Galactokinase promiscuity: a question of flexibility?

McAuley, Margaret; Kristiansson, Helena; Huang, Meilan; Pey, Ángel L.; Timson, David J.

doi:10.1042/bst20150188

Cited by 20 publications

(18 citation statements)

References 75 publications

(106 reference statements)

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“…Interestingly, the mutation giving rise to this promiscuity corresponds to the alteration of an amino acid residue far from the active site, which has led to suggestions that the flexibility of the protein may be an important factor in substrate specificity. 63,74 The same group extended the substrate diversity of the mutant with a second (M173L) mutation, this time in the active site. The doubly mutated kinase is reported to be capable of accepting a wide range of glucose and galactose derivatives, along with some L-sugars.…”

Section: Accepted Manuscriptmentioning

confidence: 95%

Functional analysis of anomeric sugar kinases

Conway

Voglmeir

2016

Carbohydrate Research

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Section: Accepted Manuscriptmentioning

confidence: 95%

Functional analysis of anomeric sugar kinases

Conway

Voglmeir

2016

Carbohydrate Research

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“…This tyrosine residue forms part of a β‐sheet structure that is distant from the active site . It has been postulated that altering the tyrosine residue results in changes to the dynamics of the active‐site residues that enable a greater range of molecules to bind, but also reduce catalytic efficiency …”

Section: Introductionmentioning

confidence: 99%

Improving the Activity and Stability of Human Galactokinase for Therapeutic and Biotechnological Applications

et al. 2018

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Galactokinase catalyses the site- and stereospecific phosphorylation of α-d-galactose. As such it has attracted interest as a biocatalyst for the introduction of phosphate groups into monosaccharides. However, attempts to broaden the substrate range of human galactokinase have generally resulted in substantially reduced activity. The enzyme also has biotechnological potential in enzyme replacement therapy (ERT) for type II galactosaemia. The return-to-consensus approach can be used to identify residues that can be altered to increase protein stability and enzyme activity. This approach identified six residues of potential interest in human galactokinase. Some of the single consensus variants (M60V, D268E, A334S and G373S) increased the catalytic turnover of the enzyme, but none resulted in improved stability. When all six changes were introduced into the protein (M60V/M180V/D268E/A334S/R366Q/G373S), thermal stability was increased. Molecular dynamics simulations suggested that these changes altered the protein's conformation at key sites. The number of salt bridges and hydrogen bonds was also increased. Combining the six consensus variations with Y379W (a variant with greater substrate promiscuity) increased the stability of this variant and its turnover towards some substrates. Thus, the six consensus variants can be used to stabilise catalytically interesting variants of human galactokinase and might also be useful if the protein were to be used in ERT.

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“…In turn, this will enable a greater range of complementary interactions with sugar molecules, but also decrease the time spent by the active site in a catalytically competent conformation. We have previously noted that combining this approach with methods to increase the overall stability of the enzyme may result in galactokinases with broader specificity and higher activities (30).…”

Section: Towards An Alternative Paradigm For Protein Engineeringmentioning

confidence: 99%

Modulating Mobility: a Paradigm for Protein Engineering?

McAuley

Timson

2016

Appl Biochem Biotechnol

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Additional Information:Question ResponsePlease select a section/category for your manuscript.Biocatalysis for organic synthesis -Novel and significant advances in biocatalysis for organic synthesis, including chemo-, regio-and enantioselective biotransformation, screening and engineering of novel enzymes for biocatalysis, biocatalyst immobilization, and nonaqueous biocatalysis.Abstract: Proteins are highly mobile structures. In addition to gross conformational changes occurring on, for example, ligand binding, they are also subject to constant thermal motion. The mobility of the protein varies through its structure and can be modulated by ligand binding and other events. It is becoming increasingly clear that this mobility plays an important role in key functions of proteins including catalysis, allostery, cooperativity and regulation. Thus, in addition to an optimum structure, proteins most likely also require an optimal dynamic state. Alteration of this dynamic state through protein engineering will affect protein function. A dramatic example of this is seen in some inherited metabolic diseases where alternation of residues distant from the active site affects the mobility of the protein and impairs function. We postulate that using molecular dynamics simulations, experimental data or a combination of the two it should be possible to engineer the mobility of active sites. This may be useful in, for example, increasing the promiscuity of enzymes. Thus, a paradigm for protein engineering is suggested in which the mobility of the active site is rationally modified. This might be combined with more "traditional" approaches such as altering functional groups in the active site. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Manuscript #ABAB-D-16-00805 Modulating mobility: a paradigm for protein engineering? Margaret McAuley and David J. TimsonReviewer: Flexibility of protein plays an important role in protein functions such as activity and stability. Up to now, a number of studies strategized for engineering the protein towards desirable properties are published. The manuscript has a great summary of recent studies based on modulating mobility of protein especially in activity aspect. I recommend its publication after addressing the following issue.Response: We thank the reviewer for his/her support and encouraging remarks.There are also many reports about the improvement of stability of protein by modulating the flexibility of protein. Such as, "Park.H.J et al, Computational approach for designing thermostable Candidaantarctica lipase B by molecular dynamics simulation; Zhu, F., et al, Rational substitution of surface acidic residues for enhancing the thermostability of thermolysin; Chen, J et al, Improving stability of nitrile hydratase by bridging the salt-bridges in specific thermal-sensitive regions, and so on". It would be attractive if the authors could incorporate some examples that explain the relationship between activity and stability modulated by flexibility of prote...

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Galactokinase promiscuity: a question of flexibility?

Cited by 20 publications

References 75 publications

Functional analysis of anomeric sugar kinases

Functional analysis of anomeric sugar kinases

Improving the Activity and Stability of Human Galactokinase for Therapeutic and Biotechnological Applications

Modulating Mobility: a Paradigm for Protein Engineering?

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