Structure-Based Engineering of E. coli Galactokinase as a First Step toward In Vivo Glycorandomization

Yang, Jie; Fu, Xun; Liao, Jianchun; Liu, Lesley; Thorson, Jon S.

doi:10.1016/j.chembiol.2005.04.009

Cited by 68 publications

(67 citation statements)

References 32 publications

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“…In humans, deficiencies of this enzyme can result in type II galactosaemia (MIM 230200), the main symptom of which is cataract development (Holton et al, 2001). Recently, Escherichia coli galactokinase has been utilized via a directed-evolution approach to create natural and unnatural sugar 1-phosphates in a process of glycorandomization, with the aim of enhancement of drug-discovery efforts (Hoffmeister et al, 2003;Yang et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase fromPyrococcus horikoshii

Inagaki¹,

Sakamoto²,

Obayashi³

et al. 2006

Acta Cryst Sect F

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Galactokinase (EC 2.7.1.6) catalyzes the ATP-dependent phosphorylation of -d-galactose to -d-galactose-1-phosphate, in an additional metabolic branch of glycolysis. The apo-form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with -d-galactose and an ATP analogue. Diffraction data sets were collected to 1.24 Å resolution for the apo form and to 1.7 Å for the ternary complex form using synchrotron radiation. The apo-form crystals belong to space group C2, with unit-cell parameters a = 108.08, b = 38.91, c = 81.57 Å , = 109.8 . The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.

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Section: Introductionmentioning

confidence: 99%

Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase fromPyrococcus horikoshii

Inagaki¹,

Sakamoto²,

Obayashi³

et al. 2006

Acta Cryst Sect F

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“…During these studies, it was observed that E. coli GalK M173L displayed moderate D-glucose activity (27). The equivalent residue in the yeast enzyme (Met-216) is located in the active site (next to Asp-217 in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Attempts to change or broaden the substrate specificity range of galactokinases have been undertaken using the Escherichia coli enzyme through the application of in vitro glyco-randomization (26,27). During these studies, it was observed that E. coli GalK M173L displayed moderate D-glucose activity (27).…”

Section: Discussionmentioning

confidence: 99%

Contribution of Amino Acid Side Chains to Sugar Binding Specificity in a Galactokinase, Gal1p, and a Transcriptional Inducer, Gal3p

Sellick

Reece

2006

Journal of Biological Chemistry

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The crystal structure of the yeast galactokinase, Gal1p, in the presence of its substrates has been solved recently. We systematically mutated each of the amino acid side chains that, from the structure, are implicated to be involved in direct contact with the hydroxyl groups of the galactose ring. One of these mutations, D62A, abolished all detectable galactokinase activity but retained the ability to use D-glucose as a substrate. Mutation of Asp-62 to either leucine, phenylalanine, or histidine resulted in the formation of protein with similar characteristics to D62A. Yeast galactokinase is highly similar to Gal3p, the ligand sensor and transcriptional inducer of the GAL genes. Equivalent mutations in Gal3p also abolished its ability to respond to galactose and uncovered its ability to respond to D-glucose. It therefore appears that Gal1p and Gal3p respond to their substrates in a similar, perhaps identical, fashion. This work also validates the approach of screening for mutants in an easily assayable system prior to mutant analysis in a more experimentally difficult transcriptional regulator.The yeast Saccharomyces cerevisiae contains within its genome two galactokinase-like genes. The first of these, GAL1, encodes Gal1p, a bona fide galactokinase whose major role in the cell is to convert ␣-Dgalactose into galactose 1-phosphate at the expense of ATP as part of the Leloir pathway, which is responsible for the conversion of ␤-Dgalactose to the more metabolically useful glucose 6-phosphate (1). The second galactokinase-like gene, GAL3, encodes Gal3p, which has no detectable galactokinase activity itself but, rather, interacts with galactose and ATP to regulate the transcription of the Leloir pathway enzyme genes (2). Gal1p and Gal3p are highly similar proteins, sharing some 70% identity and 90% similarity at the amino acid level. This similarity has been underscored by the observation that the insertion of just two amino acids into Gal3p (a serine and an alanine inserted directly after amino acid 164) imparts the resulting protein with galactokinase activity (3).The kinetic mechanism of Gal1p has been described as proceeding via an ordered, ternary complex in which ATP binds first and galactose second (4). The most likely explanation for an ordered mechanism is that the binding of the first substrate induces a conformational change in the enzyme resulting in the formation of a high affinity binding site for the second substrate. The structure of Gal1p, complexed with galactose and a nonhydrolyzable ATP analogue, has been solved recently (5). The enzyme adopts a bilobal appearance with the active site being wedged between distinct amino-and carboxyl-terminal domains. The sugar is held in the active site by a network of potential hydrogen bond interactions (Fig. 1). This network, composed of residues Arg-53, , is capable of contacting each of the five hydroxyl groups of the sugar ring. To explore the requirement of this hydrogen bond network for interaction with the sugar, we undertook a mutational analysis of th...

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“…This has been suggested in the case of galactokinase (EC 2.7.1.6), where alterations to the sequence of a β-sheet structure distant from the active site resulted in a broader range of substrates being accepted and processed by the enzyme (19,20,(55)(56)(57).…”

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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Structure-Based Engineering of E. coli Galactokinase as a First Step toward In Vivo Glycorandomization

Cited by 68 publications

References 32 publications

Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase fromPyrococcus horikoshii

Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase fromPyrococcus horikoshii

Contribution of Amino Acid Side Chains to Sugar Binding Specificity in a Galactokinase, Gal1p, and a Transcriptional Inducer, Gal3p

Modulating Mobility: a Paradigm for Protein Engineering?

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