Copyright J.J 2007Copyright J.J 2007--1010

Enzyme promiscuity: mechanism and applications

Contents

Introduction1

Mechanism2

Evolution3

Application4

HistoryEven if the term promiscuity is new inenzymology, there are numerous old examples in the literature that live up to the definition.

1898 Hill, A.C. Reversible zymohydrolysis. J. Chem. Soc. 73, 634-6581900 Kastle, J.H and Loevenhart,A.S. Concerning lipase, thefatslitting enzyme, and the reversibility of its action. Am.Chem.J 24, 491-5251921 Neuberg, C. and Hirsch, J. über ein Kohlenstoffketten knüpfendes ferment ( Carboligase ). Biochem.Z. 115, 282-3101931 Rona, P.et al. studies on the enzymatic ester formation and ester hydrolysis. Biochem.Z. 247, 113-145

Introduction

Enzyme SpecificityIntroductory courses in biochemistry teach that enzymes are specific for their substrates and reactions they catalyze.

Enzyme Promiscuity (Poly-reactivity)Refer to a protein exerting a different function using the same active site that is responsible for its original activity.

Traditional Theory‘one sequence, one structure, one function’Traditionally, it is assumed that a given sequence dictates a single 3D structure upon which function is entirely dependent.

‘New view’The ‘new view’ of proteinIt has an energy landscape with many local minima corresponding to an ensemble of pre-existing structureswith similar but discrete energy levels (plasticity).

The Landsteiner-Pauling hypothesis--a 60-year-old hypothesis revisited

Conformational diversity and protein evolution

In the 1930s, Landsteiner and

Pauling proposed that protein (antibodies in this case) can exist as an ensemble of isomers with different structures but with similarfree energy. They realized that if each isomer was capable of binding to a different ligand, then functional diversity could go far beyond sequence diversity.

Enzyme evolution mediated by conformational diversity and functional promiscuity

The classification of promiscuityTypes of enzyme promiscuity

Enzyme condition promiscuityShown by enzyme with catalytic activity in various reaction

conditions different from their natural ones, such as anhydrous media, extreme temperature or pH.Enzyme substrate promiscuity

Shown by enzymes with relaxed or broad substrate specificityEnzyme catalytic promiscuity

Shown by enzyme catalyzing distinctly different chemical transformations with different transition states. Enzyme catalytic promiscuity can be either:(i) accidental- a side reaction catalyzed by the wild-type enzyme(ii) induced- a new reaction established by one or several mutations rerouting the reaction catalyzed by the wild-type enzyme

Examples of promiscuity

Mechanism

Condition promiscuity

Both condition promiscuity and substrate promiscuity have been explored and used in enzyme-catalyzed synthesis. There are thousands of examples of these, such as reactions performed in organic solvents, in absence of solvents, at high temperature, or at extreme pH.

Here they report a variant containing 10 amino acid substitutions that was generated by sequential rounds of random mutagenesisand screening for increased activity in DMF.

1. Subtilisin is a serine endopeptidase that evolved independently of chymotrypsin.

2. Formerly EC 3.4.21.64.

Condition promiscuity

Use random mutagenesis and screeningto identify amino acid substitutions that recover the catalytic activity lost by the serine protease subtilisin E in polar organic solvents.

Condition promiscuity

Condition promiscuity

DisccusionEffective Mutations Are Located in the Variable Loops Around the Active Site, and Most Are Found in Other Natural Subtilisins.

All the amino acid substitutions found to influence catalytic activity in DMF are located at the surface of the protein and are clustered in a region that encompasses the active site and substrate binding pocket. D60N, D97G, and E156G are very close to the amino acid residues critical to catalysis, while D97G, Q103R, E156G, and N218S are located in or very close to the substrate binding pocket

Mechanism

Substrate promiscuityEnzymes with strict substrate specificity, which can be destroyed by simple point mutations, and enzymes with an intrinsic high substrate promiscuity, which can be increased even more. These examples will show that even if enzymes intrinsically have high or low substrate specificity.

The research group around Reetz showed that it was possible to evolve lipases with preference for either the R-or S-enantiomer.

1. The pancreatic enzyme acts only on an ester-water interface; the outer ester links are preferentially hydrolyzed

2. SOURCE : Pseudomonasaeruginosa Bacteria

3. EC : 3.1.1.3

Substrate promiscuityThey previously demonstrated that the method of directed evolution can be applied successfully in the quest to create enantioselective enzymes for use in synthetic organic chemistry.

The wild-type (WT) lipase has a selectivity factor of only E=1.1, in slight favor of (S)-2.

Representation of directed evolution of enzntioselective enzymes

Substrate promiscuity

Substrate promiscuity

The mechanism of lipase-catalyzed ester hydrolysis is known to involve a catalytic triadcomprising aspartate,histidine, and serine.

Discussion

MechanismCatalytic promiscuityEnzyme catalytic promiscuity refers to the ability of an enzyme active site to catalyze more than one different chemical transformation. Kazlauskas proposes that transformations are different if the types of bonds cleavedand/or made are different, and if the transition states of the two reactions are different.

Accidental catalytic promiscuityIn this work, Wu et al surprisingly found that two zinc-bindingacylases, D-aminoacylase from Escherichia coli and acylase“Amano” from Aspergillus oryzae, also possess the promiscuous activity to catalyze the Markovnikov addition of a broad range of N-heterocycles to vinyl esters and exhibit even higher activities.

1. Has a wide specificity; hydrolyzes N-acyl derivative of neutral D-amino acids.

2. Used in separating D- and L-amino acids.

Cross-references

Accidental catalytic promiscuityThe Markovnikov addition is one type of useful carbon-carbon, oxygen-carbon or nitrogen-carbon bond forming reaction. It is especially important to synthesize bioactive N-heterocycle derivatives with a nitrogen-carbon linkage which could be achieved by an addition reaction.

Accidental catalytic promiscuity

The aza-Markovnikov addition reactions of 4-nitroimidazle to vinyl acetate catalyzed by d-aminoacylase, acylase “Amano”and immobilized penicillin G acylase were up to 1260-fold, 720-fold and 320-fold faster than the respective non-enzymatic reaction.

Accidental catalytic promiscuity

DiscussionThe generally accepted acylase mechanism usually involves the polarization of a carbonyl group by the zinc ion bound in the active sites of D-aminoacylase and acylase “Amano” (or by theoxyanion hole of PGA). A highly conserved Asp (or α-amino group of N-terminal Ser B1 of PGA) plays a key role in the proton transfer from the nucleophile water to the leaving group.

Mayer, C. et al. (2000) The E358S mutant of Agrobacterium sp. β-glucosidase is greatly improved glycosynthase. FEBS Lett. 466,

40–44

Induced catalytic promiscuityGlycosynthases are nucleophile mutants of retainingglycosidases that catalyze the glycosylation of sugar acceptors using glycosyl fluoride donors, thereby synthesizingoligosaccharides.

1. Catalyze the glycosylation of sugar acceptors using glycosyl fluoride donors

2. EC 3.2.1.45

Induced catalytic promiscuitySubstitution with serine seemed to be a good alternative since the hydroxymethyl side chain should be sufficiently small to accommodate the axial fluorine and might even hydrogen bond to the departing fluoride. Moreover, its alcohol moiety should be considerably less nucleophilic than the thiol of cysteine, thus unwanted adduct formation should be less likely.

Induced catalytic promiscuityAbg E358S proved to be an effective glycosynthase when presented with α-glucosyl or α-galactosyl fluoride donors and a variety of acceptors

Induced catalytic promiscuity

No significant effect on (kcat/Km)app: was observed for either mutant over a range of pH 9~5.

DiscussionInverting glycosidases have two carboxylates in the active site: one acts as a nucleophile to form a glycosyl–enzyme intermediate, whereas the other one acts as an acid/base catalyst. Removing the nucleophilic carboxylate produces an enzyme that is unable to hydrolyze glycosidic bonds. However, the otherwise intact active site can bind a suitable activated α-glycosyl fluoride and an acceptor sugar in such a way that the remaining acid/base carboxylate can catalyze the formation of aglycosidic bond.

Because the mutant enzyme is devoid of hydrolytic activity, the product can accumulate without being hydrolyzed. A mutant in which the nucleophilic carboxylate was changed not to an alanine but to serine is especially good as glycosynthase.

EvolutionJensen, R.A. Enzyme recruitment in evolution of new function. Annu Rev Microbiol 1974, 30:409-425.

Jensen proposed that, in contrast to modern

enzymes, primitive enzymes possessed very broad specificities. This catalytic versatility enabled fewer enzymes to perform the multitude of functions that was necessary to maintain ancestral organisms. Duplication of genes and divergence led to specialized genes and increased metabolic efficiency.

Negative trade-offs and the divergence of new function

Experimental evidence favoursthe model of divergence of a ‘generalist’ progenitor enzyme to a family of ‘specialist’ enzymes.

Possible routes to new function acquisition.

Dan S Tawfik. (2005 ) The ‘evolvability’ of promiscuous protein functions. Nat Genet, 37:73-76.

Promiscuous protein functions

How proteins with new functions (e.g., drug or antibiotic resistance or degradation of man-made chemicals) evolve in a matter of months or years is still unclear. This ability is dependent on the induction of new phenotypic traits by a small number of mutations (plasticity). But mutations often have deleterious effects on functions that are essential for survival. How are these seemingly conflicting demands met at the single-protein level?

Promiscuous protein functions

Changes in activities of the newly evolved PON1 variants

Changes in activities of the newly evolved PTE variants

Changes in activities of the newly evolved CAII variants

The chemical structures of the compounds

ConclusionPromiscuous activities have another distinct inherent advantage: they have an unusual plasticity, or lack of robustness, that is not seen with the native function. Robustness of the native function can be acquired in the course of the evolutionary process, but the promiscuous functions are latent and were never under selection pressure

The Grand fir, largest of all the fir trees, produces the ultimate in "promiscuous enzymes," asesquiterpene synthase capable of producing as many as 52 different enzyme products.

Examples for variants exhibiting large improvements in promiscuous

activities and small changes in native activity

Extended contentDrug resistance

Mutations that confer drug resistance are in substrate-binding loops, rather then in the catalytic residues or the protein’s scaffold, and adaptability towards drug binding involves conformational flexibility

StandpointThe evolution depends on two critical, and seemingly conflicting, features:

(i) a reduced lethality of mutations (robustness)

(ii) the induction of new phenotypic traits by a relativelylow number of mutations (plasticity)

Application

The applied aspects of condition promiscuity and substrate promiscuity have been explored for a long time, resulting in many industrial applications. Enzyme catalytic promiscuity, however, has only recently been exploited for synthetic applications.

Scientist in this field