Egmond MR

References (31)

Title : Covalent anchoring of a racemization catalyst to CALB-beads: towards dual immobilization of DKR catalysts - Wieczorek_2011_Tetrahedron.Lett_52_1601
Author(s) : Wieczorek B , Traff A , Krumlinde P , Dijkstra HP , Egmond MR , van Koten G , Backvall JE , Klein Gebbink RJ
Ref : Tetrahedron Letters , 52 :1601 , 2011
Abstract : The preparation of a heterogeneous bifunctional catalytic system, combining the catalytic properties of an organometallic catalyst (racemization) with those of an enzyme (enantioselective acylation) is described. A novel ruthenium phosphonate inhibitor was synthesized and covalently anchored to a lipase immobilized on a solid support (CALB, Novozym 435). The immobilized bifunctional catalytic system showed activity in both racemization of (S)-1-phenylethanol and selective acylation of 1-phenylethanol.
ESTHER : Wieczorek_2011_Tetrahedron.Lett_52_1601
PubMedSearch : Wieczorek_2011_Tetrahedron.Lett_52_1601
PubMedID:
Gene_locus related to this paper: canar-LipB , fusso-cutas

Title : Solid-state structural characterization of cutinase-ECE-pincer-metal hybrids - Rutten_2009_Chemistry_15_4270
Author(s) : Rutten L , Wieczorek B , Mannie JP , Kruithof CA , Dijkstra HP , Egmond MR , Lutz M , Klein Gebbink RJ , Gros P , van Koten G
Ref : Chemistry , 15 :4270 , 2009
Abstract : The first crystal structures of lipases that have been covalently modified through site-selective inhibition by different organometallic phosphonate-pincer-metal complexes are described. Two ECE-pincer-type d(8)-metal complexes, that is, platinum (1) or palladium (2) with phosphonate esters (ECE = [(EtO)-(O=)P(-O-C(6)H(4)-(NO(2))-4)(-C(3)H(6)-4-(C(6)H(2)-(CH(2)E)(2))](-); E = NMe(2) or SMe) were introduced prior to crystallization and have been shown to bind selectively to the Ser(120) residue in the active site of the lipase cutinase to give cut-1 (platinum) or cut-2 (palladium) hybrids. For all five presented crystal structures, the ECE-pincer-platinum or -palladium head group sticks out of the cutinase molecule and is exposed to the solvent. Depending on the nature of the ECE-pincer-metal head group, the ECE-pincer-platinum and -palladium guests occupy different pockets in the active site of cutinase, with concomitant different stereochemistries on the phosphorous atom for the cut-1 (S(P)) and cut-2 (R(P)) structures. When cut-1 was crystallized under halide-poor conditions, a novel metal-induced dimeric structure was formed between two cutinase-bound pincer-platinum head groups, which are interconnected through a single mu-Cl bridge. This halide-bridged metal dimer shows that coordination chemistry is possible with protein-modified pincer-metal complexes. Furthermore, we could use NCN-pincer-platinum complex 1 as site-selective tool for the phasing of raw protein diffraction data, which shows the potential use of pincer-platinum complex 1 as a heavy-atom derivative in protein crystallography.
ESTHER : Rutten_2009_Chemistry_15_4270
PubMedSearch : Rutten_2009_Chemistry_15_4270
PubMedID: 19219875
Gene_locus related to this paper: fusso-cutas

Title : Lipase active-site-directed anchoring of organometallics: metallopincer\/protein hybrids - Kruithof_2005_Chemistry_11_6869
Author(s) : Kruithof CA , Casado MA , Guillena G , Egmond MR , van der Kerk-van Hoof A , Heck AJ , Klein Gebbink RJ , van Koten G
Ref : Chemistry , 11 :6869 , 2005
Abstract : The work described herein presents a strategy for the regioselective introduction of organometallic complexes into the active site of the lipase cutinase. Nitrophenol phosphonate esters, well known for their lipase inhibitory activity, are used as anchor functionalities and were found to be ideal tools to develop a single-site-directed immobilization method. A small series of phosphonate esters, covalently attached to ECE "pincer"-type d8-metal complexes through a propyl tether (ECE=[C6H3(CH2E)(2)-2,6]-; E=NR2 or SR), were designed and synthesized. Cutinase was treated with these organometallic phosphonate esters and the new metal-complex/protein hybrids were identified as containing exactly one organometallic unit per protein. The organometallic proteins were purified by membrane dialysis and analyzed by ESI-mass spectrometry. The major advantages of this strategy are: 1) one transition metal can be introduced regioselectively and, hence, the metal environment can potentially be fine-tuned; 2) purification procedures are facile due to the use of pre-synthesized metal complexes; and, most importantly, 3) the covalent attachment of robust organometallic pincer complexes to an enzyme is achieved, which will prevent metal leaching from these hybrids. The approach presented herein can be regarded as a tool in the development of regio- and enantioselective catalyst as well as analytical probes for studying enzyme properties (e.g., structure) and, hence, is a "proof-of-principle design" study in enzyme chemistry.
ESTHER : Kruithof_2005_Chemistry_11_6869
PubMedSearch : Kruithof_2005_Chemistry_11_6869
PubMedID: 16224766
Gene_locus related to this paper: fusso-cutas

Title : Partitioning of peptide-tagged proteins in aqueous two-phase systems using hydrophobically modified micelle-forming thermoseparating polymer - Nilsson_2002_Biochim.Biophys.Acta_1601_138
Author(s) : Nilsson A , Johansson HO , Mannesse M , Egmond MR , Tjerneld F
Ref : Biochimica & Biophysica Acta , 1601 :138 , 2002
Abstract : Genetic engineering has been used to construct hydrophobically modified fusion proteins of cutinase from Fusarium solani pisi and tryptophan-containing peptides. The aim was to enhance the partitioning of the tagged protein in a novel aqueous two-phase system formed by only one water-soluble polymer. The system was based on a hydrophobically modified random copolymer of ethylene oxide (EO) and propylene oxide (PO) units, HM-EOPO, with myristyl groups (C(14)H(29)) at both ends. The HM-EOPO polymer is strongly self-associating and has a lower critical solution temperature (cloud point) at 12 degrees C in water. At temperatures above the cloud point a two-phase system is formed with a water top phase and a polymer-enriched bottom phase. By adding a few percent of hydroxypropyl starch polymer, Reppal PES 200, to the system, it is possible to change the densities of the phases so the HM-EOPO-enriched phase becomes the top phase and Reppal-enriched phase is the bottom phase. Tryptophan-based peptides strongly preferred the HM-EOPO rich phase. The partitioning was increased with increasing length of the peptides. Full effect of the tag as calculated from peptide partitioning data was not found in the protein partitioning. When a short spacer was introduced between the protein and the tag the partitioning was increased, indicating a better exposure to the hydrophobic core of the polymer micelle. By adding a hydrophilic spacer between the protein and trp-tag, it was possible to increase the partitioning of cutinase 10 times compared to wild-type cutinase partitioning. By lowering the pH of the system and addition of NaCl, the partitioning of tagged protein was further increased towards the HM-EOPO phase. After isolating the HM-EOPO phase, the temperature was increased and the protein was back-extracted from the HM-EOPO phase to a fresh water phase.
ESTHER : Nilsson_2002_Biochim.Biophys.Acta_1601_138
PubMedSearch : Nilsson_2002_Biochim.Biophys.Acta_1601_138
PubMedID: 12445475

Title : Cutinase-peptide fusions in thermoseparating aqueous two-phase systems. Prediction of partitioning and enhanced tag efficiency by detergent addition - Nilsson_2002_J.Chromatogr.A_946_141
Author(s) : Nilsson A , Mannesse M , Egmond MR , Tjerneld F
Ref : Journal of Chromatography A , 946 :141 , 2002
Abstract : It is of increasing importance to develop efficient purification methods for recombinant proteins where the number of steps can be minimised. The aim has been to establish a method for predicting the partitioning of the wild-type target protein in an aqueous two-phase system, and with this as basis, develop fusion tags and optimise the phase system for enhanced partitioning of the target protein. The surface of the lipolytic enzyme cutinase from Fusarium solani pisi was investigated with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP). The accessible surface areas for the different amino acid residues were used together with peptide partitioning data to calculate the partition coefficient for the protein. The separation system was composed of a thermoseparating random copolymer of ethylene oxide and propylene oxide. Breox PAG 50A 1000, as top phase forming polymer and a hydroxypropyl starch polymer, Reppal PES 200, as bottom phase polymer. The calculated partition coefficient for the wild-type protein (K= 1.0) agreed reasonably well with the experimentally determined value (K=0.85). Genetic engineering was used to construct fusion proteins expressed in Saccharomyces cerevisiae based on cutinase and peptide tags containing tryptophan, to enhance the partitioning in aqueous two-phase systems. The partitioning of the cutinase constructs could qualitatively be predicted from peptide partitioning data, i.e. the trends in partitioning could be predicted. A spacer peptide introduced between protein and tag increased the partitioning of the protein towards the ethylene oxide-propylene oxide (EOPO) copolymer top phase. The aqueous two-phase system was modified by addition of detergent to increase the partitioning of the cutinase variants towards the EOPO copolymer phase. Triton and a series of C12En detergents selectively increased the partitioning of cutinase constructs with (WP)4-based tags up to 14 times compared to wild-type cutinase. The protein partition could almost quantitatively be predicted from the peptide partition data.
ESTHER : Nilsson_2002_J.Chromatogr.A_946_141
PubMedSearch : Nilsson_2002_J.Chromatogr.A_946_141
PubMedID: 11873963

Title : Cloning, purification and characterisation of Staphylococcus warneri lipase 2 - van Kampen_2001_Biochim.Biophys.Acta_1544_229
Author(s) : van Kampen MD , Rosenstein R , Gotz F , Egmond MR
Ref : Biochimica & Biophysica Acta , 1544 :229 , 2001
Abstract : A gene encoding an extracellular lipase was identified in Staphylococcus warneri 863. The deduced lipase is organised as a prepro-protein and has significant similarity to other staphylococcal lipases. The mature part of the lipase was expressed with an N-terminal histidine tag in Escherichia coli, purified and biochemically characterised. The results show that the purified lipase (named SWL2) combines the properties of the staphylococcal lipases characterised so far. It has both a high preference for short chain substrates and surprisingly, it also displays phospholipase activity. Homology alignment was used to analyse sequence-function relationships of the staphylococcal lipase family with the aim to identify the structural basis underlying the different properties of the staphylococcal lipases.
ESTHER : van Kampen_2001_Biochim.Biophys.Acta_1544_229
PubMedSearch : van Kampen_2001_Biochim.Biophys.Acta_1544_229
PubMedID: 11341932

Title : Substitutions of surface amino acid residues of cutinase probed by aqueous two-phase partitioning - Berggren_2000_Biochim.Biophys.Acta_1481_317
Author(s) : Berggren K , Egmond MR , Tjerneld F
Ref : Biochimica & Biophysica Acta , 1481 :317 , 2000
Abstract : The surface properties of a protein are often crucial for recognition and interaction with other molecules. Important functional residues can be identified by mutational analysis. There is a need for rapid methods to study protein surfaces and surface changes due to mutations. Partitioning in aqueous two-phase systems has the potential to be used in this respect since protein partitioning depends on the surface properties of the protein. The influence of surface-exposed amino acid residues in protein partitioning has been studied with cutinase variants, which differed in one or several amino acid residues as a result of site-directed mutagenesis. The solvent accessibility of the mutated residues was determined with a computer program, Graphical Representation and Analysis of Surface Properties. The aqueous two-phase system was composed of dextran and a random copolymer of ethylene oxide and propylene oxide. It was shown, for the first time, to what extent surface-exposed amino acid residues influence the partition coefficient in an aqueous two-phase system. The effect on partitioning could be described only taking into account solvent accessibility and type of residue substitution. The results demonstrate that the system can be used to detect conformational changes in mutant proteins since the expected effect on partitioning due to a mutation can be calculated. The aqueous two-phase system used here does indeed provide a rapid and convenient method to study protein surfaces and slight surface changes due to mutations.
ESTHER : Berggren_2000_Biochim.Biophys.Acta_1481_317
PubMedSearch : Berggren_2000_Biochim.Biophys.Acta_1481_317
PubMedID: 11018723

Title : Fusarium solani pisi cutinase - Egmond_2000_Biochimie_82_1015
Author(s) : Egmond MR , de Vlieg J
Ref : Biochimie , 82 :1015 , 2000
Abstract : Cutinase from Fusarium solani pisi has been studied extensively with respect to its structural and functional properties. The crystal structure of the enzyme was solved to high atomic resolution (1 angstrom), while data on structural dynamics have been obtained from detailed NMR studies. Functional data were mainly derived from kinetic studies using substrate analogues that simplify the kinetic behaviour. The properties of wild-type cutinase are reviewed and discussed in relation with the effects brought about by site-directed variants of the enzyme.
ESTHER : Egmond_2000_Biochimie_82_1015
PubMedSearch : Egmond_2000_Biochimie_82_1015
PubMedID: 11099798

Title : Extraction of peptide tagged cutinase in detergent-based aqueous two-phase systems - Rodenbrock_2000_Bioseparation_9_269
Author(s) : Rodenbrock A , Selber K , Egmond MR , Kula MR
Ref : Bioseparation , 9 :269 , 2000
Abstract : Detergent-based aqueous two-phase systems have the advantage to require only one auxiliary chemical to induce phase separation above the cloud point. In a systematic study the efficiency of tryptophan-rich peptide tags was investigated to enhance the partitioning of an enzyme to the detergent-rich phase using cutinase as an example. Up to 90% enzyme activity could be extracted in a single step from whole broth of recombinant Saccharomyces cerevisiae expressing cutinase variants carrying a (WP)4 tag. In contrast, the extraction yield of wild type cutinase was 2-3% only. The detergent concentration and the temperature are the main parameters to optimize the extraction yield. Considering availability, extraction yields, and price the detergent Agrimul NRE 1205 served best for enzyme recovery.
ESTHER : Rodenbrock_2000_Bioseparation_9_269
PubMedSearch : Rodenbrock_2000_Bioseparation_9_269
PubMedID: 11394565

Title : Modifying the substrate specificity of staphylococcal lipases - van Kampen_1999_Biochemistry_38_9524
Author(s) : van Kampen MD , Verheij HM , Egmond MR
Ref : Biochemistry , 38 :9524 , 1999
Abstract : The lipase from Staphylococcus hyicus (SHL) displays a high phospholipase activity whereas the homologous S. aureus lipase (SAL) is not active or hardly active on phospholipid substrates. Previously, it has been shown that elements within the region comprising residues 254-358 are essential for the recognition of phospholipids by SHL. To specifically identify the important residues, nine small clusters of SHL were individually replaced by the corresponding SAL sequence within region 254-358. For cloning convenience, a synthetic gene fragment of SHL was assembled, thereby introducing restriction sites into the SHL gene and optimizing the codon usage. All nine chimeras were well-expressed as active enzymes. Eight chimeras showed lipase and phospholipase activities within a factor of 2 comparable to WT-SHL in standard activity assays. Exchange of the polar SHL region 293-300 by the more hydrophobic SAL region resulted in a 32-fold increased k(cat)/K(m) value for lipase activity and a concomitant 68-fold decrease in k(cat)/K(m) for phospholipase activity. Both changes are due to effects on catalytic turnover as well as on substrate affinity. Subsequently, six point mutants were generated; G293N, E295F, T297P, K298F, I299V, and L300I. Residue E295 appeared to play a minor role whereas K298 was the major determinant for phospholipase activity. The mutation K298F caused a 60-fold decrease in k(cat)/K(m) on the phospholipid substrate due to changes in both k(cat) and K(m). Substitution of F298 by a lysine in SAL resulted in a 4-fold increase in phospholipase activity. Two additional hydrophobic to polar substitutions further increased the phospholipase activity 23-fold compared to WT-SAL.
ESTHER : van Kampen_1999_Biochemistry_38_9524
PubMedSearch : van Kampen_1999_Biochemistry_38_9524
PubMedID: 10413530

Title : Identification of a calcium binding site in Staphylococcus hyicus lipase: generation of calcium-independent variants - Simons_1999_Biochemistry_38_2
Author(s) : Simons JW , van Kampen MD , Ubarretxena-Belandia I , Cox RC , Alves dos Santos CM , Egmond MR , Verheij HM
Ref : Biochemistry , 38 :2 , 1999
Abstract : In this study we have identified the presence of a high-affinity binding site for calcium in the lipase from Staphylococcus hyicus. By means of isothermal titration calorimetry we showed that the enzyme binds one calcium per molecule of enzyme with a dissociation constant of 55 microM. The residual activity of the apoenzyme compared to the activity in the presence of calcium ions varies from 65% at 10 degreesC to nearly zero at 40 degreesC. On the basis of primary sequence alignment with other staphylococcal lipases and the lipases from Bacillus thermocatenulatus and from Pseudomonas glumae in combination with site-directed mutagenesis, aspartates 354 and 357 could be identified as calcium ligands. Kinetic measurements with the D357E variant showed that replacement of Asp357 by a glutamate decreased the affinity for calcium ions 30-fold. Introduction of a lysine, an asparagine, or an alanine at position 357 and of a lysine or an asparagine at position 354 resulted in calcium-independent variants. Isothermal titration calorimetry confirmed the loss of calcium binding. Although the D357K, D357N, and D357A variants did not bind calcium, at room temperature they were nearly as active as wild-type lipase in the presence of calcium, but at elevated temperatures these calcium-independent lipases showed a reduced activity. Over the whole temperature range the activities of the D354K and D354N variants are significantly lower than wild-type enzyme in the presence of calcium and are comparable to the activity of the wild-type apoenzyme. Our results show that binding of calcium is important for the structural stabilization of staphylococcal lipases (and possibly other lipases) and that it is possible to engineer calcium-independent variants on the basis of limited structural homology with another lipase.
ESTHER : Simons_1999_Biochemistry_38_2
PubMedSearch : Simons_1999_Biochemistry_38_2
PubMedID: 9890877
Gene_locus related to this paper: stahy-lipas

Title : Interfacial binding of cutinase rather than its catalytic activity determines the steady state interfacial tension during oil drop lipid hydrolysis - Flipsen_1999_Chem.Phys.Lipids_97_181
Author(s) : Flipsen JA , van Schaick MA , Dijkman R , van der Hijden HT , Verheij HM , Egmond MR
Ref : Chemistry & Physic of Lipids , 97 :181 , 1999
Abstract : Hydrolysis of triglycerides by cutinase from Fusarium solani pisi causes in oil drop tensiometer experiments a decrease of the interfacial tension. A series of cutinase variants with amino acid substitutions at its molecular surface yielded different values of the steady state interfacial tension. This tension value poorly correlated with the specific activity as such nor with the total activity (defined as the specific activity multiplied by the amount of enzyme bound) of the cutinase variants. Moreover, it appeared that at activity levels above 15% of that of wild type cutinase the contribution of hydrolysis to the decrease of the tension is saturating. A clear positive correlation was found between the interfacial tension plateau value and the interfacial binding of cutinase, as determined with attenuated total reflection Fourier transformed infrared spectroscopy (ATR-FTIR). These results indicate that the interfacial steady state level is not determined by the rate of hydrolysis, but mainly by the interfacial binding of cutinase.
ESTHER : Flipsen_1999_Chem.Phys.Lipids_97_181
PubMedSearch : Flipsen_1999_Chem.Phys.Lipids_97_181
PubMedID: 10192932

Title : NMR studies of Fusarium solani pisi cutinase in complex with phosphonate inhibitors - Prompers_1999_Biochemistry_38_5982
Author(s) : Prompers JJ , van Noorloos B , Mannesse ML , Groenewegen A , Egmond MR , Verheij HM , Hilbers CW , Pepermans HA
Ref : Biochemistry , 38 :5982 , 1999
Abstract : The backbone dynamics of Fusarium solani pisi cutinase in complex with a phosphonate inhibitor has been studied by a variety of nuclear magnetic resonance experiments to probe internal motions on different time scales. The results have been compared with dynamical studies performed on free cutinase. In solution, the enzyme adopts its active conformation only upon binding the inhibitor. While the active site Ser120 is rigidly attached to the stable alpha/beta core of the protein, the remainder of the binding site is very flexible in the free enzyme. The other two active site residues Asp175 and His188 as well as the oxyanion hole residues Ser42 and Gln121 are only restrained into their proper positions upon binding of the substrate-like inhibitor. The flap helix, which opens and closes the binding site in the free molecule, is also fixed in the cutinase-inhibitor complex. Our results are in contrast with the X-ray analysis results, namely that in the protein crystal, free cutinase has a well-defined active site and a preformed oxyanion hole and that it does not need any rearrangements to bind its substrate. Our solution studies show that cutinase does need conformational rearrangements to bind its substrate, which may form the rate-limiting step in catalysis.
ESTHER : Prompers_1999_Biochemistry_38_5982
PubMedSearch : Prompers_1999_Biochemistry_38_5982
PubMedID: 10320324

Title : Biochemical properties of staphylococcal (phospho)lipases - Simons_1998_Chem.Phys.Lipids_93_27
Author(s) : Simons JW , Gotz F , Egmond MR , Verheij HM
Ref : Chemistry & Physic of Lipids , 93 :27 , 1998
Abstract : Various staphylococci secrete lipases which require calcium ions for activity, and have profound preferences for substrates with different chain lengths. The lipase from Staphylococcus hyicus is exceptional since it has higher phospholipase than lipase activity. This paper gives an overview of the biochemical properties of these enzymes. It appears that chain length selectivity of these enzymes resides in the acylation step. Interfaces mainly influence the acylation step. Calcium ions do not influence the rate of acylation or deacylation although stabilise the enzyme against denaturation. Molecular modelling based on the X-ray structure of Pseudomonas glumae lipase was used to construct a model of the staphylococcal lipases. With this model the position of serveral residues involved in stubstrate selectivity was predicted. Moreover, a sequence element could be assigned that may function as the so-called lid domain in staphylococcal lipases. Sequence alignment of four staphylococcal lipases, and lipases from P. glumae and Bacillus thermocatenulatus identified several potential calcium ligands, one of which was verified by site directed mutagensesis. It is concluded that stabilisation of lipases by calcium ions might be a more general phenomenon than recognized so far.
ESTHER : Simons_1998_Chem.Phys.Lipids_93_27
PubMedSearch : Simons_1998_Chem.Phys.Lipids_93_27
PubMedID: 9720247

Title : Cloning, purification and characterisation of the lipase from Staphylococcus epidermidis--comparison of the substrate selectivity with those of other microbial lipases - Simons_1998_Eur.J.Biochem_253_675
Author(s) : Simons JW , van Kampen MD , Riel S , Gotz F , Egmond MR , Verheij HM
Ref : European Journal of Biochemistry , 253 :675 , 1998
Abstract : On the chromosome of Staphylococcus epidermidis RP62A the lipase gene (gehSE1) is immediately flanked by the icaAA'BC operon, which is involved in biofilm formation. Since lipase production might play a role in staphylococcal skin colonisation as well, we studied the biochemical properties of the staphylococcal lipases more closely. The DNA sequence and the deduced protein sequence revealed that gehSE1 is very similar to the lipase sequence of S. epidermidis strain 9. Like other staphylococcal lipases, gehSE1 is organised as a preproenzyme. The part of gehSE1 coding for the mature lipase was cloned and overexpressed as a fusion protein with an N-terminal histidine tag in Escherichia coli. The lipase was purified to homogeneity using a combination of precipitation techniques, metal-affinity chromatography and gel filtration. Biochemical characterisation showed that this lipase is closely related to the lipase from Staphylococcus aurelis NCTC8530. Both enzymes have a pH optimum around 6, are very stable at low pH, and need calcium as a cofactor for catalytic activity. The preferred substrates are small triacylglycerols, with a maximum activity toward tributyrylglycerol. Comparison of the substrate selectivity with those of other microbial lipases showed that phospholipids are generally poor substrates for lipases. An exception is the lipase from Staphylococcus hyicus, which prefers phospholipids as a substrate, distinguishing this staphylococcal lipase from other microbial lipases. These results are discussed in view of the structure/function relationships of staphylococcal lipases, and the possible involvement of these enzymes in biological processes such as skin colonisation and pathogenesis.
ESTHER : Simons_1998_Eur.J.Biochem_253_675
PubMedSearch : Simons_1998_Eur.J.Biochem_253_675
PubMedID: 9654065

Title : Substrate specificity of Staphylococcus hyicus lipase and Staphylococcus aureus lipase as studied by in vivo chimeragenesis - van Kampen_1998_Biochemistry_37_3459
Author(s) : van Kampen MD , Dekker N , Egmond MR , Verheij HM
Ref : Biochemistry , 37 :3459 , 1998
Abstract : Staphylococcus hyicus lipase (SHL) and Staphylococcus aureus lipase (SAL) are highly homologous enzymes, yet they show remarkable differences in their biochemical characteristics. SHL displays a high phospholipase activity, hydrolyses neutral lipids, and has no chain length preference, whereas SAL only degrades short-chain fatty acid esters. To identify the regions in the primary sequence of SHL responsible for phospholipase activity and chain length selectivity, a set of histidine-tagged SAL/SHL chimeras was generated by in vivo recombination in Escherichia coli. Several classes of chimeric enzymes were identified on the basis of restriction site analysis. All chimeras were well-expressed as active enzymes. They were characterized for their specific activities on both phospholipids and p-nitrophenyl esters of various chain lengths. Phospholipase activity appeared to be determined by three regions, all located in the C-terminal domain of SHL. Testing of the enzymatic activity of the chimeras toward p-nitrophenyl esters showed that chain length selectivity is defined by elements within the region of residues 180-253. Moreover, also residues along the stretch 275-358 contribute to the binding of acyl chains. Interestingly, several chimeras were even more active than the parent enzymes on long-chain p-nitrophenyl esters.
ESTHER : van Kampen_1998_Biochemistry_37_3459
PubMedSearch : van Kampen_1998_Biochemistry_37_3459
PubMedID: 9521667
Gene_locus related to this paper: stahy-lipas

Title : The phospholipase activity of Staphylococcus hyicus lipase strongly depends on a single Ser to Val mutation - van Kampen_1998_Chem.Phys.Lipids_93_39
Author(s) : van Kampen MD , Simons JW , Dekker N , Egmond MR , Verheij HM
Ref : Chemistry & Physic of Lipids , 93 :39 , 1998
Abstract : Site-directed mutagenesis and domain exchange were used to investigate the role of the C-terminal domains of Staphylococcus hyicus lipase (SHL) and S. aureus lipase (SAL) in substrate selectivity. The introduction of a single point mutation coding for the substitution of Val for Ser356 in SHL yields an enzyme which has retained full lipase activity, but with more than 12-fold lower phospholipase activity. Starting with this S356V variant of SHL the C-terminal 40 amino acids were replaced by the corresponding SAL sequence. Although 23 amino acid changes were introduced simultaneously the impact on the phospholipase/lipase activity ratio was only 4-fold. We therefore conclude that in the C-terminal domain it is Ser356 which mainly determines phospholipase activity. The introduction of a Val357 to Ser substitution in SAL did not turn SAL into a phospholipase, showing that residues from other domains contribute to this activity as well. The results are discussed in view of the sequence homology of lipases and (lyso)phospholipases.
ESTHER : van Kampen_1998_Chem.Phys.Lipids_93_39
PubMedSearch : van Kampen_1998_Chem.Phys.Lipids_93_39
PubMedID: 9720248
Gene_locus related to this paper: stahy-lipas

Title : Cutinase binding and activity at the triolein-water interface monitored by oil drop tensiometry - Flipsen_1998_Chem.Phys.Lipids_95_169
Author(s) : Flipsen JA , Kramer RA , van Duijnhoven JP , van der Hijden HT , Egmond MR , Verheij HM
Ref : Chemistry & Physic of Lipids , 95 :169 , 1998
Abstract : Changes of the oil-water interfacial tension resulting from binding of Fusarium solani pisi cutinase and subsequent lipid hydrolysis were investigated using the oil drop technique. An ELISA was developed to determine the amount of cutinase bound to the triolein-water interface after biotinylation of the enzyme. Cutinase irreversibly adsorbs to a maximum value of about 2 mg/m2. A minimal specific activity of 110 mumol/min/mg was calculated for cutinase acting on a single oil droplet, which is close to the activity found for triglyceride emulsions. At a maximum surface load cutinase could generate one monolayer of fatty acid products per second at the interface. It was found that oleic acid rapidly dissolves into the oil phase under the conditions used. The interfacial tension measured reflects the adsorption of cutinase to the oil droplet and also responds to the fate of the hydrolysis products. A model is presented that describes the catalytic events at the oil-water interface during lipid hydrolysis.
ESTHER : Flipsen_1998_Chem.Phys.Lipids_95_169
PubMedSearch : Flipsen_1998_Chem.Phys.Lipids_95_169
PubMedID: 9853365

Title : nmd, a novel gene differentially expressed in human melanoma cell lines, encodes a new atypical member of the enzyme family of lipases - van Groningen_1997_FEBS.Lett_404_82
Author(s) : van Groningen JJ , Egmond MR , Bloemers HP , Swart GW
Ref : FEBS Letters , 404 :82 , 1997
Abstract : nmd, a novel gene, was isolated by applying the differential mRNA display method to human melanoma cell lines with different metastatic capacity. In a panel of 17 other human tumor cell lines, nmd RNA expression could only be detected at low levels in T24 (bladder carcinoma) and Caco-2 (colon adenocarcinoma). Furthermore, it was found in placenta and liver, but not in skin, colon, spleen, lung, muscle, prostate and kidney. Sequence analysis classified the nmd gene product as a new member of the enzyme family of lipases (almost 30% identity in amino acid sequence with other human lipases). Active site residues of lipases were conserved in NMD, but NMD lacks the regulatory lid domain, which controls entry to the active site in classical lipases. A similar deletion was earlier reported by others in the guinea pig pancreatic (phospho)lipase GPLRP2 and the phospholipase A1 from hornet venom (DolmI).
ESTHER : van Groningen_1997_FEBS.Lett_404_82
PubMedSearch : van Groningen_1997_FEBS.Lett_404_82
PubMedID: 9074642
Gene_locus related to this paper: human-PLA1A

Title : Chiral preference of cutinase in the reaction with phosphonate inhibitors -
Author(s) : Mannesse ML , De Haas GH , van der Hijden HT , Egmond MR , Verheij HM
Ref : Biochemical Society Transactions , 25 :165 , 1997
PubMedID: 9056864

Title : Impact of structural information on understanding lipolytic function -
Author(s) : Egmond MR , van Bemmel CJ
Ref : Methods Enzymol , 284 :119 , 1997
PubMedID: 9379930

Title : Dissecting the catalytic mechanism of staphylococcal lipases using carbamate substrates: chain length selectivity, interfacial activation, and cofactor dependence - Simons_1997_Biochemistry_36_14539
Author(s) : Simons JW , Boots JW , Kats MP , Slotboom AJ , Egmond MR , Verheij HM
Ref : Biochemistry , 36 :14539 , 1997
Abstract : p-Nitrophenyl N-alkylcarbamates with different alkyl chains were used as substrates to determine separately the carbamylation and decarbamylation rates of the lipases from Staphylococcus hyicus and S. aureus. Both enzymes are reversibly inhibited by these compounds due to a rapid carbamylation of their active site serines followed by a slow decarbamylation. The carbamylation reaction is strongly pH-dependent and the pH profile suggests that an unprotonated histidine is required for this reaction. In contrast, the decarbamylation is pH-independent suggesting the presence of a hydrogen bond between the active site histidine and the carbamyl moiety. S. hyicus lipase preferably reacts with medium to long chain carbamates with an optimum for eight carbon atoms. In contrast, S. aureus lipase is highly specific for short chain carbamates. These results are in agreement with the respective substrate preferences of both lipases toward natural lipids. The decarbamylation rates of both enzymes hardly depend on the alkyl chain length, and from this it is concluded that chain length selectivity is expressed in the first step of catalysis. Both the carbamylation and decarbamylation reaction rates of S. hyicus lipase are enhanced in the presence of micelles, the activation effect being most pronounced in the first step. For the S. aureus lipase only a small influence of interfaces on both reaction steps was observed. These results are discussed in view of a possible role of a lid covering the active site. Kinetic experiments in the presence and absence of calcium strongly suggest that calcium ions are important for the structural stabilization of the unmodified as well as of the carbamylated enzymes. This structural function of calcium was supported by urea unfolding experiments, from which it appeared that for both enzymes the free energy for unfolding is significantly lower in the absence of calcium. In conclusion our results show that kinetic differences between both lipases reside in the acylation step, and that calcium is important for the structural stabilization of the unmodified, and moreover, the acylated enzymes.
ESTHER : Simons_1997_Biochemistry_36_14539
PubMedSearch : Simons_1997_Biochemistry_36_14539
PubMedID: 9398172

Title : Action of cutinase at the triolein-water interface. Characterisation of interfacial effects during lipid hydrolysis using the oil-drop tensiometer as a tool to study lipase kinetics - Flipsen_1996_Chem.Phys.Lipids_84_105
Author(s) : Flipsen JA , van der Hijden HT , Egmond MR , Verheij HM
Ref : Chemistry & Physic of Lipids , 84 :105 , 1996
Abstract : Interfacial events during lipid hydrolysis by cutinase are described as measured with the oil-drop tensiometer. A linear relation between enzyme concentration and initial decrease of oil-water interface tension (gamma o/w) due to lipolytic activity was observed. The amount of hydrolysis products showed a non-linear relation with gamma o/w. Hydrolysis is linear with time, even when the area occupied by the fatty acid molecules exceeds the drop surface by a factor 7000. At pH 9.0, fatty acids were found to partition mainly in the oil phase. Formation of calcium soaps and ionization increase the impact of fatty acids on gamma o/w without affecting enzyme activity. The presence of fatty acids at the interface, added prior to cutinase, delayed hydrolysis effects on gamma o/w. Fatty acids in the water phase almost completely abolished adsorption effects on gamma o/w, when the concentration was over the critical micellar concentration (cmc).
ESTHER : Flipsen_1996_Chem.Phys.Lipids_84_105
PubMedSearch : Flipsen_1996_Chem.Phys.Lipids_84_105
PubMedID: 9081775

Title : Cutinase from Fusarium solani pisi hydrolyzing triglyceride analogues. Effect of acyl chain length and position in the substrate molecule on activity and enantioselectivity - Mannesse_1995_Biochemistry_34_6400
Author(s) : Mannesse ML , Cox RC , Koops BC , Verheij HM , De Haas GH , Egmond MR , van der Hijden HT , de Vlieg J
Ref : Biochemistry , 34 :6400 , 1995
Abstract : Triglyceride analogues were synthesized in which one of the primary acyl ester functions has been replaced by an alkyl group and the secondary acyl ester bond has been replaced by an acyl amino bond. The chain length at either position was varied, and both (R)- and (S)-enantiomers of each compound were synthesized. These pseudo triglycerides contain only one hydrolyzable ester bond, and they are ideally suited to studying the influence of the chain length at the 1-, 2-, and 3-position on lipase activity and on stereopreference. These substrates were used to characterize cutinase from Fusarium solani pisi. Our results show that the activity of cutinase is very sensitive to the length and distribution of the acyl chains and that the highest activities are found when the chains at positions 1 and 3 contain three or four carbon atoms. The enzyme preferentially hydrolyzes the (R)-enantiomers, but this preference is strongly dependent on the acyl chain length distribution, with (R) over (S) activity ratios varying from about 30 to 1. This enantioselectivity was found in three different assay systems: a mixed micellar, a reverse micellar, and a monolayer study. Our data suggest that at least two alkyl chains of the pseudo triglycerides must be fixed during hydrolysis. Therefore, these substrates were used to characterize mutants of cutinase with mutations in putative lipid binding domains. Two mutants (A85F and A85W) have increased activities. The results obtained with these mutants suggest an interaction of the acyl chain of the scissile ester bond with a surface loop, comprising residues 80-90, in the enzyme-substrate complex.
ESTHER : Mannesse_1995_Biochemistry_34_6400
PubMedSearch : Mannesse_1995_Biochemistry_34_6400
PubMedID: 7756270
Gene_locus related to this paper: fusso-cutas

Title : Phosphonate analogues of triacylglycerols are potent inhibitors of lipase - Mannesse_1995_Biochim.Biophys.Acta_1259_56
Author(s) : Mannesse ML , Boots JW , Dijkman R , Slotboom AJ , van der Hijden HT , Egmond MR , Verheij HM , De Haas GH
Ref : Biochimica & Biophysica Acta , 1259 :56 , 1995
Abstract : 1,2-Dioctylcarbamoylglycero-3-O-p-nitrophenyl alkylphosphonates, with alkyl being methyl or octyl, were synthesised and tested as irreversible inhibitors of cutinase from Fusarium solani pisi and Staphylococcus hyicus lipase. Rapid inactivation of these enzymes occurred with a concomitant release of one mole of p-nitrophenol per mole of enzyme. With both lipases a higher reactivity was observed when the alkyl substituent on the phosphonate is a methyl rather than an octyl chain. Both lipases are highly selective for the chirality of these compounds at glycerol and at phosphorus. Rapid inactivation at an inhibitor concentration of 0.1 mol% in 100 mM NaTDOC (t 1/2 < 60 min.) occurred when the glycerol moiety had the (R) configuration, while inhibitors of the (S) configuration react 4-10-fold more slowly. The isomer with the p-nitrophenyl octylphosphonate attached to the secondary hydroxyl group of glycerol hardly inhibited (t 1/2 > 1 day) the lipases. These results reflect the known positional- and stereopreference of these enzymes which preferentially release the fatty acid at sn-3 of natural triacylglycerols. The enzymes appeared to be even more selective for the chirality at phosphorus, the differences in reactivity of the faster and slower reacting isomers being as high as about 250-fold for the methylphosphonates and about 60-fold for the octylphosphonates. These phosphonates can be regarded as true active site-directed inhibitors. The inhibited enzymes can be considered as analogues of the tetrahedral intermediate in the acylation step that occurs during triacylglycerol hydrolysis.
ESTHER : Mannesse_1995_Biochim.Biophys.Acta_1259_56
PubMedSearch : Mannesse_1995_Biochim.Biophys.Acta_1259_56
PubMedID: 7492616
Gene_locus related to this paper: fusso-cutas , stahy-lipas

Title : Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene - Kok_1995_Mol.Microbiol_15_803
Author(s) : Kok RG , van Thor JJ , Nugteren-Roodzant IM , Brouwer MB , Egmond MR , Nudel CB , Vosman B , Hellingwerf KJ
Ref : Molecular Microbiology , 15 :803 , 1995
Abstract : The extracellular lipase from Acinetobacter calcoaceticus BD413 was purified to homogeneity, via hydrophobic-interaction fast performance liquid chromatography (FPLC), from cultures grown in mineral medium with hexadecane as the sole carbon source. The enzyme has an apparent molecular mass of 32 kDa on SDS-polyacrylamide gels and hydrolyses long acyl chain p-nitrophenol (pNP) esters, like pNP palmitate (pNPP), with optimal activity between pH 7.8 and 8.8. Additionally, the enzyme shows activity towards triglycerides such as olive oil and tributyrin and towards egg-yolk emulsions. The N-terminal amino acid sequence of the mature protein was determined, and via reverse genetics the structural lipase gene was cloned from a gene library of A. calcoaceticus DNA in Escherichia coli phage M13. Sequence analysis of a 2.1 kb chromosomal DNA fragment revealed one complete open reading frame, lipA, encoding a mature protein with a predicted molecular mass of 32.1 kDa. This protein shows high similarity to known lipases, especially Pseudomonas lipases, that are exported in a two-step secretion mechanism and require a lipase-specific chaperone. The identification of an export signal sequence at the N-terminus of the mature lipase suggests that the lipase of Acinetobacter is also exported via a two-step translocation mechanism. However, no chaperone-encoding gene was found downstream of lipA, unlike the situation in Pseudomonas. Analysis of an A. calcoaceticus mutant showing reduced lipase production revealed that a periplasmic disulphide oxidoreductase is involved in processing of the lipase. Via sequence alignments, based upon the crystal structure of the closely related Pseudomonas glumae lipase, a model has been made of the secondary-structure elements in AcLipA. The active site serine of AcLipA was changed to an alanine, via site-directed mutagenesis, resulting in production of an inactive extracellular lipase.
ESTHER : Kok_1995_Mol.Microbiol_15_803
PubMedSearch : Kok_1995_Mol.Microbiol_15_803
PubMedID: 7596283
Gene_locus related to this paper: acica-este3

Title : Structural and functional aspects of an industrial lipase -
Author(s) : Egmond MR , Antheunisse WP , van Bemmel CM , Ravestein P , Frenken LG
Ref : Annals of the New York Academy of Sciences , 750 :195 , 1995
PubMedID: 7785849

Title : Analysis of the structure of Pseudomonas glumae lipase - Noble_1994_Protein.Eng_7_559
Author(s) : Noble ME , Cleasby A , Johnson LN , Egmond MR , Frenken LG
Ref : Protein Engineering , 7 :559 , 1994
Abstract : The lipase produced by Pseudomonas glumae is monomeric in the crystalline state and has a serine protease-like catalytic triad; Ser87-His285-Asp263. The largest domain of the protein resembles closely a subset of the frequently observed alpha/beta-hydrolase fold and contains a well-defined calcium site. This paper describes structural analysis of this protein, focusing on (i) structural comparison with the lipase from Geotrichum candidum, (ii) the probable nature of the conformational change involved in substrate binding and (iii) structural variations amongst the family of Pseudomonas lipases. This analysis reveals similarities between P. glumae lipase and G. candidum lipase involving secondary structural elements of the hydrolase core and the loops carrying the catalytic serine and histidine residues. A possible functional equivalence has also been identified between parts of the two molecules thought to be involved in a conformational change. In addition, determination of the structure of P. glumae lipase has allowed rationalization of previously reported protein engineering experiments, which succeeded in improving the stability of the enzyme with respect to proteolysis.
ESTHER : Noble_1994_Protein.Eng_7_559
PubMedSearch : Noble_1994_Protein.Eng_7_559
PubMedID: 8029212

Title : The crystal structure of triacylglycerol lipase from Pseudomonas glumae reveals a partially redundant catalytic aspartate - Noble_1993_FEBS.Lett_331_123
Author(s) : Noble ME , Cleasby A , Johnson LN , Egmond MR , Frenken LG
Ref : FEBS Letters , 331 :123 , 1993
Abstract : The family of lipases (triacylglycerol-acyl-hydrolases EC 3.1.1.3) constitutes an interesting class of enzymes because of their ability to interact with lipid-water interfaces, their wide range of substrate specificities, and their potential industrial applications. Here we report the first crystal structure of a bacterial lipase, from Pseudomonas glumae. The structure is formed from three domains, the largest of which contains a subset of the alpha/beta hydrolase fold and a calcium site. Asp263, the acidic residue in the catalytic triad, has previously been mutated into an alanine with only a modest reduction in activity.
ESTHER : Noble_1993_FEBS.Lett_331_123
PubMedSearch : Noble_1993_FEBS.Lett_331_123
PubMedID: 8405390
Gene_locus related to this paper: burgl-lipas

Title : Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues - Frenken_1992_Appl.Environ.Microbiol_58_3787
Author(s) : Frenken LG , Egmond MR , Batenburg AM , Bos JW , Visser C , Verrips CT
Ref : Applied Environmental Microbiology , 58 :3787 , 1992
Abstract : The lipA gene encoding the extracellular lipase produced by Pseudomonas glumae PG1 was cloned and characterized. A sequence analysis revealed an open reading frame of 358 codons encoding the mature lipase (319 amino acids) preceded by a rather long signal sequence of 39 amino acids. As a first step in structure-function analysis, we determined the Ser-Asp-His triad which makes up the catalytic site of this lipase. On the basis of primary sequence homology with other known Pseudomonas lipases, a number of putative active site residues located in conserved areas were found. To determine the residues actually involved in catalysis, we constructed a number of substitution mutants for conserved Ser, Asp, and His residues. These mutant lipases were produced by using P. glumae PG3, from which the wild-type lipase gene was deleted by gene replacement. By following this approach, we showed that Ser-87, Asp-241, and His-285 make up the catalytic triad of the P. glumae lipase. This knowledge, together with information on the catalytic mechanism and on the three-dimensional structure, should facilitate the selection of specific modifications for tailoring this lipase for specific industrial applications.
ESTHER : Frenken_1992_Appl.Environ.Microbiol_58_3787
PubMedSearch : Frenken_1992_Appl.Environ.Microbiol_58_3787
PubMedID: 1476423
Gene_locus related to this paper: burgl-lipas

Title : Crystallization and preliminary X-ray study of a lipase from Pseudomonas glumae - Cleasby_1992_J.Mol.Biol_224_281
Author(s) : Cleasby A , Garman E , Egmond MR , Batenburg M
Ref : Journal of Molecular Biology , 224 :281 , 1992
Abstract : Lipase from Pseudomonas glumae has been purified and crystallized in two forms, using the hanging drop method of vapour diffusion at 4 degrees C and 15 degrees C. Both forms grew at pH 9.0 from 0.1 M-Tris buffer in the presence of 10% (v/v) acetone. Form 1 was crystallized from 27 to 29% polyethylene glycol in the presence of less than 0.5% (v/v) n-dodecyl-beta-D-glucopyranoside. Form 2 was grown from 17 to 19% ammonium sulphate in the presence of 1% n-octyl-beta-D-glucopyranoside. Form 1 is orthorhombic with space group P2(1)2(1)2(1), and cell dimensions of a = 158.1 A, b = 158.6 A, c = 63.4 A, Form 2 is tetragonal with space group P4(1)2(1)2 (or P4(3)2(1)2) and cell dimensions of a = 89.3 A, c = 180.4 A. Form 1 probably has four molecules per asymmetric unit and diffracts to at least 2.5 A. Form 2 has two molecules per asymmetric unit and diffracts to at least 3.0 A.
ESTHER : Cleasby_1992_J.Mol.Biol_224_281
PubMedSearch : Cleasby_1992_J.Mol.Biol_224_281
PubMedID: 1548708
Gene_locus related to this paper: burgl-lipas