Altering the Substrate Specificity of Cephalosporin Acylase by Directed Evolution of the β-Subunit

Otten, Linda G.; Sio, Charles F.; Vrielink, Johanna; Cool, Robbert H.; Quax, Wim J.

doi:10.1074/jbc.m208317200

Cited by 60 publications

(44 citation statements)

References 27 publications

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“…These residues for mutagenesis may be selected through a random, rational or combined evolutionary approach. In case of a glutaryl acylase from Pseudomonas SY-77, the use of epPCR followed by saturation mutagenesis close to the catalytic core of the enzyme increased the substrate selectivity for adipyl-7-aminodesacetoxycephalosporanic acid over 200-fold (34,35).…”

Section: Discussionmentioning

confidence: 99%

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

2007

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Glycoside hydrolase family 13 (GH13) members have evolved to possess various distinct reaction specificities despite the overall structural similarity. In this study we investigated the evolutionary input required to effeciently interchange these specificities and also compared the effectiveness of laboratory evolution techniques applied, i.e., error-prone PCR and saturation mutagenesis. Conversion of our model enzyme, cyclodextrin glucanotransferase (CGTase), into an R-amylase like hydrolytic enzyme by saturation mutagenesis close to the catalytic core yielded a triple mutant (A231V/F260W/F184Q) with the highest hydrolytic rate ever recorded for a CGTase, similar to that of a highly active R-amylase, while cyclodextrin production was virtually abolished. Screening of a much larger, error-prone PCR generated library yielded far less effective mutants. Our results demonstrate that it requires only three mutations to change CGTase reaction specificity into that of another GH13 enzyme. This suggests that GH13 members may have diversified by introduction of a limited number of mutations to the common ancestor, and that interconversion of reaction specificites may prove easier than previously thought.

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Section: Discussionmentioning

confidence: 99%

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

2007

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“…Cells (a total of 250 ml pooled from 100-ml cultures) were harvested by centrifugation, sonicated in 10 ml 50 mM Tris, 2 mM EDTA, pH 8.8, and centrifuged (30 min, 20, 000 ϫ g). The PA2385 protein was purified from the resulting supernatant by column chromatography as described earlier for Pseudomonas SY-77 glutaryl acylase (33), by the subsequent use of Q-Sepharose for anion exchange chromatography and phenyl-Sepharose for hydrophobic interaction chromatography, and finally by a polishing step on Q-Sepharose columns (from Amersham Biosciences). Fractions were analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and pooled based on the presence of the appropriate bands.…”

Section: Methodsmentioning

confidence: 99%

Quorum Quenching by an N -Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1

et al. 2006

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The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3 position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-Lhomoserine lactone (C 4 -HSL) and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C 12 -HSL), only 3-oxo-C 12 -HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C 12 -HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.Pseudomonas aeruginosa PAO1 is an opportunistic pathogen which causes disease mainly in individuals who are immunocompromised, have cystic fibrosis, or suffer from serious burn wounds. It utilizes two N-acyl-homoserine lactone (AHL)-dependent quorum-sensing systems, termed las and rhl, which together regulate an extensive set of cell population density and growth-phase-dependent virulence factors (7,22). The las and the rhl quorum-sensing systems employ N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C 12 -HSL) and N-butanoyl-L-homoserine lactone (C 4 -HSL), which function by activating the response regulator proteins LasR and RhlR, respectively. In diverse gram-negative bacteria, many different AHL signal molecules have been characterized. These all consist of a homoserine lactone (HSL) ring or nucleus which is connected via an amide bond to a fatty acid side chain of 4 to 14 carbon atoms in length that may contain an oxo or hydroxyl group at the 3Ј position and unsaturated bonds (Fig. 1).In P. aeruginosa PAO1, swarming motility, biofilm maturation, and the expression of virulence factors such as exoproteases, hemolysins, exotoxin A, exoenzyme S, pyocyanin, cyanide, and the cytotoxic lectins PA-IL and PA-IIL, as w...

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“…Other recent similar examples of single forward mutations leading to a catalytic shift include the Arabidopsis thaliana cycloartenol synthase, yeast lanosterol synthase (46)(47)(48)(49), and the adipyl acylase evolved from a Pseudomonas glutaryl acylase (50). From an analysis of the GalK substrate specificity profiles, one can begin to construct a loose structure-activity requirement for both wild-type enzyme and the corresponding Y371H mutant.…”

Section: Characterization Of the Galk Variant (Y371h)mentioning

confidence: 99%

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

Hoffmeister

Yang²,

Liu³

et al. 2003

Proceedings of the National Academy of Sciences

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Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates. Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides. Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase GalK. From this approach, one particular GalK mutant carrying a single amino acid exchange (Y371H) displayed a surprisingly substantial degree of kinase activity toward sugars as diverse as D-galacturonic acid, D-talose, L-altrose, and L-glucose, all of which failed as wild-type GalK substrates. Furthermore, this mutant provides enhanced turnover of the small pool of sugars converted by the wild-type enzyme. Comparison of this mutation to the recently solved structure of Lactococcus lactis GalK begins to provide a blueprint for further engineering of this vital class of enzyme. In addition, the rapid access to such promiscuous sugar C-1 kinases will significantly enhance accessibility to natural and unnatural sugar-1-phosphates and thereby impact both in vitro and in vivo glycosylation methodologies, such as natural product glycorandomization.galactokinase ͉ glycorandomization ͉ in vitro evolution ͉ enzyme M any clinically important medicines are derived from glycosylated natural products, the D-or L-sugar substituents of which often dictate their overall biological activity. This paradigm is found throughout the anticancer and antiinfective arenas with representative clinical examples (Fig. 1a), including enediynes (calicheamicin, 1), polyketides (doxorubicin, 2; erythromycin, 3), indolocarbazoles (staurosporine, 4), nonribosomal peptides (vancomycin, 5), polyenes (nystatin, 6), coumarins (novobiocin, 7), and cardiac glycosides (digitoxin, 8) (1-7). Given the importance of the sugars attached to these and other biologically significant metabolites, extensive effort has been directed in recent years toward altering sugars as a means to enhance or alter natural product-based therapeutics by both in vivo and in vitro approaches (ref. 8 and references therein). Among these, in vitro glycorandomization (IVG) makes use of the inherent or engineered substrate promiscuity of nucleotidylyltransferases and glycosyltransferases to activate and attach chemically synthesized sugar precursors to various natural product scaffolds (6,7,(9)(10)(11)(12)(13)(14)(15), the advantage of which is the ability to efficiently incorporate highly functionalized ''unnatural'' sugar substitutions into the corresponding natural product scaffold (Fig. 1b). In a recent demonstration of IVG, Ͼ50 analogs of 5 were generated, some of which displayed enhanced and distinct antibacterial profiles from the parent natural product (13).As starting materials, sugar phosphates play a key role in the entire IVG process. Thus, the ability to rapidly construct sugar phosphate ...

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Altering the Substrate Specificity of Cephalosporin Acylase by Directed Evolution of the β-Subunit

Cited by 60 publications

References 27 publications

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

Quorum Quenching by an N -Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

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