Restriction enzymes evolution

Restriction enzymes constitute one of the largest families of related enzymes known so far and therefore exemplify how natural evolution has generated many different specificities from one (or a few) ancestral protein(s). A detailed comparison of the... 

The application of directed evolution approaches for the change or extension of the specificity of a restriction enzyme is hampered by the fact that an in vivo selection assay is not available and that examination of endonuclease activity in vitro usually requires purification of the enzymes to avoid background activity by other nucleases prevalent in all cells. This means that an altered or extended specificity can only be observed with sufficiently purified protein preparations, thereby unfortunately separating genotype and phenotype. As neither a reliable in vivo test nor the secretion of restriction endo-... 

Before we describe the chemistry of the compartments involved, note that like prokaryotes, a number of oxidative enzymes are found in the cytoplasm but they do not release damaging chemicals (see Section 6.10). We also observed that such kinds of kinetic compartments are not enclosed by physical limitations such as membranes. We have also mentioned that increased size itself makes for kinetic compartments if diffusion is restricted. In this section, we see many additional advantages of eukaryotes from those given in Section 7.4. How deceptive it can be to use just the DNA, the all-embracing proteome, metabolome or metallome in discussing evolution without the recognition of the thermodynamic importance of compartments and their concentrations These data could be useful both here and in simpler studies of single-compartment bacteria even in the analysis of species but not much information is available. 

The first high-throughput ee assay used in the directed evolution of enantioselective enzymes was based on UV/Vis spectroscopy (16,74). It is a crude but useful screening system that is restricted to the hydrolytic kinetic resolution of racemic / -nitrophenyl esters catalyzed by lipases or esterases. The development of this assay arose from the desire to evolve highly enantioselective mutants of the lipase from Pseudomonas aeruginosa as potential biocatalysts in the hydrolytic kinetic resolution of the chiral ester rac-. The wild type leads to an E value of only 1.1 in slight... 

Metalloenzymes or metal ion-activated enzymes catalyze an enormous variety of organic reactions that are not restricted to any particular reaction class, but appear as catalysts for all types of reactions. Thus neither the presence of the metal ion nor the reaction type seems to be restrictive as far as metal-assisted enzyme catalysis is concerned. In some cases the metal ion appears to function as an electron acceptor or donor, but flavin cofactors have substituted as redox centers during evolution in some enzymes. 

The nature of the metal ion that is used by an enzyme may have several origins. Of course, the first reason for the selection of a certain metal is the availability of the various metals at the time and place of evolution of the particular enzyme or protein. When this restriction has been met, the choice of metal is determined by the role that it has to play in the activity of the enzyme if it is just needed to act as a Lewis acid to bind and activate the substrate, then a metal with limited redox activity such as zinc may be used. If, on the other hand, apart from substrate binding, redox activity is needed to perform the catalytic function of the enzyme, then other metals, which have different oxidation states readily available, are needed. 

It is well understood that the synthesis and modihcation of metabolites is under enzymatic control. The enzymes may function as catalysts, and the reactions themselves are not restricted only to living systems. So the evolution of biosynthetic capacity is largely the result of changes in enzymes by mutation, gene duplication, and other familiar processes. The organisms synthesize and modify secondary metabolites in a stepwise fashion, much as organic chemists do, and in neither case are the laws of nature violated. 

At present, 16 cysteine-containing subtilisin-type enzymes are known and the position of the cysteine residues is restricted to the nine corresponding sites described above.42 Of the 16 enzymes, six enzymes other than aqualysin I and proteinase K have cysteine residues at positions where the cysteine residues are able to form disulfide bond(s) like the two enzymes. Although these disulfide bonds seem to have been acquired to increase protein stability, only four kinds of disulfide bonds are found in the subtilisin-type enzymes, suggesting that the positions of the disulfide bonds have been selected strictly in the process of molecular evolution of the enzyme. 

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