Engineering by homologous recombination: exploring sequence and function within a conserved fold

Carbone, Martina; Arnold, Frances H.

doi:10.1016/j.sbi.2007.08.005

Cited by 42 publications

(31 citation statements)

References 31 publications

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“…The present review focuses primarily on the effect of point mutations (change of a single nucleotide) and will consider only proteins but not RNA, although many general principles of evolution are applicable to both classes of biomolecules. We refer to other authors for the evolution of protein structures via sequence re-arrangements such as domain-wise evolution [2][3][4], the fusion of small peptide fragments [5] or the 'chimeric' recombination of fragments that is also exploited in protein engineering [6][7][8][9].…”

Section: Introductionmentioning

confidence: 99%

Biophysics of protein evolution and evolutionary protein biophysics

Sikosek

Chan

2014

J. R. Soc. Interface.

224

207

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The study of molecular evolution at the level of protein-coding genes often entails comparing large datasets of sequences to infer their evolutionary relationships. Despite the importance of a protein's structure and conformational dynamics to its function and thus its fitness, common phylogenetic methods embody minimal biophysical knowledge of proteins. To underscore the biophysical constraints on natural selection, we survey effects of protein mutations, highlighting the physical basis for marginal stability of natural globular proteins and how requirement for kinetic stability and avoidance of misfolding and misinteractions might have affected protein evolution. The biophysical underpinnings of these effects have been addressed by models with an explicit coarse-grained spatial representation of the polypeptide chain. Sequence-structure mappings based on such models are powerful conceptual tools that rationalize mutational robustness, evolvability, epistasis, promiscuous function performed by 'hidden' conformational states, resolution of adaptive conflicts and conformational switches in the evolution from one protein fold to another. Recently, protein biophysics has been applied to derive more accurate evolutionary accounts of sequence data. Methods have also been developed to exploit sequence-based evolutionary information to predict biophysical behaviours of proteins. The success of these approaches demonstrates a deep synergy between the fields of protein biophysics and protein evolution.

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Section: Introductionmentioning

confidence: 99%

Biophysics of protein evolution and evolutionary protein biophysics

Sikosek

Chan

2014

J. R. Soc. Interface.

224

207

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“…In other words, point mutation can change protein function without changing their domain composition. It is well known that proteins with identical folds can diverge greatly not only in sequence but also in function [26].…”

Section: Discussionmentioning

confidence: 99%

“…This indicates that duplicates are more likely to diversify the biological process they participate into and the cellular compartment to which they belong rather than their molecular function. Secondly, they do so at domain score nearly fixed to a value close to one, indicating that on average the function of duplicates migrates within the same fold structure, presumably by sequence mutations or recombinations maintaining the same structural domains [26].…”

Section: Functional Divergence and Duplication Agementioning

confidence: 99%

Identity and divergence of protein domain architectures after the yeast whole-genome duplication event

et al. 2010

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Analyzing the properties of duplicate genes during evolution is useful to understand the development of new cell functions. The yeast S. cerevisiae is a useful testing ground for this problem, because its duplicated genes with different evolutionary birth and destiny are well distinguishable. In particular, there is a clear detection for the occurrence of a Whole Genome Duplication (WGD) event in S. cerevisiae, and the genes derived from this event ("WGD paralogs") are known. We studied WGD and non-WGD duplicates by two parallel analysis based on structural protein domains and on Gene Ontology annotation scheme respectively. The results show that while a large number of "duplicable" structural domains is shared in local and global duplications, WGD and non-WGD paralogs tend to have different functions. The reason for this is the existence of WGD and non-WGD specific domains with largely different functions. In agreement with the recent findings of Wapinski and collaborators (Nature 449, 2007), WGD paralogs often perform "core" cell functions, such as translation and DNA replication, while local duplications associate with "peripheral" functions such as response to stress. Our results also support the fact that domain architectures are a reliable tool to detect homology, as the domains of duplicates are largely invariant with date and nature of the duplication, while their sequences and also their functions might migrate.

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“…However, a homologous family is required to use gene shuffling to keep a conserved fold of an enzyme (Carbone and Arnold, 2007). …”

Section: Measurement Of Hydrolytic Activities Of the Homologous Protementioning

confidence: 99%

“…Finding and studying homologous enzymes of interest will likely provide not only an evolutionary knowledge but also an opportunity of creating a new enzyme by gene shuffling. Gene shuffling is one of the strong tools to produce diversity of an enzyme and then to create a new enzyme through directed evolution (Crameri et al, 1998;Carbone and Arnold, 2007). This paper describes the discovery of the homologous enzymes of CAL-B by comparison of protein sequences in a pool of protein sequences and the functional expression of the homologous enzymes in E. coli.…”

mentioning

confidence: 99%

Exploration and functional expression of homologous lipases of Candida antarctica lipase B

Park

2015

The Korean Journal of Microbiology

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Candida (also known as Pseudozyma) antarctica lipase B (CAL-B) has been intensely studied in academic and industrial fields. However, the research related to its homologous enzymes has been rarely reported. In the current investigation, protein sequence similarity search of CAL-B has been conducted and six homologous protein sequences were identified. After the syntheses of their codon-optimized genes, the synthetic genes have been cloned into a periplasmic expression vector to express in Escherichia coli. Among six homologous sequences, four sequences were successfully expressed in E. coli. The hydrolytic activities of the expressed proteins towards 4-nitrophenyl acetate and 4-nitrophenyl butyrate were measured and compared with those of CAL-B to identify whether the expressed proteins work as a hydrolase. It has been revealed that the expressed proteins can hydrolyze the substrates and the specific activities were determined as (1.3-30) × 10 -2 μmol/min/mg, which are lower than those of CAL-B. Among these homologous enzymes, Pseudozyma hubeiensis SY62 exhibits the comparable enantioselectivity to that of CAL-B towards the hydrolysis of (±)-1-phenylethyl acetate.

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Engineering by homologous recombination: exploring sequence and function within a conserved fold

Cited by 42 publications

References 31 publications

Biophysics of protein evolution and evolutionary protein biophysics

Biophysics of protein evolution and evolutionary protein biophysics

Identity and divergence of protein domain architectures after the yeast whole-genome duplication event

Exploration and functional expression of homologous lipases of Candida antarctica lipase B

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