Enzyme promiscuity

Enzymes are evolved to catalyze a particular reaction on a particular substrate with high catalytic efficiency (k_cat/K_M, cf. Michaelis–Menten kinetics). However, in addition to this main activity, they possess other activities that are generally several orders of magnitude lower, and that are not a result of evolutionary selection and therefore do not partake in the physiology of the organism.

This phenomenon allows new functions to be gained as the promiscuous activity could confer a fitness benefit under a new selective pressure leading to its duplication and selection as a new main activity.

Several theoretical models exist to predict the order of duplication and specialisation events, but the actual process is more intertwined and fuzzy (§ Reconstructed enzymes below).{{cite journal | vauthors = Voordeckers K, Brown CA, Vanneste K, van der Zande E, Voet A, Maere S, Verstrepen KJ | title = Reconstruction of ancestral metabolic enzymes reveals molecular mechanisms underlying evolutionary innovation through gene duplication | journal = PLOS Biology | volume = 10 | issue = 12 | pages = e1001446 | year = 2012 | pmid = 23239941 | pmc = 3519909 | doi = 10.1371/journal.pbio.1001446 | editor1-last = Thornton | editor1-first = Joseph W | doi-access = free }} On one hand, gene amplification results in an increase in enzyme concentration, and potentially freedom from a restrictive regulation, therefore increasing the reaction rate (v) of the promiscuous activity of the enzyme making its effects more pronounced physiologically ("gene dosage effect").{{cite journal | vauthors = Patrick WM, Quandt EM, Swartzlander DB, Matsumura I | title = Multicopy suppression underpins metabolic evolvability | journal = Molecular Biology and Evolution | volume = 24 | issue = 12 | pages = 2716–22 | date = December 2007 | pmid = 17884825 | pmc = 2678898 | doi = 10.1093/molbev/msm204 }} On the other, enzymes may evolve an increased secondary activity with little loss to the primary activity ("robustness") with little adaptive conflict (§ Robustness and plasticity below).{{cite journal | vauthors = Aharoni A, Gaidukov L, Khersonsky O, McQ Gould S, Roodveldt C, Tawfik DS | title = The 'evolvability' of promiscuous protein functions | journal = Nature Genetics | volume = 37 | issue = 1 | pages = 73–6 | date = January 2005 | pmid = 15568024 | doi = 10.1038/ng1482 | s2cid = 8245673 }}

A study of four distinct hydrolases (human serum paraoxonase (PON1), pseudomonads phosphotriesterase (PTE), Protein tyrosine phosphatase(PTP) and human carbonic anhydrase II (CAII)) has shown the main activity is "robust" towards change, whereas the promiscuous activities are weak and more "plastic". Specifically, selecting for an activity that is not the main activity (via directed evolution), does not initially diminish the main activity (hence its robustness), but greatly affects the non-selected activities (hence their plasticity).

The phosphotriesterase (PTE) from Pseudomonas diminuta was evolved to become an arylesterase (P–O to C–O hydrolase) in eighteen rounds gaining a 10⁹ shift in specificity (ratio of K_M), however most of the change occurred in the initial rounds, where the unselected vestigial PTE activity was retained and the evolved arylesterase activity grew, while in the latter rounds there was a little trade-off for the loss of the vestigial PTE activity in favour of the arylesterase activity.{{cite journal | vauthors = Tokuriki N, Jackson CJ, Afriat-Jurnou L, Wyganowski KT, Tang R, Tawfik DS | title = Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme | journal = Nature Communications | volume = 3 | pages = 1257 | year = 2012 | pmid = 23212386 | doi = 10.1038/ncomms2246 | bibcode = 2012NatCo...3.1257T | doi-access = free | hdl = 1885/69835 | hdl-access = free }}

This means firstly that a specialist enzyme (monofunctional) when evolved goes through a generalist stage (multifunctional), before becoming a specialist again—presumably after gene duplication according to the IAD model—and secondly that promiscuous activities are more plastic than the main activity.

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,Pauling, L. and E. Zuckerkandl, Chemical Paleogenetics Molecular Restoration Studies of Extinct Forms of Life. Acta Chemica Scandinavica, 1963. 17: p. 9-&. which has had a recent revival thanks to improved inference techniques{{cite journal | vauthors = Williams PD, Pollock DD, Blackburne BP, Goldstein RA | title = Assessing the accuracy of ancestral protein reconstruction methods | journal = PLOS Computational Biology | volume = 2 | issue = 6 | pages = e69 | date = June 2006 | pmid = 16789817 | pmc = 1480538 | doi = 10.1371/journal.pcbi.0020069 | bibcode = 2006PLSCB...2...69W | doi-access = free }} and low-cost artificial gene synthesis,{{cite journal | vauthors = Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL | title = Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides | journal = Gene | volume = 164 | issue = 1 | pages = 49–53 | date = October 1995 | pmid = 7590320 | doi = 10.1016/0378-1119(95)00511-4 }} resulting in several ancestral enzymes—dubbed "stemzymes" by some{{cite journal | vauthors = Wouters MA, Liu K, Riek P, Husain A | title = A despecialization step underlying evolution of a family of serine proteases | journal = Molecular Cell | volume = 12 | issue = 2 | pages = 343–54 | date = August 2003 | pmid = 14536074 | doi = 10.1016/s1097-2765(03)00308-3 | doi-access = free | hdl = 10536/DRO/DU:30038994 | hdl-access = free }}—to be studied.{{cite journal | vauthors = Thornton JW | title = Resurrecting ancient genes: experimental analysis of extinct molecules | journal = Nature Reviews Genetics | volume = 5 | issue = 5 | pages = 366–75 | date = May 2004 | pmid = 15143319 | doi = 10.1038/nrg1324 | s2cid = 205482979 | url = http://pages.uoregon.edu/joet/thornton-NRG-2004.pdf | url-status = live | archive-url = https://web.archive.org/web/20120327220730/http://pages.uoregon.edu/joet/thornton-NRG-2004.pdf | archive-date = 2012-03-27 }}

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, 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.{{cite journal | vauthors = Thornton JW, Need E, Crews D | title = Resurrecting the ancestral steroid receptor: ancient origin of estrogen signaling | journal = Science | volume = 301 | issue = 5640 | pages = 1714–7 | date = September 2003 | pmid = 14500980 | doi = 10.1126/science.1086185 | bibcode = 2003Sci...301.1714T | s2cid = 37628350 }}

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.

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.{{cite journal | vauthors = Jensen RA | title = Enzyme recruitment in evolution of new function | journal = Annual Review of Microbiology | volume = 30 | pages = 409–25 | year = 1976 | pmid = 791073 | doi = 10.1146/annurev.mi.30.100176.002205 }} As a consequence, many central-metabolic enzymes have structural homologues that diverged before the last universal common ancestor.{{cite journal | vauthors = Fondi M, Brilli M, Emiliani G, Paffetti D, Fani R | title = The primordial metabolism: an ancestral interconnection between leucine, arginine, and lysine biosynthesis | journal = BMC Evolutionary Biology | volume = 7 | pages = S3 | year = 2007 | issue = Suppl 2 | pmid = 17767731| pmc = 1963480| doi = 10.1186/1471-2148-7-S2-S3 | doi-access = free | bibcode = 2007BMCEE...7S...3F }}

Promiscuity is not only a first trait, but also a 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{{cite journal | vauthors = Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H | title = Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection | journal = Molecular Systems Biology | volume = 2 | pages = 2006.0008 | year = 2006 | pmid = 16738554 | pmc = 1681482 | doi = 10.1038/msb4100050 }}) could be rescued by overexpressing a noncognate E. coli protein (using a pooled set of plasmids of the ASKA collection{{cite journal | vauthors = Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H, Mori H | title = 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 | journal = DNA Research | volume = 12 | issue = 5 | pages = 291–9 | year = 2006 | pmid = 16769691 | doi = 10.1093/dnares/dsi012 | doi-access = free }}). 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. Similarly, overexpressing the ORF collection allowed E. coli to gain over an order of magnitude in resistance in 86 out 237 toxic environment.{{cite journal | vauthors = Soo VW, Hanson-Manful P, Patrick WM | title = Artificial gene amplification reveals an abundance of promiscuous resistance determinants in Escherichia coli | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 108 | issue = 4 | pages = 1484–9 | date = January 2011 | pmid = 21173244| pmc = 3029738| doi = 10.1073/pnas.1012108108 | bibcode = 2011PNAS..108.1484S | doi-access = free }}

Homologues are sometimes known to display promiscuity towards each other's main reactions.{{cite journal | vauthors = O'Brien PJ, Herschlag D | title = Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of Escherichia coli alkaline phosphatase | journal = Biochemistry | volume = 40 | issue = 19 | pages = 5691–9 | date = May 2001 | pmid = 11341834 | doi = 10.1021/bi0028892 | citeseerx = 10.1.1.322.8876 }}

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.{{cite journal | vauthors = Zhao C, Kumada Y, Imanaka H, Imamura K, Nakanishi K | title = Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli | journal = Protein Expression and Purification | volume = 47 | issue = 2 | pages = 607–13 | date = June 2006 | pmid = 16546401 | doi = 10.1016/j.pep.2006.01.002 }} Despite the separation the homologues have a varying degree of reciprocal promiscuity: the differences in promiscuity are due to mechanisms involved, particularly the intermediate required.

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).{{cite journal | vauthors = Tokuriki N, Tawfik DS | title = Stability effects of mutations and protein evolvability | journal = Current Opinion in Structural Biology | volume = 19 | issue = 5 | pages = 596–604 | date = October 2009 | pmid = 19765975 | doi = 10.1016/j.sbi.2009.08.003 }} 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.{{cite journal | vauthors = Daumann LJ, McCarthy BY, Hadler KS, Murray TP, Gahan LR, Larrabee JA, Ollis DL, Schenk G | title = Promiscuity comes at a price: catalytic versatility vs efficiency in different metal ion derivatives of the potential bioremediator GpdQ | journal = Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics | volume = 1834 | issue = 1 | pages = 425–32 | date = January 2013 | pmid = 22366468 | doi = 10.1016/j.bbapap.2012.02.004 }}

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).{{cite journal | vauthors = Schmidt DM, Mundorff EC, Dojka M, Bermudez E, Ness JE, Govindarajan S, Babbitt PC, Minshull J, Gerlt JA | title = Evolutionary potential of (beta/alpha)8-barrels: functional promiscuity produced by single substitutions in the enolase superfamily | journal = Biochemistry | volume = 42 | issue = 28 | pages = 8387–93 | date = July 2003 | pmid = 12859183 | doi = 10.1021/bi034769a }} 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.{{cite journal | vauthors = Yoshikuni Y, Ferrin TE, Keasling JD | title = Designed divergent evolution of enzyme function | journal = Nature | volume = 440 | issue = 7087 | pages = 1078–82 | date = April 2006 | pmid = 16495946 | doi = 10.1038/nature04607 | bibcode = 2006Natur.440.1078Y | s2cid = 4394693 }}

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.{{cite journal | vauthors = Ma W, Tang C, Lai L | title = Specificity of trypsin and chymotrypsin: loop-motion-controlled dynamic correlation as a determinant | journal = Biophysical Journal | volume = 89 | issue = 2 | pages = 1183–93 | date = August 2005 | pmid = 15923233 | pmc = 1366603 | doi = 10.1529/biophysj.104.057158 | arxiv = q-bio/0505037 | bibcode = 2005BpJ....89.1183M }}{{cite journal | vauthors = Yasutake Y, Yao M, Sakai N, Kirita T, Tanaka I | title = Crystal structure of the Pyrococcus horikoshii isopropylmalate isomerase small subunit provides insight into the dual substrate specificity of the enzyme | journal = Journal of Molecular Biology | volume = 344 | issue = 2 | pages = 325–33 | date = November 2004 | pmid = 15522288 | doi = 10.1016/j.jmb.2004.09.035 }}

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. For example, if a tRNA synthesis 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.{{cite journal | vauthors = Perona JJ, Hadd A | title = Structural diversity and protein engineering of the aminoacyl-tRNA synthetases | journal = Biochemistry | volume = 51 | issue = 44 | pages = 8705–29 | date = November 2012 | pmid = 23075299 | doi = 10.1021/bi301180x }} Similar in reaction to tRNA synthesis, 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.{{cite journal | vauthors = Villiers BR, Hollfelder F | title = Mapping the limits of substrate specificity of the adenylation domain of TycA | journal = ChemBioChem | volume = 10 | issue = 4 | pages = 671–82 | date = March 2009 | pmid = 19189362 | doi = 10.1002/cbic.200800553 | s2cid = 21536526 }} 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.

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.{{cite journal | vauthors = Vasu K, Nagamalleswari E, Nagaraja V | title = Promiscuous restriction is a cellular defense strategy that confers fitness advantage to bacteria | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 20 | pages = E1287–93 | date = May 2012 | pmid = 22509013 | pmc = 3356625 | doi = 10.1073/pnas.1119226109 | bibcode = 2012PNAS..109E1287V | doi-access = free }}

File:Delphinidin.svg (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 to 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.{{cite journal | vauthors = Weng JK, Philippe RN, Noel JP | title = The rise of chemodiversity in plants | journal = Science | volume = 336 | issue = 6089 | pages = 1667–70 | date = June 2012 | pmid = 22745420 | doi = 10.1126/science.1217411 | bibcode = 2012Sci...336.1667W | s2cid = 206539148 }}

{{main|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.{{cite journal | vauthors = Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K | title = Engineering the third wave of biocatalysis | journal = Nature | volume = 485 | issue = 7397 | pages = 185–94 | date = May 2012 | pmid = 22575958 | doi = 10.1038/nature11117 | bibcode = 2012Natur.485..185B | s2cid = 4379415 }}

An example of a commonly evolved enzyme is ω-transaminase which can replace a ketone with a chiral amine{{cite journal | vauthors = Shin JS, Kim BG | title = Comparison of the omega-transaminases from different microorganisms and application to production of chiral amines | journal = Bioscience, Biotechnology, and Biochemistry | volume = 65 | issue = 8 | pages = 1782–8 | date = August 2001 | pmid = 11577718 | doi = 10.1271/bbb.65.1782 | doi-access = free }} and consequently libraries of different homologues are commercially available for rapid biomining (eg. Codexis{{Cite web | url=https://www.facebook.com/notes/682716209027329/ | title=Codexis® Enzyme Platforms | website=www.facebook.com | date=2021-03-14 | url-access=registration | access-date=2024-06-11}}).

Another example is the possibility of using the promiscuous activities of cysteine synthase (cysM) towards nucleophiles to produce non-proteinogenic amino acids.{{cite journal | vauthors = Maier TH | title = Semisynthetic production of unnatural L-alpha-amino acids by metabolic engineering of the cysteine-biosynthetic pathway | journal = Nature Biotechnology | volume = 21 | issue = 4 | pages = 422–7 | date = April 2003 | pmid = 12640465 | doi = 10.1038/nbt807 | s2cid = 22280900 }}

Similarity between enzymatic reactions ([http://www.chem.qmul.ac.uk/iubmb/enzyme/ EC]) can be calculated by using bond changes, reaction centres or substructure metrics ([http://www.ebi.ac.uk/thornton-srv/software/rbl/ EC-BLAST] {{Webarchive|url=https://web.archive.org/web/20190530132101/http://www.ebi.ac.uk/thornton-srv/software/rbl/ |date=2019-05-30 }}).{{cite journal | vauthors = Rahman SA, Cuesta SM, Furnham N, Holliday GL, Thornton JM | title = EC-BLAST: a tool to automatically search and compare enzyme reactions | journal = Nature Methods | volume = 11 | issue = 2 | pages = 171–4 | date = February 2014 | pmid = 24412978 | doi = 10.1038/nmeth.2803 | pmc=4122987}}

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. 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.{{cite journal | vauthors = Jakoby WB, Ziegler DM | title = The enzymes of detoxication | journal = The Journal of Biological Chemistry | volume = 265 | issue = 34 | pages = 20715–8 | date = December 1990 | doi = 10.1016/S0021-9258(17)45272-0 | pmid = 2249981 | doi-access = free }}

• Evolution by gene duplication
• Michaelis–Menten kinetics
• Molecular promiscuity
• Protein moonlighting
• Susumu Ohno

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{{#tag:ref|Most authors refer to as promiscuous activities the non-evolved activities and not secondary activities that have been evolved. Consequently, glutathione S-transferases (GSTs) and cytochrome P450 monooxygenases (CYPs) are termed multispecific or broad-specificity enzymes.

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.{{OED|promiscuity}}|name=foot}}

}}

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