بنام خداوند جان و خرد
كزاين برترانديشه برنگذرد
Enzyme Evolution and Directed Enzyme Evolution
By : MOHAMMAD SADEGH HIRANIAN
Supervised By : Dr MAHMODI
Contents
I. Enzyme Evolution
Introduction
Phylogeny
the Hot Spots and the Cold Spots
Explaining the Hot Spots and the Cold Spots
II. Directed Enzyme Evolution
Introduction
Evolving novel activity
Evolving specific enzymes
Directed evolution usually goes through single amino acid improvements
The challenge of directing evolution
III. Conclusions
Introduction
Importants:
1- reconstructing the historical relationships among various taxa
2- provides an all-important window through which we can glimpse the action of underlying evolutionary mechanisms
continued
Rates of molecular evolution : rates of mutation
rates of spontaneous nucleotide substitution (per site per generation)
low of 2 x 10 -11 in Neurospora crassa
through 5.4 x 10 -10 for Escherichia coli
high of 1.5 x 10 -3 in Qβ.
continued
rates of molecular evolution are greatly modified from the underlying rates of mutation
testify - rates of evolution are presented as substitutions per site per year; rates of mutation are presented as substitutions per site per generation.
Precisely why the rate of molecular evolution should appear so regular on an annual basis, rather than on a per generation basis, remains a mystery.
pseudogenes, evolve slightly faster than introns, 3’- regions of genes and the degenerate sites of coding regions.
The nondegenerate are considered the most constrained because they determine the amino acid sequences of proteins whose functions are directly subject to natural selection.
The frequent replacement of one hydrophobic amino acid by another in the lipid binding domains of mammalian apolipoproteins has been taken as evidence of a lack of structural constraints
The concept of constraint carries force only when supported by evidence garnered independently of evolutionary argument
Constraints are not the only mechanism offered in explanation of rate heterogeneity. Fisher long ago argued that mutations with smaller phenotypic effects are more likely to be selectively advantageous.
rapid evolution seen in degenerate sites within coding regions might be related with smaller phenotypic effects
slow evolution seen at nondegenerate sites is merely a consequence of mutations with larger phenotypic effects being less likely to be advantageous.
Phylogeny
Isocitrate dehydrogenases (IDH) are ubiquitous in nature, catalyzing the oxidation of isocitrate to α -ketoglutarate and CO2 with concomitant reduction of either NAD or NADP.
Many organisms lacking Krebs’ cycle for energy production nevertheless retain its IDH to produce the α-ketoglutarate so essential for glutamate biosynthesis
continued
IDHs belong to an ancient and diverse family of enzymes which include isopropylmalate dehydrogenases, tartrate dehydrogenases, homoisocitrate dehydrogenases
Eubacterial IDHs are approximately 400 amino acid residues long, share at least 40% amino acid sequence identity
Phylogenetic trees have similar topologies.
Most eubacterial IDHs belong to a single monophyletic clade (Figure 1)
the only exceptions being those of Mycobacteria, which more closely resemble the eukaryotic cytosolic IDHs (not shown),
that of Rickettsia prowazekii which more closely resembles the eukaryotic mitochondrial IDHs.
The X-ray structures of Escherichia coli IDH are extraordinarily similar to those of isopropylmalate dehydrogenase, a distantly related member within the protein superfamily.
secondary structure has not evolved within the eubacterial IDH clade, is robustly justified.
the Hot Spots and the Cold Spots
many regions that significant heterogeneity. The most abnormal region is a 10A diameter sphere centered at amino acid residue 153 (Figure 2).
This region evolves at a rate that is 2.8 times slower than the overall average of the molecule. The 59 residue region encompasses much of the active site
Explaining the Hot Spots and the Cold Spots
Directed enzyme evolution Introduction
Unlike rational design, directed evolution does not rely on a detailed understanding of the relationship between enzyme structure and function; rather, it relies on the simple yet powerful Darwinian principles of mutation and selection
Directed evolution is now well established as highly effective for protein engineering and optimization. Directed evolution entails accumulation of beneficial mutations in iterations of mutagenesis and screening or selection
it can be thought of as an uphill climb on a‘fitness landscape’, a multidimensional plot of fitness versus sequence.
Fitness in a directed evolution experiment is defined by the experimenter, who also controls the relationship between fitness and reproduction.
There are an enormous number of ways to mutate any given protein
most mutational paths lead downhill and eventually to unfolded, the challenge lies in identifying an efficient path to the desired function.
Whereas negative epistatic effects are pervasive in natural evolution , such effects have played a major role in facilitating directed evolution.
The vast majority of evolutionary engineering studies over the past ten years involve simple uphill walks, one step at a time.
many ways to create sequence diversity, and this is an important part of the search strategy for molecular optimization.
The goal in choosing a mutagenesis strategy is to minimize the screening requirement and increase the chances of finding beneficial mutations
There is no single ‘best’ mutagenesis method. multiple methods will work (although some are far more efficient than others).
Evolving novel activity
challenge approach to obtaining a new activity was used by Fasan et al. who converted a cytochrome P450 fatty acid hydroxylase into a highly efficient propane hydroxylase, an activity absent in the native enzyme.
Early steps often create ‘generalists’ that are active on a much broader range of substrates. These have been used in a more serendipitous approach to obtaining new activities.
New activities can also arise during ‘neutral drifts’. mutations that do not abolish the native activity are accumulated in multiple rounds of high-error-rate mutagenesis and screening
Evolving specific enzymes
enzyme with high specificity for a new substrate can be generated with a single amino acid substitution.
it may be possible to eliminate variants using negative selection.
Further mutagenesis may also be required to obtain the desired specificity ,with positive selection to improve the desired activity and negative selection to remove the undesired one (s).
Directed evolution usually goes through single amino acid improvements
Analysis of the directed evolution literature shows that a wide range of problems can be solved by uphill walks involving single amino acid changes.
Often, single mutations are responsible for the functional change ,even when multiple mutations are made . Or, when multiple beneficial mutations are found, they all contribute and could have been found separately
Low error-rate random mutagenesis by error-prone PCR is very simple to implement, but only accesses a limited set of (mostly conservative) amino acid changes.
Other mutagenesis methods, including saturation mutagenesis, can effectively generate additional amino acid possibilities in targeted residues .
When the mutation rate is high, beneficial mutations are quickly masked by the much more frequent deleterious ones. Low error rates – 1–2 amino acid substitutions per gene – are therefore preferred if the entire gene is mutated.
Statistical methods to identify beneficial mutations in variants containing multiple mutations have been described and allow simultaneous examination of substitutions at more positions.
The challenge of directing evolution
Even with high-quality mutant libraries and screening, not all bad enzymes can become good ones via a simple uphill walk.
Some problems are more difficult, and coupled mutations might be necessary for the desired functional changes. Beneficial mutations are rare, but combinations of beneficial mutations that only work together are even rarer.
While making targeted libraries may help, it is not clear that we know how to choose the amino acids to target.
It is likely that not all protein folds are ideal for catalyzing desired reactions. Some starting points might require major changes (perhaps even to the protein architecture) in order to improve catalysis and these changes are not accessible by directed evolution.
conclusion
Heterogeneity in rates of molecular evolution
differences in rates of evolution constraints
rates of molecular evolution are greatly modified from the underlying rates of mutation
Directed evolution is now well established as highly effective for protein engineering and optimization
Unlike rational design, directed evolution does not rely on a detailed understanding of the relationship between enzyme structure and function; rather, it relies on the simple yet powerful Darwinian principles of mutation and selection
هیچ نظری موجود نیست:
ارسال یک نظر