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酶催化混杂（enzyme promiscuity）是指一种酶除了主要反应外，还能偶然催化一种副反应。尽管酶是专一性强的催化剂，但它们除了具有主要的天然催化活性外，还经常会发生副反应。这些混杂的反应通常比主要反应慢，在中性选择下。尽管这些反应在生理上通常是不相关的，但在新的自然选择压力下，这些活动可能会带来适应效益，从而促使以前的混杂反应演变成新的主要反应。[1] 酶催化混杂的一个例子是来自假单胞菌属ADP的阿特拉津氯水解酶（编码atzA），其由三聚氰胺脱氨酶（编码triA）演化而来，其对人造化学品阿特拉津具有非常小的混杂活性。[2]

介绍

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酶进化为在特定底物上以高催化效率（kcat/KM，参见米-门二氏动力学）催化特定反应的催化剂。然而，除了这一主要活性外，它们还具有其他活性，这些活性通常要低几个数量级，并且不是进化选择的结果，因此不参与生物体的生理活动。[nb 1] 该现象允许新功能的获得，因为混杂的活性可以在新的选择压力下赋予适应效益，导致其作为新的主要活性的重复和选择。

酶的进化

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重复和专门化

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有几种理论模型可以预测重复和专门化事件的顺序，但实际过程更为复杂和模糊（§下文重构酶）。[3] 一方面，基因扩增导致酶浓度增加，并且可能不受限制性调节，因此增加了酶混杂活性的反应速率（v），使其作用在生理上更加明显（“基因剂量效应”）。[4] 另一方面，酶可能会增加次要活性，而几乎没有损失主要活性（“稳健性”），适应性冲突也很小（下文的稳健性和可塑性）。[5]

稳健性（robustness）和可塑性（plasticity）

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对4种不同水解酶（人血清对氧磷酶（PON1）、假单胞菌磷酸三酯酶（PTE）、蛋白酪氨酸磷酸酶（PTP）和人碳酸酐酶II（CAII））的研究表明，其主要活性对变化具有“稳健性”，而混杂活性较弱，更具“可塑性”。具体来说，选择一个非主要反应（通过定向进化），最初不会减弱主要反应（因此它具有稳健性），但会极大地影响未选择的反应（因此它们具有可塑性）。[5]

来自微小假单胞菌（Pseudomonas diminuta）的磷酸三酯酶（PTE）在十八轮中进化为芳基酯酶（P-O到 C-O水解酶），特异性（KM比值）发生了109次变化，但大部分变化发生在初始阶段，保留了未经选择的残留PTE活性，进化出的芳基酯酶活性增加，对于残留PTE活性的丧失与芳基酯酶活性的提高有一点权衡。[6]

这意味着根据IAD模型，进化时，专门的酶（功能单一）经历了通用（功能多样）的阶段，然后又可能在基因重复后再次成为专门的酶，而混杂活动比主要活动更具可塑性。

重构酶（reconstructed enzymes）

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酶进化的最新、最清晰易懂的例子是过去60年中生物修复酶技术的兴起。其中氨基酸变化的数量非常少，为研究自然界中的酶进化提供了一个极好的模型。但通过使用现存的酶来确定酶家族是如何进化拥有缺点，就是将新进化的酶和同源酶相比较时不知道两个基因分化之前祖先的真实身份。由于祖先重构，这个问题可以得到解决。祖先重建是由莱纳斯·鲍林（Linus Pauling）和埃米尔·扎克坎德尔（Emile Zuckerkandl）于1963年首次提出的，是从一组基因的祖先形式推断和合成一个基因The most recent and most clear cut example of enzyme evolution is the rise of bioremediating enzymes in the past 60 years. Due to the very low number of amino acid changes, these provide an excellent model to investigate enzyme evolution in nature. However, using extant enzymes to determine how the family of enzymes evolved has the drawback that the newly evolved enzyme is compared to paralogues without knowing the true identity of the ancestor before the two genes diverged. This issue can be resolved thanks to ancestral reconstruction. First proposed in 1963 by Linus Pauling and Emile Zuckerkandl, ancestral reconstruction is the inference and synthesis of a gene from the ancestral form of a group of genes,[7] which has had a recent revival thanks to improved inference techniques[8] and low-cost artificial gene synthesis,[9] resulting in several ancestral enzymes—dubbed "stemzymes" by some[10]—to be studied.[11]

Evidence gained from reconstructed enzyme suggests that the order of the events where the novel activity is improved and the gene is duplication is not clear cut, unlike what the theoretical models of gene evolution suggest.

One study showed that the ancestral gene of the immune defence protease family in mammals had a broader specificity and a higher catalytic efficiency than the contemporary family of paralogues,[10] whereas another study showed that the ancestral steroid receptor of vertebrates was an oestrogen receptor with slight substrate ambiguity for other hormones—indicating that these probably were not synthesised at the time.[12]

This variability in ancestral specificity has not only been observed between different genes, but also within the same gene family. In light of the large number of paralogous fungal α-glucosidase genes with a number of specific maltose-like (maltose, turanose, maltotriose, maltulose and sucrose) and isomaltose-like (isomaltose and palatinose) substrates, a study reconstructed all key ancestors and found that the last common ancestor of the paralogues was mainly active on maltose-like substrates with only trace activity for isomaltose-like sugars, despite leading to a lineage of iso-maltose glucosidases and a lineage that further split into maltose glucosidases and iso-maltose glucosidases. Antithetically, the ancestor before the latter split had a more pronounced isomaltose-like glucosidase activity.[3]

原始代谢（Primordial metabolism）

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Roy Jensen in 1976 theorised that primordial enzymes had to be highly promiscuous in order for metabolic networks to assemble in a patchwork fashion (hence its name, the patchwork model). This primordial catalytic versatility was later lost in favour of highly catalytic specialised orthologous enzymes.[13] As a consequence, many central-metabolic enzymes have structural homologues that diverged before the last universal common ancestor.[14]

Distribution

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Promiscuity is however not only a primordial trait, in fact it is very widespread property in modern genomes. A series of experiments have been conducted to assess the distribution of promiscuous enzyme activities in E. coli. In E. coli 21 out of 104 single-gene knockouts tested (from the Keio collection[15]) could be rescued by overexpressing a noncognate E. coli protein (using a pooled set of plasmids of the ASKA collection[16]). The mechanisms by which the noncognate ORF could rescue the knockout can be grouped into eight categories: isozyme overexpression (homologues), substrate ambiguity, transport ambiguity (scavenging), catalytic promiscuity, metabolic flux maintenance (including overexpression of the large component of a synthase in the absence of the amine transferase subunit), pathway bypass, regulatory effects and unknown mechanisms.[4] Similarly, overexpressing the ORF collection allowed E. coli to gain over an order of magnitude in resistance in 86 out 237 toxic environment.[17]

同源

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Homologues are sometimes known to display promiscuity towards each other's main reactions.[18] This crosswise promiscuity has been most studied with members of the alkaline phosphatase superfamily, which catalyse hydrolytic reaction on the sulfate, phosphonate, monophosphate, diphosphate or triphosphate ester bond of several compounds.[19] Despite the divergence the homologues have a varying degree of reciprocal promiscuity: the differences in promiscuity are due to mechanisms involved, particularly the intermediate required.[19]

Degree of promiscuity

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Enzymes are generally in a state that is not only a compromise between stability and catalytic efficiency, but also for specificity and evolvability, the latter two dictating whether an enzyme is a generalist (highly evolvable due to large promiscuity, but low main activity) or a specialist (high main activity, poorly evolvable due to low promiscuity).[20] Examples of these are enzymes for primary and secondary metabolism in plants (§ Plant secondary metabolism below). Other factors can come into play, for example the glycerophosphodiesterase (gpdQ) from Enterobacter aerogenes shows different values for its promiscuous activities depending on the two metal ions it binds, which is dictated by ion availability.[21] In some cases promiscuity can be increased by relaxing the specificity of the active site by enlarging it with a single mutation as was the case of a D297G mutant of the E. coli L-Ala-D/L-Glu epimerase (ycjG) and E323G mutant of a pseudomonad muconate lactonizing enzyme II, allowing them to promiscuously catalyse the activity of O-succinylbenzoate synthase (menC).[22] Conversely, promiscuity can be decreased as was the case of γ-humulene synthase (a sesquiterpene synthase) from Abies grandis that is known to produce 52 different sesquiterpenes from farnesyl diphosphate upon several mutations.[23]

Studies on enzymes with broad-specificity—not promiscuous, but conceptually close—such as mammalian trypsin and chymotrypsin, and the bifunctional isopropylmalate isomerase/homoaconitase from Pyrococcus horikoshii have revealed that active site loop mobility contributes substantially to the catalytic elasticity of the enzyme.[24][25]

Toxicity

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A promiscuous activity is a non-native activity the enzyme did not evolve to do, but arises due to an accommodating conformation of the active site. However, the main activity of the enzyme is a result not only of selection towards a high catalytic rate towards a particular substrate to produce a particular product, but also to avoid the production of toxic or unnecessary products.[1] For example, if a tRNA syntheses loaded an incorrect amino acid onto a tRNA, the resulting peptide would have unexpectedly altered properties, consequently to enhance fidelity several additional domains are present.[26] Similar in reaction to tRNA syntheses, the first subunit of tyrocidine synthetase (tyrA) from Bacillus brevis adenylates a molecule of phenylalanine in order to use the adenyl moiety as a handle to produce tyrocidine, a cyclic non-ribosomal peptide. When the specificity of enzyme was probed, it was found that it was highly selective against natural amino acids that were not phenylalanine, but was much more tolerant towards unnatural amino acids.[27] Specifically, most amino acids were not catalysed, whereas the next most catalysed native amino acid was the structurally similar tyrosine, but at a thousandth as much as phenylalanine, whereas several unnatural amino acids where catalysed better than tyrosine, namely D-phenylalanine, β-cyclohexyl-L-alanine, 4-amino-L-phenylalanine and L-norleucine.[27]

One peculiar case of selected secondary activity are polymerases and restriction endonucleases, where incorrect activity is actually a result of a compromise between fidelity and evolvability. For example, for restriction endonucleases incorrect activity (star activity) is often lethal for the organism, but a small amount allows new functions to evolve against new pathogens.[28]

Plant secondary metabolism

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Anthocyanins (delphinidin pictured) confer plants, particularly their flowers, with a variety of colours to attract pollinators and a typical example of plant secondary metabolite.

Plants produce a large number of secondary metabolites thanks to enzymes that, unlike those involved in primary metabolism, are less catalytically efficient but have a larger mechanistic elasticity (reaction types) and broader specificities. The liberal drift threshold (caused by the low selective pressure due the small population size) allows the fitness gain endowed by one of the products to maintain the other activities even though they may be physiologically useless.[29]

Biocatalysis

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主条目：biocatalysis

In biocatalysis, many reactions are sought that are absent in nature. To do this, enzymes with a small promiscuous activity towards the required reaction are identified and evolved via directed evolution or rational design.[30]

An example of a commonly evolved enzyme is ω-transaminase which can replace a ketone with a chiral amine[31] and consequently libraries of different homologues are commercially available for rapid biomining (eg. Codexis[32]).

Another example is the possibility of using the promiscuous activities of cysteine synthase (cysM) towards nucleophiles to produce non-proteinogenic amino acids.[33]

Reaction similarity

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Similarity between enzymatic reactions (EC) can be calculated by using bond changes, reaction centres or substructure metrics (EC-BLAST).[34]

Drugs and promiscuity

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Whereas promiscuity is mainly studied in terms of standard enzyme kinetics, drug binding and subsequent reaction is a promiscuous activity as the enzyme catalyses an inactivating reaction towards a novel substrate it did not evolve to catalyse.[5] This could be because of the demonstration that there are only a small number of distinct ligand binding pockets in proteins.

Mammalian xenobiotic metabolism, on the other hand, was evolved to have a broad specificity to oxidise, bind and eliminate foreign lipophilic compounds which may be toxic, such as plant alkaloids, so their ability to detoxify anthropogenic xenobiotics is an extension of this.[35]

参见

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脚注

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  1. ^ Most authors refer to as promiscuous activities the non-evolved activities and not secondary activities that have been evolved.[1] Consequently, glutathione S-transferases (GSTs) and cytochrome P450 monooxygenases (CYPs) are termed multispecific or broad-specificity enzymes.[1] The ability to catalyse different reactions is often termed catalytic promiscuity or reaction promiscuity, whereas the ability to act upon different substrates is called substrate promiscuity or substrate ambiguity. The term latent has different meanings depending on the author, namely either referring to a promiscuous activity that arises when one or two residues are mutated or simply as a synonym for promiscuous to avoid the latter term. Promiscuity here means muddledom, not lechery —the latter is a recently gained meaning of the word.[36]

引用

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  1. ^ 1.0 1.1 1.2 1.3 Khersonsky O, Tawfik DS. Enzyme promiscuity: a mechanistic and evolutionary perspective. Annual Review of Biochemistry. 2010, 79: 471–505. PMID 20235827. doi:10.1146/annurev-biochem-030409-143718. 
  2. ^ Scott C, Jackson CJ, Coppin CW, Mourant RG, Hilton ME, Sutherland TD, Russell RJ, Oakeshott JG. Catalytic improvement and evolution of atrazine chlorohydrolase. Applied and Environmental Microbiology. April 2009, 75 (7): 2184–91. PMC 2663207 可免费查阅. PMID 19201959. doi:10.1128/AEM.02634-08. 
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  12. ^ Thornton JW, Need E, Crews D. Resurrecting the ancestral steroid receptor: ancient origin of estrogen signaling. Science. September 2003, 301 (5640): 1714–7. Bibcode:2003Sci...301.1714T. PMID 14500980. doi:10.1126/science.1086185. 
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  15. ^ Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology. 2006, 2: 2006.0008. PMC 1681482 可免费查阅. PMID 16738554. doi:10.1038/msb4100050. 
  16. ^ Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H, Mori H. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Research. 2006, 12 (5): 291–9. PMID 16769691. doi:10.1093/dnares/dsi012. 
  17. ^ Soo VW, Hanson-Manful P, Patrick WM. Artificial gene amplification reveals an abundance of promiscuous resistance determinants in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America. January 2011, 108 (4): 1484–9. Bibcode:2011PNAS..108.1484S. PMC 3029738 可免费查阅. PMID 21173244. doi:10.1073/pnas.1012108108. 
  18. ^ O'Brien PJ, Herschlag D. Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of Escherichia coli alkaline phosphatase. Biochemistry. May 2001, 40 (19): 5691–9. CiteSeerX 10.1.1.322.8876 可免费查阅. PMID 11341834. doi:10.1021/bi0028892. 
  19. ^ 19.0 19.1 Zhao C, Kumada Y, Imanaka H, Imamura K, Nakanishi K. Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli. Protein Expression and Purification. June 2006, 47 (2): 607–13. PMID 16546401. doi:10.1016/j.pep.2006.01.002. 
  20. ^ Tokuriki N, Tawfik DS. Stability effects of mutations and protein evolvability. Current Opinion in Structural Biology. October 2009, 19 (5): 596–604. PMID 19765975. doi:10.1016/j.sbi.2009.08.003. 
  21. ^ Daumann LJ, McCarthy BY, Hadler KS, Murray TP, Gahan LR, Larrabee JA, Ollis DL, Schenk G. Promiscuity comes at a price: catalytic versatility vs efficiency in different metal ion derivatives of the potential bioremediator GpdQ. Biochimica et Biophysica Acta. January 2013, 1834 (1): 425–32. PMID 22366468. doi:10.1016/j.bbapap.2012.02.004. 
  22. ^ Schmidt DM, Mundorff EC, Dojka M, Bermudez E, Ness JE, Govindarajan S, Babbitt PC, Minshull J, Gerlt JA. Evolutionary potential of (beta/alpha)8-barrels: functional promiscuity produced by single substitutions in the enolase superfamily. Biochemistry. July 2003, 42 (28): 8387–93. PMID 12859183. doi:10.1021/bi034769a. 
  23. ^ Yoshikuni Y, Ferrin TE, Keasling JD. Designed divergent evolution of enzyme function. Nature. April 2006, 440 (7087): 1078–82. Bibcode:2006Natur.440.1078Y. PMID 16495946. doi:10.1038/nature04607. 
  24. ^ Ma W, Tang C, Lai L. Specificity of trypsin and chymotrypsin: loop-motion-controlled dynamic correlation as a determinant. Biophysical Journal. August 2005, 89 (2): 1183–93. Bibcode:2005BpJ....89.1183M. PMC 1366603 可免费查阅. PMID 15923233. arXiv:q-bio/0505037 可免费查阅. doi:10.1529/biophysj.104.057158. 
  25. ^ Yasutake Y, Yao M, Sakai N, Kirita T, Tanaka I. Crystal structure of the Pyrococcus horikoshii isopropylmalate isomerase small subunit provides insight into the dual substrate specificity of the enzyme. Journal of Molecular Biology. November 2004, 344 (2): 325–33. PMID 15522288. doi:10.1016/j.jmb.2004.09.035. 
  26. ^ Perona JJ, Hadd A. Structural diversity and protein engineering of the aminoacyl-tRNA synthetases. Biochemistry. November 2012, 51 (44): 8705–29. PMID 23075299. doi:10.1021/bi301180x. 
  27. ^ 27.0 27.1 Villiers BR, Hollfelder F. Mapping the limits of substrate specificity of the adenylation domain of TycA. ChemBioChem. March 2009, 10 (4): 671–82. PMID 19189362. doi:10.1002/cbic.200800553. 
  28. ^ Vasu K, Nagamalleswari E, Nagaraja V. Promiscuous restriction is a cellular defense strategy that confers fitness advantage to bacteria. Proceedings of the National Academy of Sciences of the United States of America. May 2012, 109 (20): E1287–93. Bibcode:2012PNAS..109E1287V. PMC 3356625 可免费查阅. PMID 22509013. doi:10.1073/pnas.1119226109. 
  29. ^ Weng JK, Philippe RN, Noel JP. The rise of chemodiversity in plants. Science. June 2012, 336 (6089): 1667–70. Bibcode:2012Sci...336.1667W. PMID 22745420. doi:10.1126/science.1217411. 
  30. ^ Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K. Engineering the third wave of biocatalysis. Nature. May 2012, 485 (7397): 185–94. Bibcode:2012Natur.485..185B. PMID 22575958. doi:10.1038/nature11117. 
  31. ^ Shin JS, Kim BG. Comparison of the omega-transaminases from different microorganisms and application to production of chiral amines. Bioscience, Biotechnology, and Biochemistry. August 2001, 65 (8): 1782–8. PMID 11577718. doi:10.1271/bbb.65.1782. 
  32. ^ http://www.codexis.com/pdf/Codexis_EnzymePlatforms.pdf[永久失效連結]
  33. ^ Maier TH. Semisynthetic production of unnatural L-alpha-amino acids by metabolic engineering of the cysteine-biosynthetic pathway. Nature Biotechnology. April 2003, 21 (4): 422–7. PMID 12640465. doi:10.1038/nbt807. 
  34. ^ Rahman SA, Cuesta SM, Furnham N, Holliday GL, Thornton JM. EC-BLAST: a tool to automatically search and compare enzyme reactions. Nature Methods. February 2014, 11 (2): 171–4. PMC 4122987 可免费查阅. PMID 24412978. doi:10.1038/nmeth.2803. 
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  36. ^ promiscuity. 牛津英語詞典 (第三版). 牛津大學出版社. 2005-09 （英语）. 
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