Evolution Of Enzyme Function Research Articles

Evolution of Enzyme Function and the Development of Catalytic Efficiency: Triosephosphate Isomerase, Jeremy R. Knowles, and W. John Albery.

Every reader knows that an enzyme accelerates a reaction by reducing the activation-energy barrier. However, understanding how this is achieved by the structure of the enzyme and its interactions with stable complexes and transition states and, then, using this to (re)design enzymes to catalyze novel reactions remain the "holy grail" of mechanistic enzymology. The necessary foundation is the free-energy profile that specifies the energies of the bound substate, product, and intervening intermediates as well as the transition states by which they are interconverted. When this free-energy profile is compared to that for the uncatalyzed reaction, strategies for establishing and enhancing catalysis can be identified. This Perspective reminds readers that the first free-energy profile determined for an enzyme-catalyzed reaction, that for triosephosphate isomerase, was published in Biochemistry in 1976 by Jeremy R. Knowles, W. John Albery, and co-workers. They used the profile to propose three steps of increasing "subtlety" that can be influenced by evolutionary pressure to increase the flux through the reaction coordinate: (1) "uniform binding" of the substrate, product, and intermediates; (2) "differential binding" of complexes so that these are isoenergetic (to minimize the energy of the intervening transition states); and (3) "catalysis of an elementary step" in which the transition state for the kinetically significant chemical step is stabilized so that flux can be determined by the rate of substrate binding or product dissociation. These papers continue to guide mechanistic studies of enzyme-catalyzed reactions and provide principles for the (re)design of novel enzymes.

A Paradigm for CH Bond Cleavage: Structural and Functional Aspects of Transition State Stabilization by Mandelate Racemase.

Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg2+-dependent, 1,1-proton transfer reaction that racemizes (R)- and (S)-mandelate. MR shares a partial reaction (i.e., the metal ion-assisted, Brønsted base-catalyzed proton abstraction of the α-proton of carboxylic acid substrates) and structural features ((β/α)7β-barrel and N-terminal α + β capping domains) with a vast group of homologous, yet functionally diverse, enzymes in the enolase superfamily. Mechanistic and structural studies have developed this enzyme into a paradigm for understanding how enzymes such as those of the enolase superfamily overcome kinetic and thermodynamic barriers to catalyze the abstraction of an α-proton from a carbon acid substrate with a relatively high pKa value. Structural studies on MR bound to intermediate/transition state analogues have delineated those structural features that MR uses to stabilize transition states and enhance reaction rates of proton abstraction. Kinetic, site-directed mutagenesis, and structural studies have also revealed that the phenyl ring of the substrate migrates through the hydrophobic cavity within the active site during catalysis and that the Brønsted acid-base catalysts (Lys 166 and His 297) may be utilized as binding determinants for inhibitor recognition. In addition, structural studies on the adduct formed from the irreversible inhibition of MR by 3-hydroxypyruvate revealed that MR can form and deprotonate a Schiff-base with 3-hydroxypyruvate to yield an enol(ate)-aldehyde adduct, suggesting a possible evolutionary link between MR and the Schiff-base forming aldolases. As the archetype of the enolase superfamily, mechanistic and structural studies on MR will continue to enhance our understanding of enzyme catalysis and furnish insights into the evolution of enzyme function.

Co-occurrence of analogous enzymes determines evolution of a novel (βα)8-isomerase sub-family after non-conserved mutations in flexible loop.

We investigate the evolution of co-occurring analogous enzymes involved in L-tryptophan and L-histidine biosynthesis inActinobacteria Phylogenetic analysis oftrpFhomologues, a missing gene in certain clades of this lineage whose absence is complemented by a dual-substrate HisA homologue, termed PriA, found that they fall into three categories: (i) trpF-1, an L-tryptophan biosynthetic gene horizontally acquired by certain Corynebacteriumspecies; (ii) trpF-2, a paralogue known to be involved in synthesizing a pyrrolopyrrole moiety and (iii) trpF-3, a variable non-conserved orthologue of trpF-1 We previously investigated the effect of trpF-1 upon the evolution of PriA substrate specificity, but nothing is known about the relationship between trpF-3 and priA After invitro steady-state enzyme kinetics we found that trpF-3encodes a phosphoribosyl anthranilate isomerase. However,mutation of this gene inStreptomyces sviceusdid not lead to auxothrophy, as expected from the biosynthetic role of trpF-1 Biochemical characterization of a dozen co-occurring TrpF-2 or TrpF-3, with PriA homologues, explained the prototrophic phenotype, and unveiled an enzyme activity trade-off between TrpF and PriA. X-ray structural analysis suggests that the function of these PriA homologues is mediated by non-conserved mutations in the flexible L5 loop, which may be responsible for different substrate affinities. Thus, the PriA homologues that co-occur with TrpF-3 represent a novel enzyme family, termed PriB, which evolved in response to PRA isomerase activity. The characterization of co-occurring enzymes provides insights into the influence of functional redundancy on the evolution of enzyme function, which could be useful for enzyme functional annotation.

Evolution of Substrate Specificity in a Recipient’s Enzyme Following Horizontal Gene Transfer

Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole l-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in l-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA's substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (βα)8 isomerase subfamily with a specialized function in l-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism.

Harnessing Promiscuity Patterns to Map Evolution in the Alkaline Phosphatase Superfamily

In the past 30 yeas scientists have demonstrated that many enzymes are capable of promiscuous catalytic activity, in which one enzyme catalyses the turnover of chemically distinct substrates. It has been suggested that such promiscuity can play an important role in the evolution of enzyme function. This phenomenon is particularly pronounced in the alkaline phosphatase superfamily, in which the native reaction for one member of the superfamily is often a promiscuous side reaction for another. Moreover, despite the similarity of the substrates involved, the reactions catalysed (cleavage of P-O and S-O bonds) proceed with very different solvation and protonation requirements. We present here a detailed study of the promiscuous catalytic activity of three evolutionarily related but structurally different members of this superfamily: an arylsulfatase from Pseudonomas aeruginosa (PAS) and two phosphonate monoester hydrolases (PMH) from Rhizobium leguminosarum and Burkholderia caryophylli. The latter enzymes are particularly interesting due to the fact that, in addition to their broad promiscuity, they are also capable of catalysing a xenobiotic substrate for which no natural catalyst is known. We demonstrate that subtle changes in active site residues can have a significant impact on mechanism. However, despite such mechanistic variation, the promiscuity simply arises out of the ability of the non-native substrates to exploit the pre-existing electrostatic pre-organization of the active site towards the native substrate. We believe this provides an example of chemically-driven protein evolution within an enzyme superfamily.

DNA cleavage function of seryl-histidine dipeptide and its application

The double-stranded DNA or circular plasmid DNA can be cleaved by the Ser-His dipeptide by hydrolysis of the DNA-phospho-diester bond. The proper sequential order of the amino acids serine and histidine is apparently crucial in its unique cleavage activity as compared to the other di- or tri-peptides containing one of these amino acids. An inverted sequence of this dipeptide to a His-Ser linkage renders the peptide ineffective in the cleavage of DNA. In addition to the DNA cleavage function, Ser-His is also capable of cleaving other molecules, e.g., proteins, esters and RNAs. The cooperative actions of the hydroxyl group and the basic groups in the serine and histidine or related amino acids can be found in contemporary enzymes, such as DNase, serine proteases, lipases, esterases, chymotrypsin, trypsin, and elastase, etc. The Ser-His and related oligopeptides might have played important roles in the evolution of enzyme functions.

Mechanistic Analogies Amongst Carbohydrate Modifying Enzymes

Carbohydrates are known to play essential roles in a myriad of biological processes. The enormous complexity of the various oligosaccharide structures found in biology is derived from a rational orchestration of the enzymatic formation and breakdown of glycosidic linkages achieved by glycosyltransferases, glycosidases and phosphorylases. A detailed understanding of the chemical mechanisms by which these classes of enzymes function not only provides a rational basis for their engineering and application in both the development and synthesis of new classes of therapeutic agents, but also provides insight into the role of convergence in the natural evolution of enzyme function.

Evolution of enzyme function and the development of catalytic efficiency.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEvolution of enzyme function and the development of catalytic efficiencyW. John Albery and Jeremy R. KnowlesCite this: Biochemistry 1976, 15, 25, 5631–5640Publication Date (Print):December 1, 1976Publication History Published online1 May 2002Published inissue 1 December 1976https://doi.org/10.1021/bi00670a032RIGHTS & PERMISSIONSArticle Views3616Altmetric-Citations471LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts