The tarnished plant bug has become increasingly resistant to organophosphates in recent years. To better understand acephate resistance mechanisms, biological, biochemical, and molecular experiments were systematically conducted with susceptible (LLS) and acephate-selected (LLR) strains. Selection of a field population with acephate significantly increased resistance ratio to 5.9-fold, coupled with a significant increase of esterase activities by 2-fold. Microarray analysis of 6,688 genes revealed 329 up- and 333 down-regulated (≥2-fold) genes in LLR. Six esterase, three P450, and one glutathione S-transferase genes were significantly up-regulated, and no such genes were down-regulated in LLR. All vitellogenin and eggshell protein genes were significantly down-regulated in LLR. Thirteen protease genes were significantly down-regulated and only 3 were up-regulated in LLR. More than twice the number of catalysis genes and more than 3.6-fold of metabolic genes were up-regulated, respectively, as compared to those down-regulated with the same molecular and biological functions. The large portion of metabolic or catalysis genes with significant up-regulations indicated a substantial increase of metabolic detoxification in LLR. Significant increase of acephate resistance, increases of esterase activities and gene expressions, and variable esterase sequences between LLS and LLR consistently demonstrated a major esterase-mediated resistance in LLR, which was functionally provable by abolishing the resistance with esterase inhibitors. In addition, significant elevation of P450 gene expression and reduced susceptibility to imidacloprid in LLR indicated a concurrent resistance risk that may impact other classes of insecticides. This study demonstrated the first association of down-regulation of reproductive- and digestive-related genes with resistance to conventional insecticides, suggesting potential fitness costs associated with resistance development. This study shed new light on the understanding of the molecular basis of insecticide resistance, and the information is highly valuable for development of chemical control guidelines and tactics to minimize resistance and cross-resistance risks.

The genome sequence of Streptomyces coelicolor A3(2) contains more than 50 genes coding for putative lipolytic enzymes. Many studies have shown the capacity of this actinomycete to store important reserves of intracellular triacylglycerols in nutrient depletion situations. In the present study, we used genome mining of S. coelicolor to identify genes coding for putative, non-secreted esterases/lipases. Two genes were cloned and successfully overexpressed in E. coli as His-tagged fusion proteins. One of the recombinant enzymes, EstC, showed interesting cold-active esterase activity with a strong potential for the production of valuable esters. The purified enzyme displayed optimal activity at 35°C and was cold-active with retention of 25% relative activity at 10°C. Its optimal pH was 8.5-9 but the enzyme kept more than 75% of its maximal activity between pH 7.5 and 10. EstC also showed remarkable tolerance over a wide range of pH values, retaining almost full residual activity between pH 6-11. The enzyme was active toward short-chain p-nitrophenyl esters (C2-C12), displaying optimal activity with the valerate (C5) ester (k(cat)/K(m) = 737±77 s(-1) mM(-1)). The enzyme was also very active toward short chain triglycerides such as triacetin (C2:0) and tributyrin (C4:0), in addition to showing good primary alcohol and organic solvent tolerance, suggesting it could function as an interesting candidate for organic synthesis of short-chain esters such as flavors.

In the work, molecular docking method was applied to extensively predict the enantioselectivity of lipases and esterases. A ligand library consisted of 69 chiral substrates was docked to four lipases and two esterases to set up the prediction model. During the docking process, necessary modification was carried out on van de Waals and hydrogen bond parameters of enzyme/substrate pair so that the ligands were able to adopt productive geometry in the enzymes. The docking results correctly indicated the enantiopreference for 91%(63/69) of docking pairs and the docking energy difference between substrate enantiomers (Delta Delta G(docking)) was significantly (correlation coefficient = 0.72, P < 0.05) correlated with the activation free energy difference (Delta Delta G( not equal)) that was quantitatively correlated with enantioselectivity of the enzymes. The prediction method was further validated by docking with another 12 enzyme/substrate pairs. Moreover, the prediction error was susceptible to the size of groups bonded to substrate's chiral center and expected Delta Delta G( not equal) values but was not related to the substrate type and reaction medium. The possible reasons of observed error were discussed. It is demonstrated that the docking method has great application potential in high performance prediction of enzyme enantioselectivity.

The esterase/lipase family (EC 3.1.1.3/EC 3.1.1.1) represents a diverse group of hydrolases that catalyze the cleavage of ester bonds and are widely distributed in animals, plants and microorganisms. Among these enzymes, hormone-sensitive lipases, play a critical role in the regulation of rodent fat cell lipolysis and are regarded as adipose tissue-specific enzymes. Recently, we reported the structural and biological characterization of EstE5 from the metagenome library [K.H. Nam, M.Y. Kim, S.J. Kim, A. Priyadarshi, W.H. Lee, K.Y. Hwang, Structural and functional analysis of a novel EstE5 belonging to the subfamily of hormone-sensitive lipase, Biochem. Biophys. Res. Commun. 379 (2009) 553-556]. The structure of this protein revealed that it belongs to the HSL-family. Here, we report the inhibition of the activity of the HSL-homolog EstE5 protein as determined by the use of esterase/lipase inhibitors. Our results revealed that the EstE5 protein is significantly inhibited by PMSF. In addition, this is the first study to identify the crystal structures of EstE5-PMSF at 2.4 and 2.5A among the HSL-homolog structures. This structural configuration is similar to that adopted when serine proteases are inhibited by PMSF. The results presented here provide valuable information regarding the properties of the HSL-family.

The structural elucidations of microbial lipases have been of prime interest since the 1980s. Knowledge of structural features plays an important role in designing and engineering lipases for specific purposes. Significant structural data have been presented for few microbial lipases, while, there is still a structure-deficit, that is, most lipase structures are yet to be resolved. A search for 'lipase structure' in the RCSB Protein Data Bank (http://www.rcsb.org/pdb/) returns only 93 hits (as of September 2007) and, the NCBI database (http://www.ncbi.nlm.nih.gov) reports 89 lipase structures as compared to 14719 core nucleotide records. It is therefore worthwhile to consider investigations on the structural analysis of microbial lipases. This review is intended to provide a collection of resources on the instrumental, chemical and bioinformatics approaches for structure analyses. X-ray crystallography is a versatile tool for the structural biochemists and is been exploited till today. The chemical methods of recent interests include molecular modeling and combinatorial designs. Bioinformatics has surged striking interests in protein structural analysis with the advent of innumerable tools. Furthermore, a literature platform of the structural elucidations so far investigated has been presented with detailed descriptions as applicable to microbial lipases. A case study of Candida rugosa lipase (CRL) has also been discussed which highlights important structural features also common to most lipases. A general profile of lipase has been vividly described with an overview of lipase research reviewed in the past.

Solvent-tolerant microbes are a newly emerging class that possesses the unique ability to thrive in the presence of organic solvents. Their enzymes adapted to mediate cellular and metabolic processes in a solvent-rich environment and are logically stable in the presence of organic solvents. Enzyme catalysis in non-aqueous/low-water media is finding increasing applications for the synthesis of industrially important products, namely peptides, esters, and other trans-esterification products. Solvent stability, however, remains a prerequisite for employing enzymes in non-aqueous systems. Enzymes, in general, get inactivated or give very low rates of reaction in non-aqueous media. Thus, early efforts, and even some recent ones, have aimed at stabilization of enzymes in organic media by immobilization, surface modifications, mutagenesis, and protein engineering. Enzymes from solvent-tolerant microbes appear to be the choicest source for studying solvent-stable enzymes because of their unique ability to survive in the presence of a range of organic solvents. These bacteria circumvent the solvent's toxic effects by virtue of various adaptations, e.g. at the level of the cytoplasmic membrane, by degradation and transformation of solvents, and by active excretion of solvents. The recent screening of these exotic microbes has generated some naturally solvent-stable proteases, lipases, cholesterol oxidase, cholesterol esterase, cyclodextrin glucanotransferase, and other important enzymes. The unique properties of these novel biocatalysts have great potential for applications in non-aqueous enzymology for a range of industrial processes.

During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll --> pheophytin --> pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.

An extracellular sterol esterase from Ophiostoma piceae efficiently hydrolyzes sterol esters, triglycerides and p-nitrophenol esters. cDNA was screened with a probe obtained by PCR using as primers oligonucleotides corresponding to the N-terminal and internal mature enzyme sequences and complete sequence was obtained by 3' rapid amplification of cDNA end (RACE) and inverse PCR. The O. piceae esterase gene had a length of 1.8 kbp and lacked introns. A search for proteins with related amino acid sequences revealed around 40% identity with lipases from Candida rugosa and Geotrichum candidum. Modelling the O. piceae enzyme, using the crystal structures of Lip1 and Lip3 from C. rugosa as templates, revealed a similar substrate-binding site, but some changes affecting the flap zone and the aromatic region of the tunnel may be responsible for the wide substrate specificity of this interesting sterol esterase. The ability of the new fungal esterase to hydrolyze triglycerides and esters of p-nitrophenol and cholesterol was compared with those of commercial lipases and cholesterol esterases showing the new enzyme the highest efficiency hydrolyzing triglycerides and sterol esters in the conditions assayed (in presence of Genapol X-100). Finally, the O. piceae gene was successfully expressed in Pichia pastoris, as a model organism to express fungal enzymes, resulting in higher levels of esterase activity than those obtained in the O. piceae cultures. In spite of its higher glycosylation degree, the recombinant enzyme was able to hydrolyze more efficiently than native enzyme the assayed substrates.

To better understand the ligand-binding mechanism of protein Pir7b, important part in detoxification of a pathogen-derived compound against Pyricularia oryzae, a 3D structure model of protein Pir7b was constructed based on the structure of the template SABP2. Three substrates were docking to this protein, two of them were proved to be active, and some critical residues are identified, which had not been confirmed by the experiments. His87 and Leu17 considered as 'oxyanion hole' contribute to initiating the Ser86 nucleophilic attack. Gln187 and Asp139 can form hydrogen bonds with the anilid group to maintain the active binding orientation with the substrates. The docking model can well interpret the specificity of protein Pir7b towards the anilid moiety of the substrates and provide valuable structure information about the ligand binding to protein Pir7b.

The effect of solvent hydrophobicity on activation of Candida rugosa lipase (CRL) was investigated by performing molecular dynamics simulations for four nano seconds (ns). The closed/inactive conformer of CRL (PDB code 1TRH) was solvated in three alkane-aqueous environments. The alkanes aggregated in a predominantly aqueous environment and by 1 ns a stable spherical alkane-aqueous interface had formed. This led to the interfacial activation of CRL. On analyzing the simulated conformers with the closed conformer of CRL, the flap was found to have opened from a closed state by 7.7 A, 10.2 A, 13.1 A at hexane-aqueous, octane-aqueous, and decane-aqueous interfaces. Further, essential dynamics analysis revealed that major anharmonic fluctuations were confined to residues 64-81, the flap of CRL.

The protein surface is the interface through which a protein senses the external world. Its composition of charged, polar and hydrophobic residues is crucial for the stability and activity of the protein. The charge state of seven of the twenty naturally occurring amino acids is pH dependent. A total of 95% of all titratable residues are located on the surface of soluble proteins. In evolutionary related families of proteins such residues are particularly prone to substitutions, insertions and deletions. We present here an analysis of the residue composition of 4038 proteins, selected from 125 protein families with < 25% identity between core members of each family. Whereas only 16.8% of the residues were truly buried, 40.7% were > 30% exposed on the surface and the remainder were < 30% exposed. The individual residue types show distinct differences. The data presented provides an important new approach to protein engineering of protein surfaces. Guidelines for the optimization of solvent exposure for a given residue are given. The cutinase family of enzymes has been investigated. The stability of native cutinase has been studied as a function of pH, and has been compared with the cutinase activity towards tributyrin. Whereas the onset of enzymatic activity is linked with the deprotonation of the active site HIS188, destabilization of the 3D structure as determined by differential scanning calorimetry is coupled with the loss of activity at very basic pH values. A modeling investigation of the pH dependence of the electrostatic potentials reveals that the activity range is accompanied by the development of a highly significant negative potential in the active site cleft. The 3D structures of three mutants of the Fusarium solani pisi cutinase have been solved to high resolution using X-ray diffraction analysis. Preliminary X-ray data are presented.

Here we present an investigation of the contacts that cysteines make with residues in their three-dimensional environment and a comprehensive analysis of the conformational features of 351 disulphide bridges in 131 non-homologous single-chain protein structures. Upstream half-cystines preferentially have downstream neighbours, whereas downstream half-cystines have mainly upstream neighbours. Non-disulphide bridged cysteines (free cysteines) have no preference for upstream or downstream neighbours. Free cysteines have more contacts to non-polar residues and fewer contacts to polar/charged residues than half-cystines, which correlates with our observation that free cysteines are more buried than half-cystines. Free cysteines prefer to be located in alpha-helices while no clear preference is observed for half-cystines. Histidine and methionine are preferentially seen nearby free cysteines. Tryptophan is found preferentially nearby half-cystines. We have merged sequential and spatial information, and highly interesting novel patterns have been discovered. The number of cysteines per protein is typically an even number, peaking at four. The number of residues separating two half-cystines is preferentially 11 and 16. Left-handed and right-handed disulphide bridges display different conformational parameters. Here we present side chain torsion angle information based on a 5-12 times larger number of disulphide bridges than has previously been published. Considering the importance of cysteines for maintaining the 3D-structural scaffold of proteins, it is essential to have as accurate information as possible concerning the packing and conformational preferences. The present work may provide key information for engineering the protein environment around cysteines.

We present an atomic force microscopy (AFM) study of a supported triacylglyceride multilayer phase and its interaction with lipolytic enzyme cutinase from Fusarium solani pisi. The multilayer triacylglyceride phase of coconut oil showed a rippled surface structure in the AFM images. Upon enzymatic degradation of the triacylglyceride phase, the ripple structure vanished rapidly. The apparent catalytic rate constants could be estimated based on the AFM image information. Interestingly, in one sample we observed what we interpret as a recurrent structural collapse of the cavity dug out by the protein. We interpret the cavities seen in the AFM images as molten surfaces or surface holes filled with liquidified phase containing product molecules, which appear transparent during the image recording.

By analysing the surface composition of a set of protein 3D structures, complemented with predicted surface compositional information for homologous proteins, we have found significant evidence for a layer composition of protein structures. In the innermost and outermost parts of proteins there is a net negative charge, while the middle has a net positive charge. In addition, our findings indicate that the concept of conservative mutation needs substantial revision, e.g. very different spatial preferences were found for glutamic acid and aspartic acid. The alanine screening often used in protein engineering projects involves the substitution of residues to alanine, based on the assumption that alanine is a "neutral" residue. However, alanine has a high negative correlation with all but the non-polar residues. We therefore propose the use of, for example, serine as a substitute for the residues that are negatively correlated with alanine.

The optimisation of enzymes for particular purposes or conditions remains an important target in virtually all protein engineering endeavours. Here, we present a successful strategy for altering the pH-optimum of the triglyceride lipase cutinase from Fusarium solani pisi. The computed electrostatic pH-dependent potentials in the active site environment are correlated with the experimentally observed enzymatic activities. At pH-optimum a distinct negative potential is present in all the lipases and esterases that we studied so far. This has prompted us to propose the "The Electrostatic Catapult Model" as a model for product release after cleavage of the ester bond. The origin of the negative potential is associated with the titration status of specific residues in the vicinity of the active site cleft. In the case of cutinase, the role of Glu44 was systematically investigated by mutations into Ala and Lys. Also, the neighbouring Thr45 was mutated into Proline, with the aim of shifting the spatial location of Glu44. All the charge mutants displayed altered titration behaviour of active site electrostatic potentials. Typically, the substitution of the residue Glu44 pushes the onset of the active site negative potential towards more alkaline conditions. We, therefore, predicted more alkaline pH optima, and this was indeed the experimentally observed. Finally, it was found that the pH-dependent computed Coulombic energy displayed a strong correlation with the observed melting temperatures of native cutinase.

This work explores the role of one of the factors explaining lipase/esterase activity: the contribution of electrostatic interactions to lipase/esterase activity. The electrostatic potential distribution on the molecular surface of an enzyme as a function of pH determines, to a large extent, the enzyme's pH activity profile. Other important factors include the presence and distribution of polar and hydrophobic residues in the active cleft. We have mapped the electrostatic potential distribution as a function of pH on the molecular surface of nine lipases/esterases for which the 3D structure is experimentally known. A comparison of these potential maps at different pH values with the corresponding pH-activity profile, pH optimum or pH range where the activity displayed by the enzyme is maximum, has revealed a considerable correlation. A negative potential in the active site appears correlated with maximum activity towards triglycerides, which has prompted us to propose a model for product release ('The electrostatic catapult model') after cleavage of an ester bond. At the same time as the bottom of the active site cleft becomes negatively charged, other nearby regions also titrate and become negatively charged when pH becomes more alkaline, for some of the studied lipases. If such lipases also show phospholipase activity (such as guinea pig lipase-related proteins 2 chimera) we raise the hypothesis that such other titratable regions after becoming negatively charged might stabilise the positive charge present in the polar head of phospholipids, such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The distribution of polar, weak polar and non-polar residues on the molecular surface of each studied lipase, in particular the active site region, was compared for all the lipases studied. The combination of graphical visualisation of the electrostatic potential maps and the polarity maps combined with knowledge about the location of key residues on the protein surface allows us to envision atomic models for lipolytic activity.

The fungal lipolytic enzyme cutinase, incorporated into sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles has been investigated using dynamic light scattering. The reversed micelles form spontaneously when water is added to a solution of sodium bis-(2ethylhexyl) sulfosuccinate in isooctane. When an enzyme is previously dissolved in the water before its addition to the organic phase, the enzyme will be incorporated into the micelles. Enzyme encapsulation in reversed micelles can be advantageous namely to the conversion of water insoluble substrates and to carry out synthesis reactions. However protein unfolding occurs in several systems as for cutinase in sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles. Dynamic light scattering measurements of sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles with and without cutinase were taken at different water to surfactant ratios. The results indicate that cutinase was attached to the micellar wall and that might cause cutinase unfolding. The interactions between cutinase and the bis-(2ethylhexyl) sulfosuccinate interface are probably the driving force for cutinase unfolding at room temperature. Twenty-four hours after encapsulation, when cutinase is unfolded, a bimodal distribution was clearly observed. The radii of reversed micelles with unfolded cutinase were determined and found to be considerable larger than the radii of the empty reversed micelles. The majority of the reversed micelles were empty (90-96% of mass) and the remainder (4-10%) containing unfolded cutinase were larger by 26-89 A.

The trypsin family of serine proteases is one of the most studied protein families, with a wealth of amino acid sequence information available in public databases. Since trypsin-like enzymes are widely distributed in living organisms in nature, likely evolutionary scenarios have been proposed. A novel methodology for Fourier transformation of biological sequences (FOTOBIS) is presented. The methodology is well suited for the identification of the size and extent of short repeats in protein sequences. In the present paper the trypsin family of enzymes is analyzed with FOTOBIS and strong evidence for tandem gene duplication is found. A likely evolutionary path for the development of present-day trypsins involved an intrinsic extensive tandem gene duplication of a small DNA fragment of 15-18 nucleotides, corresponding to five or six amino acids. This ancestral trypsin gene was subsequently duplicated, leading to the earliest version of a full-sized trypsin, from which the contemporary trypsins have developed.

Pseudozyma antarctica lipase B (CALB) shows activity in the acrylation of hydroxypropylcarbamate, a racemic mixture of enantiomers of primary and secondary alcohols. However, full conversion is hampered by the slowly reacting S enantiomer of the secondary alcohol. The same is true for a wide range of secondary alcohols, for example, octan-2- and -3-ol. In order to get high conversion in these reactions in a short time, the stereospecificity pocket of CALB was redesigned by using predictions from molecular modeling. Positions 278, 104, and 47 were targeted, and a library for two-site saturation mutagenesis at positions 104 and 278 was constructed. The library was then screened for hydrolysis of acrylated hydroxypropylcarbamates. The best mutants L278A, L278V, L278A/W104F, and L278A/W104F/S47A showed an increased conversion in hydrolysis and transesterification of more than 30 %. While the wild-type showed only 73 % conversion in the acrylation of hydroxypropylcarbamate after 6 h, 97 % conversion was achieved by L278A in this time. Besides this, L278A/W104F reached >96 % conversion in the acrylation of octan-2- and -3-ol within 48 h and showed a significant decrease in stereoselectivity, while the wild-type reached only 68 and 59 % conversion, respectively. Thus the new biocatalysts can be used for efficient transformation of racemic alcohols and esters with high activity when the high stereoselectivity of the wild-type hampers complete conversion of racemic substrates in a short time.

A bacterial strain which can be grown in a medium containing water-miscible organic solvents and can secrete an esterase was isolated and identified as Lysinibacillus sphaericus. The enzyme was stable in the presence of water-miscible organic solvents. The activity of the enzyme was enhanced 3-fold after incubating the enzyme with 80% acetonitrile. The solvent-stable esterase hydrolyses short chain p-nitrophenyl esters. The secreted enzyme undergoes temperature and time dependent aggregation. The enzyme was purified to homogeneity with an approximate molecular weight of 28kDa. Ca(2+) and Mg(2+) ions enhanced and EDTA and PMSF inhibited the enzyme activity. The optimum pH and temperature for its activity was 8.0 and 40 degrees C, respectively. Stability in organic solvent makes it an attractive enzyme and provides advantage for stereospecific ester bond hydrolysis and synthesis. The isolated strain's similarity to extremophiles could be the reason for stability in organic solvents.

Comparing the phase transitions of metal clusters with those of dielectric clusters shows that although the relative energies of the phase transitions for metals is typically less than those of dielectrics, other parameters of the phase transitions such as the entropy jumps and the relative widths of the coexistence bands are comparable for the two kinds of clusters. The dominating special characteristic of metal clusters is the large number of isomers with low excitation energy, in contrast with dielectric clusters, whose liquid aggregate states, generated by configurational excitation requiring fairly significant energies (relative to the atomic bond energy), have a significantly smaller density. This is partially due to the contribution of low-lying electronic excited states of the metal clusters. We analyze hysteresis in the phase transitions of large metal clusters as a result of cluster heating and cooling. The experimental and theoretical aspects of metal clusters as catalysts are considered.

The conformation of a surface loop, the lid, controls activity of pancreatic triglyceride lipase (PTL) by moving from a position that sterically hinders substrate access to the active site into a new conformation that opens and configures the active site. Movement of the lid is accompanied by a large change in steady state tryptophan fluorescence. Although a change in the microenvironment of Trp-253, a lid residue, could account for the increased fluorescence, the mechanism and tryptophan residues have not been identified. To identify the tryptophan residues responsible for the increased fluorescence and to gain insight into the mechanism of lid opening and the structure of PTL in aqueous solution, we examined the effects of mutating individual tryptophan residues to tyrosine, alanine, or phenylalanine on lipase activity and steady state fluorescence. Substitution of tryptophans 86, 107, 253, and 403 reduced activity against tributyrin with the largest effects caused by substituting Trp-86 and Trp-107. Trp-107 and Trp-253 fluorescence accounts for the increased fluorescence emissions of PTL that is stimulated by tetrahydrolipstatin and sodium taurodeoxycholate. The largest contribution is from Trp-107. Contrary to the prediction from the crystal structure of PTL, Trp-107 is likely exposed to solvent. Both tetrahydrolipstatin and sodium taurodeoxycholate are required to produce the increased fluorescence in PTL. Alone, neither is sufficient. Colipase does not significantly influence the conformational changes leading to increased emission fluorescence. Thus, Trp-107 and Trp-253 contribute to the change in steady state fluorescence that is triggered by mixed micelles of inhibitor and bile salt. Furthermore, the results suggest that the conformation of PTL in solution differs significantly from the conformation in crystals.

An alkali stable polyamidase was isolated from a new strain of Nocardia farcinica. The enzyme consists of four subunits with a total molecular weight of 190 kDa. The polyamidase cleaved amide and ester bonds of water insoluble model substrates like adipic acid bishexylamide and bis(benzoyloxyethyl)terephthalate and hydrolyzed different soluble amides to the corresponding acid. Treatment of polyamide 6 with this amidase led to an increased hydrophilicity based on rising height and tensiometry measurements and evidence of surface hydrolysis of polyamide 6 is shown. In addition to amidase activity, the enzyme showed activity on p-nitrophenylbutyrate. On hexanoamide the amidase exhibited a K(m) value of 5.5 mM compared to 0.07 mM for p-nitroacetanilide. The polyamidase belongs to the amidase signature family and is closely related to aryl acylamidases from different strains/species of Nocardia and to the 6-aminohexanoate-cyclic dimer hydrolase (EI) from Arthrobacter sp. KI72.

A recent study challenges the oft-held notion that ester bonds in prodrug molecules are cleaved rapidly and completely inside cells by endogenous, nonspecific esterases. Structure-activity relationship studies on acylated sugars reveal that regioisomeric compounds display disparate biological activity, suggesting that ester bonds can persist in a cellular context.

Based on a recent ternary complex crystal structure of human DNA polymerase beta with a G:A mismatch in the active site, we carried out a theoretical investigation of the catalytic mechanism of incorrect nucleotide incorporation using molecular dynamics simulation, quantum mechanics, combined quantum mechanics, and molecular mechanics methods. A two-stage mechanism is proposed with a nonreactive active-site structural rearrangement prechemistry step occurring before the nucleotidyl transfer reaction. The free energy required for formation of the prechemistry state is found to be the major factor contributing to the decrease in the rate of incorrect nucleotide incorporation compared with correct insertion and therefore to fidelity enhancement. Hence, the transition state and reaction barrier for phosphodiester bond formation after the prechemistry state are similar to that for correct insertion reaction. Key residues that provide electrostatic stabilization of the transition state are identified.

AIMS To screen novel micro-organisms and enzymes capable of degrading 3-hydroxypalmitic acid methyl ester (3-OH PAME), the quorum-sensing signal molecule (quormone), which regulates the virulence of Ralstonia solanacearum. METHODS AND RESULTS Ideonella sp. 0-0013, a betaproteobacterium isolated from soil using the selective-enrichment culture method, was grown on plates containing 3-OH PAME as its main carbon source. beta-Hydroxypalmitate methyl ester hydrolase (betaHPMEH) purified from the supernatant of the Ideonella sp. 0-0013 culture exhibited high hydrolysing activity towards the ester bond of 3-OH PAME and eliminated the 3-OH PAME activity, thereby reducing the virulence of R. solanacearum. An Escherichia coli transformant of the betahpmeh gene expression vector degraded 3-OH PAME, and the crude enzyme from the transformant inhibited in vitro production of the R. solanacearum exopolysaccharide (EPS). CONCLUSIONS The ability of betaHPMEH to hydrolyse 3-OH PAME inhibited the production of EPS by the R. solanacearum wild-type strain, indicating that betaHPMEH inhibits the effects of activation of virulence genes. This ability will be potentially useful for pest control of the wilt disease caused by this bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY This enzyme is the first protein that has been found to degrade a quormone other than N-acyl homoserine lactone.

Strain Pseudomonas sp. CR-611, previously isolated from a subtropical forest soil on tributyrine-supplemented plates, displays phenotypic and physiological properties consistent with those described for Pseudomonas fluorescens. However, no complete match to this species could be found after 16S rDNA comparison. Zymographic analysis of the strain revealed a complex lipolytic system, showing the presence of at least two enzymes with activity on MUF-butyrate. Alignment of Pseudomonas fluorescens lipase/esterase-coding sequences allowed the design of specific primers for family VI lipases, and the isolation and cloning of the resulting gene estA6. The recombinant clone obtained displayed high activity on fatty acid-derivative substrates, indicating that one of the lipolytic enzymes of the strain had been cloned. The enzyme, named EstA6, was then purified and characterized, showing maximum activity on short chain-length substrates under conditions of high temperature and neutral pH. Amino acid sequence alignment of EstA6 with other family VI esterases allowed identification of a highly conserved beta-/gamma-protobacterial cluster in family VI lipases, to which EstA6 belongs.

Using the classical emulsified system and the monomolecular film technique, we compared the interfacial properties of the scorpion digestive lipase (SDL) with those of higher animals'. In the absence of bile slats, SDL does not hydrolyse efficiently pure tributyrin, as well as dicaprin films maintained at low surface pressure. The preincubation of bile salts with tributyrin seems to be a better substrate for SDL than the pure tributyrin. A kinetic study on the surface pressure dependency, stereospecificity and regioselectivity of SDL was performed using monomolecular films of either three dicaprin isomers or three pairs of didecanoyl-deoxyamino-O-methyl glycerol enantiomers (DDG) containing a single hydrolysable decanoyl ester bond. With all diacylglycerol isomers, SDL has a surface pressure threshold of about 15 m Nm(-1), below which enzymatic activity is undetectable. SDL seems to prefer vicinal ester groups of the diacylglycerol isomers, with preference for sn-1 position at both 15 and 23 m Nm(-1). Furthermore, the maximum SDL activity is measured with DDG having a primary ester bond (1,3DDG, SII). This shows that SDL has a preference for the sn-1 position of this diacylglycerol analogue. Moreover, this was in line with the fact that SDL is inactive on sn-2 position of both DDG isomers and a triacylglycerol. With diacylglycerol analogue isomers, SDL shows a preference for distal isomers contrary to what has been observed with diacylglycerol isomers. SDL interacts with egg-phosphatidyl choline (egg-PC) monomolecular films. The critical surface pressure value (13 m Nm(-1)) is comparable to those of pancreatic lipases.