Directed evolution of a glutaryl acylase into an adipyl acylase

Sio, Charles F.; Riemens, Anja; Laan, Jan-Metske van der; Verhaert, R.M.D.; Quax, Wim J.

doi:10.1046/j.1432-1033.2002.03143.x

Cited by 45 publications

(35 citation statements)

References 45 publications

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“…Chromosomal DNA was isolated from an overnight culture of P. aeruginosa PAO1 (Holloway collection) as described by Chen and Kuo (4). The open reading frame (ORF) encoding the acylase gene (bases 2638805 to 2636517, obtained from www.pseudomonas.com) (47) was amplified from chromosomal DNA by PCR and cloned into plasmid pMcTNde (45) under the control of the tac promoter, resulting in plasmid pMc-PA2385. For this procedure, the three bases preceding the putative start codon were altered to create an NdeI restriction site.…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

“…A putative acylase gene (the PA2385 gene) was located in the PAO1 genome sequence (47) by homology searching using the sequence encoding the ␤-lactam acylase from Pseudomonas SY-77 (45). This gene is also known as qsc112 (52) and pvdQ (21).…”

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confidence: 99%

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

et al. 2006

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“…However, due to precipitation phenomena, it was decided to use the DC protein assay with these purified acylase samples. This resulted in ϳ35% higher protein concentration values and thus in a decreased k cat value for SY-77 WT compared with an earlier study (16).…”

Section: Table II K M and K Cat Values Of Purified Enzymes With An Admentioning

confidence: 56%

“…It has a 5 times better ratio than the wild type enzyme and even a 2 times better ratio than our positive control SY-77 Y178H (16 M271V/Q291K/T374S . We genetically separated the multiple mutants and measured the AD/GL activity ratio of the resulting mutants in order to pinpoint the mutation that contributes most to the altered activity.…”

Section: Table Imentioning

confidence: 85%

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

Otten

Sio

Vrielink

et al. 2002

Journal of Biological Chemistry

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Using directed evolution, we have selected an adipyl acylase enzyme that can be used for a one-step bioconversion of adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) to 7-ADCA, an important compound for the synthesis of semisynthetic cephalosporins. The starting point for the directed evolution was the glutaryl acylase from Pseudomonas SY-77. The gene fragment encoding the ␤-subunit was divided into five overlapping parts that were mutagenized separately using error-prone PCR. Mutants were selected in a leucine-deficient host using adipyl-leucine as the sole leucine source. In total, 24 out of 41 plate-selected mutants were found to have a significantly improved ratio of adipyl-7-ADCA versus glutaryl-7-ACA hydrolysis. Several mutations around the substrate-binding site were isolated, especially in two hot spot positions: residues Phe-375 and Asn-266. Five mutants were further characterized by determination of their Michaelis-Menten parameters. Strikingly, mutant SY-77 N266H shows a nearly 10-fold improved catalytic efficiency (k cat /K m ) on adipyl-7-ADCA, resulting from a 50% increase in k cat and a 6-fold decrease in K m , without decreasing the catalytic efficiency on glutaryl-7-ACA. In contrast, the improved adipyl/glutaryl activity ratio of mutant SY-77 F375L mainly is a consequence of a decreased catalytic efficiency toward glutaryl-7-ACA. These results are discussed in the light of a structural model of SY-77 glutaryl acylase.

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Industrial Biotechnology

Soetaert¹,

Vandamme²

2010

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RESUMEN: La Biotecnología Industrial, como campo tecnológico de aprovechamiento industrial de materiales biológi-cos, se conforma como un área muy amplia con aplicaciones nuevas y tradicionales, que ha incorporado los nuevos desarrollos iniciados con el conocimiento del material genético, así como las nuevas tecnologías precisas para la obtención eficiente de los productos. En este trabajo se lleva a cabo una descripción de distintos productos de base biológica con interés comercial, así como unos breves comentarios sobre los procesos para su obtención.PALABRAS CLAVE: Biotecnología industrial; productos comerciales; biocatalizadores; procesos; biorreacciones; separaciones.ABSTRACT: Industrial biotechnology, is a broad area involving the industrial benefit of biological materials, that is applying efficiently the new developments following the genetic material knowledge, and the new technologies necessary for product development. A short description of the different products of commercial interest are here presented, followed by brief comments on the processes used to be obtained.

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Directed evolution of a glutaryl acylase into an adipyl acylase

Cited by 45 publications

References 45 publications

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

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

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

Industrial Biotechnology

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