Mutagenesis of Escherichia coli uridine phosphorylase by random pentapeptide insertions

Oliva, Ilaria; Zuffi, Gabriele; Orsini, Gaetano; Tonon, Giancarlo; Gioia, Luca De; Ghisotti, Daniela

doi:10.1016/j.enzmictec.2004.04.017

Cited by 6 publications

(4 citation statements)

References 23 publications

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“…It should be expected that not all binding-site residues contribute equally to a protein's function. Experimentally, it has been observed for several binding sites (23)(24)(25)(26) that the different residues comprising each site can contribute vastly different amounts to the functional processes of the protein (i.e., catalysis or allosteric response).…”

Section: Resultsmentioning

confidence: 99%

Functional residues serve a dominant role in mediating the cooperativity of the protein ensemble

Liu

Whitten

Hilser

2007

Proc. Natl. Acad. Sci. U.S.A.

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Conformational fluctuations in proteins have emerged as a potentially important aspect of biological function, although the precise relationship and the implications have yet to be fully explored. Numerous studies have reported that the binding of ligand can influence fluctuations. However, the role of the binding site in mediating these fluctuations is not known. Of particular interest is whether in addition to serving as structural scaffolds for recognition and catalysis, active-site residues may also play a role in modulating the cooperative network. To address this question, we employ an experimentally validated ensemble-based description of proteins to elucidate the extent to which perturbations at different sites can influence the cooperative network in the protein. Applying this method to a database of test proteins, it is found statistically that binding sites are located in regions most able to affect the cooperative network, even for cooperative interactions between residues distant to the binding sites. This indicates that the conformational manifold under native conditions is determined by the network of cooperative interactions within the protein and suggests that proteins have evolved to use these conformational fluctuations in carrying out their functions. Furthermore, because the energetic coupling pattern calculated for each protein is robust and relatively insensitive to sequence, these studies further suggest that binding sites evolved in regions of the protein that are inherently poised to take advantage of the fluctuations in the native structure.COREX analysis ͉ energetic coupling ͉ native state ensemble ͉ thermodynamic linkage B iological work in living organisms is mediated primarily through the use of protein molecules. Knowledge of the means by which proteins facilitate this work is of paramount importance to an understanding of function as well as the diseases that result from aberrant function. Of particular interest in recent years has been the observation that proteins are highly dynamic molecules that experience dramatic conformational excursions from the canonical high-resolution structure (1-3). In addition, it has also become clear that conformational dynamics play an important role in determining the ability of proteins to perform such diverse tasks as catalysis, allosterism, and signal transduction (4-6).The observation that protein conformational fluctuations play an important role in function suggests that functional sites in proteins may be uniquely coupled to structural fluctuations (7-9) and thus identifiable by how perturbations at these sites affect the conformational manifold. Here we use an ensemblebased model of the protein to explore this issue. By identifying a statistically significant difference in the response of the ensemble to perturbations at active sites of proteins, as determined from a representative test set, we find that binding sites play a unique role in influencing the cooperative network within proteins. This suggests that binding sites have been pre...

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Section: Resultsmentioning

confidence: 99%

Functional residues serve a dominant role in mediating the cooperativity of the protein ensemble

Liu

Whitten

Hilser

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Briefly, LacZ 8 -PNP 20 -Kex-1-Met-G-CSF hybrid fusion protein (where LacZ8 represents the first 8 aminoacids of the LacZ protein fragment [UniProt/SwissProt accession Q37953], PNP 20 is a sequence coding for the first twenty amino acids of E.coli purine nucleoside phosphorylase [9] and the N-terminal methionine residue of filgrastim is immediately preceded by a sequence coding for the short flexible peptide -Glu-Ser-Ser-Met-Ser-Gly-Leu-Phe-Lys-Arg- ending with a Lys-Arg basic dipeptide which in vitro is specifically cleaved at C-terminal site of arginine by the endoprotease ss-Kex-1-C 611 to leave the mature sequence of filgrastim) was expressed in E.coli as cytoplasmic inclusion bodies. After isolation of inclusion bodies, the fusion protein was dissolved in 7 M guanidine, renaturated by dilution, dialyzed and cleaved by treatment with the patented recombinant endoprotease ss-Kex1-C 611 [10] and the released filgrastim was purified to homogeneity by column chromatography to obtain a clinical grade preparation coded BK0023.…”

Section: Methodsmentioning

confidence: 99%

Preclinical and clinical phase I studies of a new recombinant Filgrastim (BK0023) in comparison with Neupogen®

Crobu

Spinetti

Schrepfer

et al. 2014

BMC Pharmacol Toxicol

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BackgroundFilgrastim or methionyl-granulocyte colony-stimulating factor (Met-G-CSF), is a recombinant therapeutic protein widely used to treat severe neutropenia caused by myelosuppressive drugs in patients with nonmyeloid malignancies. In addition to its role in the regulation of granulopoiesis, treatment with G-CSF is considered the standard approach to mobilize CD34 positive (CD34+) mononuclear cells for reconstituting hemopoietic ability for bone marrow transplantation. An intended biosimilar filgrastim (coded BK0023) was produced in GMP conditions by E.coli fermentation according to an original recombinant process and showed physico-chemical properties and purity profile similar to Neupogen®, a commercial preparation of filgrastim. The aim of the present study was to demonstrate the comparability of BK0023 to Neupogen® in terms of both in vitro biological activities and in vivo toxicology, pharmacokinetics and pharmacodynamics.MethodsCell proliferation and radioligand binding assays were conducted in NFS-60 cells to compare the biological activity and functional interaction with the G-CSF receptor in vitro, while preclinical in vivo studies, including pharmacokinetics and pharmacodynamics after repeated dose were performed in normal and neutropenic rats. A phase I study was carried out in healthy male volunteers treated by multiple-dose subcutaneous administration of BK0023 and Neupogen® to evaluate their pharmacodynamic effects as well as their pharmacokinetic and safety profile and to demonstrate their pharmacodynamic equivalence and pharmacokinetic bioequivalence.ResultsThe results reported in this work demonstrate that BK0023 is comparable in terms of biological activity, efficacy and safety to Neupogen®.ConclusionsBK0023 has the same pharmacokinetic profile, efficacy and safety as the reference commercial filgrastim Neupogen® and therefore could be further developed to become a convenient option to treat neutropenia in oncological patients.Trial registrationTrial registration number (TRN): NCT01933971. Date of registration: Sept 6th 2013.

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“…Both ribose and phosphate binding pockets of UP are similar to those of E. coli PNP [69], and the higher affinity of UP for ribosides over 2'-deoxyribosides may be explained by the three hydrogen bonds formed by the OH2', Fig. (4c).…”

Section: B Quaternary Structure and Active Sitesmentioning

confidence: 93%

“…Recently [98], Oliva et al used pentapeptide scanning mutagenesis to cause random insertion of a 5 aminoacid cassette in the UP from E. coli. This mutagenesis approach appears to be useful for the rapid preparation of mutants that present altered enzymatic activities.…”

Section: Screening and Immobilisationmentioning

confidence: 99%

Nucleoside Phosphorylases

Lewkowicz

Iribarren

2006

COC

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Nucleoside phosphorylases (NPs) are transferases that catalyse the reversible cleavage of the glycosidic bond of ribo-or deoxyribo nucleosides, in the presence of inorganic phosphate, to generate the base and ribose-or deoxyribose-1-phosphate.Since pyrimidine as well as purine nucleoside phosphorylases exist, the combination of both enzymes makes possible the generation of purine nucleosides from pyrimidine ones. As a consequence, NPs from different sources, mainly bacterial, have been exploited as tools for the enzymatic synthesis of nucleoside analogues. These molecules are extensively used as antiviral and anticancer agents because of their ability to act as reverse transcriptase inhibitors or chain terminators in RNA or DNA synthesis.This review covers literature reports from 2000 on, focused mainly on the synthesis of nucleosides by free and immobilised microbial whole cells, along with some examples of modified nucleosides obtained by coupling transglycosylation to other enzymatic reactions.The biological aspects of NPs are also discussed since they became an interesting target for clinical applications due to their key role in nucleotide metabolism. Finally, brief comments about their structures and catalytic mechanisms are included.

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Mutagenesis of Escherichia coli uridine phosphorylase by random pentapeptide insertions

Cited by 6 publications

References 23 publications

Functional residues serve a dominant role in mediating the cooperativity of the protein ensemble

Functional residues serve a dominant role in mediating the cooperativity of the protein ensemble

Preclinical and clinical phase I studies of a new recombinant Filgrastim (BK0023) in comparison with Neupogen®

Nucleoside Phosphorylases

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