Enantiomeric perylene-glycerolipids as fluorogenic substrates for a dual wavelength assay of lipase activity and stereoselectivity

Zandonella, Gerhild; Haalck, Lutz; Spener, Fritz; Faber, Kurt; Paltauf, Fritz; Hermetter, Albin

doi:10.1002/(sici)1520-636x(1996)8:7<481::aid-chir4>3.0.co;2-e

Cited by 26 publications

(6 citation statements)

References 18 publications

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“…Consequently, either the carbonyl oxygen (O7) of the compound or the side chains of the appropriate residues must reorient to prevent unfavourable proteinϪligand interactions. before for other lipases [38,40]. the HA pocket, where the sn-3 fatty acid chain binds (Fig.…”

Section: Resultsmentioning

confidence: 91%

Structural basis of the chiral selectivity of Pseudomonas cepacia lipase

Lang

Mannesse

Haas

et al. 1998

European Journal of Biochemistry

141

102

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To investigate the enantioselectivity of Pseudomonas cepacia lipase, inhibition studies were performed with S C -and R C -(R P ,S P )-1,2-dialkylcarbamoylglycero-3-O-p-nitrophenyl alkylphosphonates of different alkyl chain lengths. P. cepacia lipase was most rapidly inactivated by R C -(R P ,S P )-1,2-dioctylcarbamoylglycero-3-O-p-nitrophenyl octylphosphonate (R C-trioctyl) with an inactivation half-time of 75 min, while that for the S C -(R P ,S P )-1,2-dioctylcarbamoylglycero-3-O-p-nitrophenyl octyl-phosphonate (S C -trioctyl) compound was 530 min. X-ray structures were obtained of P. cepacia lipase after reaction with R Ctrioctyl to 0.29-nm resolution at pH 4 and covalently modified with R C-(RP ,SP)-1,2-dibutylcarbamoylglycero-3-O-p-nitrophenyl butyl-phosphonate (R C -tributyl) to 0.175-nm resolution at pH 8.5. The three-dimensional structures reveal that both triacylglycerol analogues had reacted with the active-site Ser87, forming a covalent complex. The bound phosphorus atom shows the same chirality (S P) in both complexes despite the use of a racemic (R P ,S P ) mixture at the phosphorus atom of the triacylglycerol analogues. In the structure of R C -tributyl-complexed P. cepacia lipase, the diacylglycerol moiety has been lost due to an aging reaction, and only the butyl phosphonate remains visible in the electron density. In the R C-trioctyl complex the complete inhibitor is clearly defined; it adopts a bent tuning fork conformation. Unambiguously, four binding pockets for the triacylglycerol could be detected : an oxyanion hole and three pockets which accommodate the sn-1, sn-2, and sn-3 fatty acid chains. Van der Waals' interactions are the main forces that keep the radyl groups of the triacylglycerol analogue in position and, in addition, a hydrogen bond to the carbonyl oxygen of the sn-2 chain contributes to fixing the position of the inhibitor.Keywords : crystal structure; transition-state analog; enantioselectivity; lipase; stereospecificity.Lipases are lipolytic enzymes, which hydrolyze ester bonds fold, a structural motif common to a wide variety of hydrolases of triacylglycerols. However, their substrate specificity is not [3]. Their active sites contain a catalytic triad, Ser-His-Asp/Glu, limited to triacylglycerols. They may also hydrolyze ester bonds similar to those of serine proteases [4Ϫ6]. Studies with lipases of other compounds such as acetyl-arylpropionic acid esters, covalently complexed with organosulfates [7], organophosphates which are precursors for the nonsteroidal anti-inflammatory [8, 9], or organophosphonates [10Ϫ12] demonstrated that, in the agents naproxen and ibuprofen [1]. Because of this broad sub-presence of lipid-like compounds or organic solvents, their strate specificity, and because of their distinct stereopreferences, active-site regions may undergo drastic conformational changes, lipases have found widespread application in the enantioselec-exposing the catalytic residues and the surrounding hydrophobic tive synthesis of organic compounds, and in the resolution of ...

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Section: Resultsmentioning

confidence: 91%

Structural basis of the chiral selectivity of Pseudomonas cepacia lipase

Lang

Mannesse

Haas

et al. 1998

European Journal of Biochemistry

141

102

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“…All experiments were performed in triplicate. According to a modified method by Zandonella et al (1996) with 25 mM DGGR, 2.5 mg of h1Lip1 were incubated for 20 min at 25 1C, and the formation of methylresorufine was analysed spectrophotometrically at 580 nm (Panteghini et al, 2001). The initial hydrolysis velocity of h1Lip1 (1.5 mg) at various concentrations of substrate was determined spectrophotometrically by monitoring the formation of p-nitrophenol at 400 nm at 22 1C.…”

Section: Characterization Of H1lip1mentioning

confidence: 99%

“…To determine the presence of lipase activity, the triglyceride derivative 1,2-di-O-lauryl-rac-glycero-3glutaric acid 6 0 -methylresorufin ester (DGGR) (Sigma Aldrich) (Panteghini et al, 2001) was used as a chromogenic substrate . According to a modified method by Zandonella et al (1996) with 25 mM DGGR, 2.5 mg of h1Lip1 were incubated for 20 min at 25 1C, and the formation of methylresorufine was analysed spectrophotometrically at 580 nm (Panteghini et al, 2001). Candida rugosa lipase (Sigma Aldrich) was used as a positive control.…”

Section: Characterization Of H1lip1mentioning

confidence: 99%

Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment

Hårdeman¹,

Sjöling²

2007

FEMS Microbiology Ecology

154

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A novel lipase was isolated from a metagenomic library of Baltic Sea sediment bacteria. Prokaryotic DNA was extracted and cloned into a copy control fosmid vector (pCC1FOS) generating a library of >7000 clones with inserts of 24-39 kb. Screening for clones expressing lipolytic activity based on the hydrolysis of tributyrin and p-nitrophenyl esters, identified 1% of the fosmids as positive. An insert of 29 kb was fragmented and subcloned. Subclones with lipolytic activity were sequenced and an open reading frame of 978 bp encoding a 35.4-kDa putative lipase/esterase h1Lip1 (DQ118648) with 54% amino acid similarity to a Pseudomonas putida esterase (BAD07370) was identified. Conserved regions, including the putative active site, GDSAG, a catalytic triad (Ser148, Glu242 and His272) and a HGG motif, were identified. The h1Lip1 lipase was over expressed, (pGEX-6P-3 vector), purified and shown to hydrolyse p-nitrophenyl esters of fatty acids with chain lengths up to C14. Hydrolysis of the triglyceride derivative 1,2-di-O-lauryl-rac-glycero-3-glutaric acid 6'-methylresorufin ester (DGGR) confirmed that h1Lip1 was a lipase. The apparent optimal temperature for h1Lip1, by hydrolysis of p-nitrophenyl butyrate, was 35 degrees C. Thermal stability analysis showed that h1Lip1 was unstable at 25 degrees C and inactivated at 40 degrees C with t1/2 <5 min.

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“…The highest activities of enzyme assay using the substrate (i. e. p -NP-caprylate) was defined as the 100%. To determine the presence of esterase activity, the triglyceride derivative 1,2-di- O -lauryl- rac -glycero-3-glutaric acid 6'-methylresorufin ester (DGGR) (Sigma Aldrich) was used as a chromogenic substrate, and the formation of methylresorufin was analyzed spectrophotometrically at 580 nm [32-34]. Candida rugosa lipase (Sigma Aldrich) was used as a positive control.…”

Section: Methodsmentioning

confidence: 99%

Cloning and biochemical characterization of a novel lipolytic gene from activated sludge metagenome, and its gene product

Zhang

Han

2010

Microb Cell Fact

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In this study, a putative esterase, designated EstMY, was isolated from an activated sludge metagenomic library. The lipolytic gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene estMY contained a 1,083 bp open reading frame (ORF) encoding a polypeptide of 360 amino acids with a molecular mass of 38 kDa. Sequence analysis indicated that it showed 71% and 52% amino acid identity to esterase/lipase from marine metagenome (ACL67845) and Burkholderia ubonensis Bu (ZP_02382719), respectively; and several conserved regions were identified, including the putative active site, GDSAG, a catalytic triad (Ser203, Asp301, and His327) and a HGGG conserved motif (starting from His133). The EstMY was determined to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤C8). This EstMY exhibited the highest activity at 35°C and pH 8.5 respectively, by hydrolysis of p-NP caprylate. It also exhibited the same level of activity over wide temperature and pH spectra and in the presence of metal ions or detergents. The high level of stability of esterase EstMY with unique substrate specificities makes it highly valuable for downstream biotechnological applications.

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Enantiomeric perylene-glycerolipids as fluorogenic substrates for a dual wavelength assay of lipase activity and stereoselectivity

Cited by 26 publications

References 18 publications

Structural basis of the chiral selectivity of Pseudomonas cepacia lipase

Structural basis of the chiral selectivity of Pseudomonas cepacia lipase

Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment

Cloning and biochemical characterization of a novel lipolytic gene from activated sludge metagenome, and its gene product

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