enzymes of diacylglyceryl-O-4′-(N,N,N,-trimethyl)homoserine biosynthesis are encoded by btaA and btaB in the purple bacterium Rhodobacter sphaeroides. Proc. Natl. Acad. Sci. USA. 98 (10), 5910-5915 (2001).
- Zavaleta-Pastor, M., et al. Sinorhizobium meliloti phospholipase C required for lipid remodeling duringphosphorus limitation. Proc. Natl. Acad. Sci. USA. 107 (1), 302-307 (2010).
- Pech-Canul, A., et al. FadD is required for utilization of endogenous fatty acids released from membrane lipids. J. Bacteriol. 193 (22), 6295-6304 (2011).
- Banerji, S., Flieger, A. Patatin-like proteins: a new family of lipolytic enzymes present in bacteria?. Microbiology. 150 (Pt 3), 522-525 (2004).
- Sahonero-Canavesi, D. X., et al. Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase. Environ. Microbiol. 17 (9), 3391-3406 (2015).
- Fischer, M., Pleiss, J. The Lipase Engineering Database: a navigation and analysis tool for protein families. Nucl. Acid. Res. 31 (1), 319-321 (2003).
- Sigrist, C. J. A., et al. New and continuing developments at PROSITE. Nucleic Acids Res. 41 (Database issue), D344-D347 (2013).
- Lorenz, T. C. Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies. J. Vis. Exp. (63), e3998 (2012).
- Untergasser, A., et al. Primer3 – new capabilities and interfaces. Nucl. Acids Res. 40 (15), e115 (2012).
- Studier, F. W., Rosenberg, A. H., Dunn, J. J., Dubendorff, J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60-89 (1990).
- Miller, J. H. . Experiments in Molecular Genetics. , (1972).
- Desjardins, P., Hansen, J. B., Allen, M. Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer. J. Vis. Exp. (33), e1610 (2009).
- Bligh, E. G., Dyer, W. J. A rapid method for total lipid extraction and purification. Can. J. Biochem. Physiol. 37 (8), 911-917 (1959).
- Molecular Dynamics. . Phosphorimager SI User´s Guide. , (1994).
- Dixon, M., Webb, E. . Enzymes: Third Edition. , (1979).
- Rudolph, A. E., et al. Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member. J. Biol. Chem. 274 (17), 11824-11831 (1999).
- Kato, S., Yoshimura, T., Hemmi, H., Moriyama, R. Biochemical analysis of a novel lipolytic enzyme YvdO from Bacillus subtilis. Biosci. Biotechnol. Biochem. 74 (4), 701-706 (2010).
- Peppelenbosch, M. P. Kinome profiling. Scientifica (Cairo). , (2012).
- Manafi, M., Kneifel, W., Bascomb, S. Fluorogenic and chromogenic substrates used in bacterial diagnostics. Microbiol. Rev. 55 (3), 335-348 (1991).
- Kuznetsova, E., et al. Enzyme genomics: Application of general enzymatic screens to discover new enzymes. FEMS Microbiol. Rev. 29 (2), 263-279 (2005).
- Scopes, R. K. . Protein Purification, Principles and Practice. , (2010).
- Geiger, O., Lopez-Lara, I. M., Sohlenkamp, C. Phosphatidylcholine biosynthesis and function in bacteria. Biochim. Biophys. Acta. 1831 (3), 503-513 (2013).
