A role for pectin de-methylesterification in a developmentally regulated growth acceleration in dark-grown Arabidopsis hypocotyls.

• We focused on a developmentally regulated growth acceleration in the dark-grown Arabidopsis hypocotyl to study the role of changes in cell wall metabolism in the control of cell elongation. • To this end, precise transcriptome analysis on dissected dark-grown hypocotyls, Fourier transform infrared (FT-IR) microspectroscopy and kinematic analysis were used. • Using a cellulose synthesis inhibitor, we showed that the growth acceleration marks a developmental transition during which growth becomes uncoupled from cellulose synthesis. We next investigated the cellular changes that take place during this transition. FT-IR microspectroscopy revealed significant changes in cell wall composition during, but not after, the growth acceleration. Transcriptome analysis suggested a role for cell wall remodeling, in particular pectin modification, in this growth acceleration. This was confirmed by the overexpression of a pectin methylesterase inhibitor, which caused a delay in the growth acceleration. • This study shows that the acceleration of cell elongation marks a developmental transition in dark-grown hypocotyl cells and supports a role for pectin de-methylesterification in the timing of this event.

Inhibition of pneumococcal choline-binding proteins and cell growth by esters of bicyclic amines.

Streptococcus pneumoniae is one of the major pathogens worldwide. The use of currently available antibiotics to treat pneumococcal diseases is hampered by increasing resistance levels; also, capsular polysaccharide-based vaccination is of limited efficacy. Therefore, it is desirable to find targets for the development of new antimicrobial drugs specifically designed to fight pneumococcal infections. Choline-binding proteins are a family of polypeptides, found in all S. pneumoniae strains, that take part in important physiologic processes of this bacterium. Among them are several murein hydrolases whose enzymatic activity is usually inhibited by an excess of choline. Using a simple chromatographic procedure, we have identified several choline analogs able to strongly interact with the choline-binding module (C-LytA) of the major autolysin of S. pneumoniae. Two of these compounds (atropine and ipratropium) display a higher binding affinity to C-LytA than choline, and also increase the stability of the protein. CD and fluorescence spectroscopy analyses revealed that the conformational changes of C-LytA upon binding of these alkaloids are different to those induced by choline, suggesting a different mode of binding. In vitro inhibition assays of three pneumococcal, choline-dependent cell wall lytic enzymes also demonstrated a greater inhibitory efficiency of those molecules. Moreover, atropine and ipratropium strongly inhibited in vitro pneumococcal growth, altering cell morphology and reducing cell viability, a very different response than that observed upon addition of an excess of choline. These results may open up the possibility of the development of bicyclic amines as new antimicrobials for use against pneumococcal pathologies.

The relationship between the carboxylesterase and monoacylglycerol lipase activities of chicken liver microsomes.

The carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) and monoacylglycerol lipase (glycerol-monoester acylhydrolase, EC 3.1.1.23) activities, measured against ethyl butyrate and emulsified monooleoylglycerol respectively, were determined for chicken liver microsomes and highly purified chicken liver carboxylesterase. The activity ratio (ethyl butyrate activity/monooleoylglycerol activity) was approx. 5 for microsomes and approx. 400 for carboxylesterase. Homogenization of microsomes in 0.1 M Tris-HCl buffer (pH 7.92) released all of the ethyl butyrate activity and about half of the monooleoylglycerol activity into a soluble form. Both activities eluted from a Sephadex G-200 column with the same elution volume as that of pure carboxylesterase. This fraction (fraction B) had an activity ratio of approx. 15, an average pI of 5.01 (cf. 4.75 for carboxylesterase), and ran on polyacrylamide gel electrophoresis at pH 8.6 as a number of closely spaced esterase bands with mobilities considerably less than those of the esterase bands present in the carboxylesterase. Fraction B activities against both substrates were completely inhibited by diethyl p-nitrophenyl phosphate and completely precipitated by antibody to carboxylesterase. The remaining half of the monoacylglycerol lipase activity of microsomes was solubilized by treatment with 1.5% (w/v) Triton X-100. This solubilized monoacylglycerol lipase was completely inhibited by diethyl p-nitrophenyl phosphate, showing it to be a serine-dependent enzyme like the carboxylesterases. However, it had no detectable activity against ethyl butyrate, indicating that it is not closely related to the carboxylesterases.

Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.

The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T1 has two disulfide bonds, one of which appears to be necessary for the full enzyme activity. This depolymerase hydrolyzed not only hydrophobic poly(3-hydroxybutyrate) but also water-soluble trimer and larger oligomers of D-(-)-3-hydroxybutyrate, regardless of their solubilities in water. Kinetic analyses with oligomers of various sizes indicated that the substrate cleaving site of the enzyme consisted of four subsites with individual affinities for monomer units of the substrate. Analyses of the hydrolytic products of oligomers, which had labeled D-(-)-3-hydroxybutyrate at the hydroxy terminus, showed that the enzyme cleaved only the second ester linkage from the hydroxy terminus of the trimer and tetramer, and acted as an endo-type hydrolase toward the pentamer and higher oligomers. The enzyme appeared to have a hydrophobic site which interacted with poly(3-hydroxybutyrate) and determined the affinity of the enzyme toward the hydrophobic substrate.

Inhibition of rat liver cholesterol esterase by local anaesthetics.

1. A number of local anaesthetics was shown to inhibit rat liver cholesterol esterase activity towards radioactively labelled cholesterol oleate. The anaesthetics inhibited in the order dibucaine greater than chlorpromazine greater than tetracaine greater than benzocaine greater than procaine greater than lidocaine greater than cocaine. 2. The mode of inhibition was seen to be non-competitive with respect to the substrate and is probably independent of any involvement of Ca2+. 3. The inhibition by tetracaine is partially reversed by sodium deoxycholate. However, all ionic and non-ionic detergents studied, sodium deoxycholate, sodium taurocholate, Triton X-100, and cetyltrimethylammonium bromide are capable of inhibiting the rat liver cholesterol esterase in a concentration dependent manner. Only sodium taurocholate stimulates enzymic activity.

Inactivation of chlorophyllase by negatively charged plant membrane lipids.

Chlorophyllide combines spontaneously not only with phosphatidylcholine (PC) liposomes but also with various other (plant) lipids dispersed in an aqueous medium. The lipid-associated chlorophyllide is highly fluorescent and the fluorescence yield is virtually independent of the nature of the lipid. Chlorophyllase (chlorophyll chlorophyllidohydrolase, EC 3.1.1.14) activity assays that are based on the determination of this chlorophyllide fluorescence show that phosphatidylglycerol (PG), and also sulphoquinovosyldiacylglycerol (SQDG), associate with isolated chlorophyllase, thereby inactivating the enzyme in a co-operative way. The extent of this inactivation depends on the pH and ionic strength of the reaction medium and can be completely reversed by divalent cations (Mg2+). The inhibition of chlorophyllase effected by free PG liposomes can be counteracted by electrically neutral lipids at relatively high concentration (PC and also chloroplast lipids). Digalactosyldiacylglycerol (DGDG) is not effective in this respect. When PG has been incorporated in PC or DGDG liposomes, its ability to inhibit chlorophyllase activity is reduced. Whereas the remaining chlorophyllase-inactivating effect of PG, incorporated in PC, can still be reversed by Mg2+, this is not found when enzyme inactivation is caused by PG incorporated in DGDG. The results reported here are consistent with those obtained earlier concerning the stabilization of chlorophyllase by PG and PG/galactolipid mixtures (Lambers, J.W.J., Verkleij, A.J. and Terpstra, W. (1984) Biochim. Biophys. Acta 786, 1-8). They are discussed in terms of the regulation of chlorophyllase activity by lipids surrounding the enzyme and by divalent cations.

Formation of soman (1,2,2-trimethylpropyl methylphosphonofluoridate) via fluoride-induced reactivation of soman-inhibited aliesterase in rat plasma.

After incubation (37 degrees) of rat blood or plasma with the nerve agent soman, (CH3)3C(CH3)C(H)O(CH3)P(O)F (7.7 microM), for 10 min, only a small amount of this organophosphate (7 or 1%, respectively) is left, as determined enzymatically (acetylcholinesterase) and gas chromatographically. Comparison of the results obtained with both analyses shows that this residual soman consists only of its P(-)-isomers. Incubation (25 degrees) at pH 4.8-6.1 of such soman-treated rat blood or plasma with sodium fluoride (2.5 mM) for 0.5 min leads to (i) a substantial increase of the P(-)-soman concentration, and (ii) a (partial) reactivation of the soman-inhibited aliesterase, proportional to the amount of generated P(-)-soman. These results indicate strongly that added fluoride ions regenerate soman by a reversal of the inhibition reaction. From the relationship between percentage of reactivation and increase of soman concentration the aliesterase concentration in rat plasma is calculated as 2.6 microM. Sodium fluoride has a similar effect in blood taken from rats to which soman was administered intravenously. The increase of the P(-)-soman concentration is higher with higher sodium fluoride concentrations and at lower pH values. In accordance with the absence of aliesterase, addition of sodium fluoride does not induce an increase of the P(-)-soman concentration in soman-treated human plasma.

Inhibitor studies of purified haemopoietic (myeloid) cell esterases. Evidence for the existence of distinct enzyme species.

Human myeloid cells synthesize and express two major species of esterase, defined by isoelectric focusing (IEF). The first of these (MonEst) is specifically associated with haemopoietic cells of monocytic lineage, whereas the other species (ComEst) is common to all myeloid cells (granulocytes and monocytes) irrespective of lineage affiliation. Having recently purified these two species of human myeloid cell esterase, this present study extensively investigated the effects of 17 different inhibitors on their ability to hydrolyse the synthetic substrate alpha-naphthyl acetate (alpha NA). Significant inhibition of both ComEst and MonEst was exerted by 1% sodium dodecyl sulphate (SDS) and 1.0 mM diethyl pyrocarbonate (DEPC), but the patterns of inhibition for the two esterase species with the remaining compounds studied differed considerably; for example, 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 5.0 x 10(-3) M dichloroisocoumarin (DCIC) and 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) all inhibited MonEst but not ComEst. Mechanisms of inhibition were also examined and these studies established that SDS, PMSF, DCIC and TPCK irreversibly inactivated MonEst whilst the inhibition of ComEst by SDS was reversible. Analysis of inhibition kinetics further showed that (a) the reversible inhibition of both ComEst and MonEst by sodium fluoride (NaF) was noncompetitive (with Ki values of 1.28 and 0.01 mM, respectively, indicating a marked difference in sensitivity); (b) the inhibition of MonEst by PMSF was of 'mixed' noncompetitive-competitive type; and (c) that DEPC exerted noncompetitive inhibition with similar Ki values (0.05 mM) for both esterase species. These observations unequivocably demonstrate that ComEst and MonEst are unrelated enzyme species, with a common ability to hydrolyse alpha NA, and that these esterase show marked differences with respect to their active sites as adjudged by inhibitor sensitivities. These observations are particularly relevant to the histochemical analysis of these enzymes and to the elucidation of their in vivo functions.

Purification and properties of D-(-)-3-hydroxybutyrate oligomer hydrolase of Paracoccus denitrificans.

D-(-)-3-Hydroxybutyrate (3HB) oligomer hydrolase was purified from Paracoccus denitrificans. The enzyme was a monomeric protein with an approximate molecular mass of 31 kDa. The isoelectric point of the enzyme was 5.2. Optimum temperature and pH were 35-40 degrees C and 8.0, respectively. The enzyme activity was not affected by sulfhydryl reagents but strongly inhibited by serine proteinase inhibitors. Both 3HB trimer and 3HB dimer were hydrolyzed by the enzyme, indicating that the enzyme is not 3HB dimer hydrolase but 3HB oligomer hydrolase. para-Nitrophenyl esters of short-chain fatty acids were also hydrolyzed by the enzyme. 3HB dimer was hydrolyzed somewhat faster than 3HB trimer. The level of the enzyme activity was almost constant, irrespective of carbon sources for the bacterial growth and of the cultivation conditions.

Methyl methacrylate toxicity in rat nasal epithelium: studies of the mechanism of action and comparisons between species.

Female F344 rats exposed to 200 ppm methyl methacrylate for 6 h developed a lesion in the nasal olfactory epithelium which was characterised by degeneration and atrophy. The severity of the lesion was markedly reduced by pre-treatment of the rats with an intraperitoneal dose of 100 mg/kg bis-(p-nitrophenyl)phosphate, an inhibitor of carboxylesterase enzymes, thus demonstrating that the lesion is caused by the carboxylesterase mediated metabolism of methyl methacrylate to methacrylic acid, an irritant and corrosive metabolite. The distribution of the carboxylesterases in nasal tissues has been investigated and the metabolism of methyl methacrylate to methacrylic acid has been compared in rat, hamster and human nasal tissue fractions in vitro. Histocytochemistry showed that the carboxylesterases are heavily localised in the sustentacular cells and Bowman's glands of the rat olfactory region, but are more generally distributed in human olfactory epithelium. Consistent with this, the enzyme activity in all three species was higher in fractions prepared from olfactory tissue than from respiratory tissue, 3-fold in rat and human and 12-fold in the hamster. The maximum rates (V(max)) of metabolism in rat and hamster olfactory tissue fractions were comparable, whereas those in human olfactory tissue fractions were at least 13-fold lower. The rate of metabolism in rat olfactory tissue was also comparable to that in rat liver whereas in humans, the rate in olfactory tissue was 500-fold lower than that in the liver. In respiratory tissues, the rate in humans was at least 6-fold lower than that in the rat. These results suggest that humans are significantly less sensitive than rodents to the nasal toxicity of methyl methacrylate.

Extracellular poly(hydroxyalkanoate) depolymerases and their inhibitor from Pseudomonas lemoignei.

Enzymatic degradation processes of microbial copolyesters, poly(3-hydroxybutyrate-co-3-hydroxyvalerate): P(3HB-co-3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate): P(3HB-co-4HB), were studied by the weight loss (erosion) of copolyester films. These studies employed three extracellular depolymerases which degrade poly(3-hydroxybutyrate): P(3HB). Two enzymes were purified from the culture supernatant of Pseudomonas lemoignei and one from Alcaligenes faecalis T1. The rate of enzymatic degradation of microbial copolyester films with various compositions showed an almost similar tendency to three different P(3HB) depolymerases, and decreased in the following order: P(3HB-co-4HB) greater than P(3HB) greater than P(3HB-co-3HV). An inhibitory protein of P(3HB) depolymerases in the succinate culture medium of P. lemoignei was isolated and characterized. The molecular weight of P(3HB) depolymerase inhibitor was 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This inhibitor of a single polypeptide chain may reversibly bind the serine residues at the active site of P(3HB) depolymerase. This inhibitory protein was not induced in the culture medium when P. lemoignei was grown on P(3HB) as the sole carbon source.

Enzymatic degradation of poly(hydroxyalkanoates) by Pseudomonas pickettii.

A bacterium capable of degrading poly(3-hydroxybutyrate) [P(3HB)] was isolated from laboratory media and identified as Pseudomonas pickettii. The strain P. pickettii excreted an extracellular poly(hydroxyalkanoate) (PHA) depolymerase and grew on P(3HB) as the sole carbon source. P. pickettii also grew on 3-hydroxybutyrate, glucose, fructose, citrate or succinate. However, only 3-hydroxybutyrate apart from P(3HB) induced the secretion of PHA depolymerase. The PHA depolymerase was purified from the culture medium containing 3-hydroxybutyrate at the sole carbon source by hydrophobic column chromatography and gel filtration, and its molecular weight was determined as about 40,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The optimum activity of degrading P(3HB) by the depolymerase was observed at pH 5.5 and 40 degrees C. The enzymatic degradation of microbial copolyesters was studied by the weight loss (erosion) of solution-cast films of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate). 1H-n.m.r. analysis of the water-soluble products after the hydrolysis of P(3HB) film by PHA depolymerase revealed that the main product was the monomer 3-hydroxybutyric acid.

A human antibody fragment with high affinity for biodegradable polymer film.

Antibodies with high affinity for the surface of a solid material would be advantageous in biomaterial science as a protein device. A human antibody fragment that binds to poly(hydroxybutyrate) (PHB), a biodegradable polymer matter, was generated by a phage display system. Clone PH7-3d3 was isolated after several rounds of selection and prepared as a fragment of immunoglobulin variable regions (Fv). The quartz crystal microbalance technique showed that PH7-3d3 Fv completely inhibited PHB enzymatic degradation by competing with PHB depolymerase. Kinetic analysis based on surface plasmon resonance demonstrated that PH7-3d3 Fv bound to the PHB film with an equilibrium dissociation constant of 14 nM. The three-dimensional structure of PH7-3d3 Fv was resolved to 1.7 A, revealing that the complementarity determining regions (CDRs) in the Fv fragment form a relatively flat surface on which uncharged polar and aromatic amino acids are distributed in clusters. The structure of PH7-3d3 Fv was similar to that of PHB depolymerase in the orientation of aromatic residues in the binding sites. Alanine scanning mutagenesis demonstrated that these aromatic residues, especially tryptophan residues in CDRs, were critical in the interaction between PH7-3d3 Fv and PHB. Our results suggest the possible selection of an antibody fragment that binds a material surface in a manner similar to protein-ligand interaction.

Stereospecific and regioselective hydrolysis of cannabinoid esters by ES46.5K, an esterase from mouse hepatic microsomes, and its differences from carboxylesterases of rabbit and porcine liver.

The properties of ES46.5K, an esterase from mouse hepatic microsomes, were compared with those of carboxylesterases from rabbit and porcine liver. The inhibitory profile with a serine hydrolase inhibitor (bis-p-nitrophenylphosphate) and detergents (sodium dodecylsulfate, Emulgen 911) was different between ES46.5K and the carboxylesterases. Bis-p-nitrophenylphosphate (0.1 mM) markedly inhibited the catalytic activity of the carboxylesterases but not that of ES46.5K. Emulgen 911 (0.05-0.25%) inhibited the catalytic activity of the carboxylesterases, whereas the detergent conversely stimulated that of ES46.5K by 150%. The two carboxylesterases catalyzed the hydrolysis of acetate esters of tetrahydrocannabinol (THC) analogues with different side chain lengths (C1-C5), although ES46.5K showed marginal activity only against the acetate of Delta8-tetrahydrocannabiorcol, a methyl side chain derivative of Delta8-THC. ES46.5K hydrolyzed cannabinoid esters stereospecifically and regioselectively. The esterase hydrolyzed 8alpha-acetoxy-Delta9-tetrahydrocannabinol (8alpha-acetoxy-Delta9-THC, 5.62 nmol/min/mg protein), while the enzyme did not hydrolyze 8beta-acetoxy-Delta9-THC, 7alpha-acetoxy-, and 7beta-acetoxy-Delta8-THC at all. In contrast, the carboxylesterases from rabbit and porcine liver hydrolyzed 8beta-acetoxy-Delta9-THC efficiently but not 8alpha-acetoxy-Delta9-THC. ES46.5K hydrolyzed side chain acetoxy derivatives of Delta8-THC at the 3'- and 4'-positions, and a methyl ester of 5'-nor-Delta8-THC-4'-oic acid. The enzyme, however, could not hydrolyze methyl esters of Delta8- and Delta9-THC-11-oic acid, while both carboxylesterases hydrolyzed side chain acetoxy derivatives of Delta8-THC and three methyl esters of THC-oic acids. These differences in stereospecificity and regioselectivity between ES46.5K and carboxylesterases suggest that the configurations of important amino acids for the catalytic activities of these enzymes are different from each other.

The benzoylarginine peptidase from Treponema denticola (strain ASLM), a human oral spirochaete: evidence for active-site carboxyl groups.

The benzoylarginine peptidase of Treponema denticola (strain ASLM; a human oral spirochaete) was progressively and irreversibly inactivated by 1-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline, a carboxyl-group reagent. At acidic pH values, reaction of one mole of the modifier per active site of the enzyme resulted in total inactivation of the enzyme. Assuming that this modifier is a specific carboxyl reagent, the data suggest that the inactivation of the T. denticola benzoylarginine peptidase was caused by the modification of one carboxyl group located close to the active site of the enzyme. Results obtained with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulphonate) supported these findings. Carbethoxylation with diethylpyrocarbonate effectively inactivated the enzyme, and addition of hydroxylamine at pH 7.0 restored the activity almost totally, suggesting that the pyrocarbonate had reacted with tyrosyl or histidyl residues.

The "PHB depolymerase inhibitor" of Paucimonas lemoignei is a PHB depolymerase.

A approximately 35 kDa protein that has been described to be secreted by Paucimonas lemoignei during growth on succinate and to inhibit hydrolysis of denatured (crystalline) poly(3-hydroxybutyrate) (dPHB) by extracellular PHB depolymerases of P. lemoignei (PHB depolymerase inhibitor (PDI)) was purified and characterized. Purified PDI (M(r), 36 199 +/- 45 Da) inhibited hydrolysis of dPHB by two selected purified PHB depolymerases (PhaZ2 and PhaZ5) but did not inhibit the hydrolysis of water-soluble substrates such as p-nitrophenylbutyrate by PhaZ5 and PhaZ2. PDI revealed a high binding affinity to dPHB although it was not able to hydrolyze the crystalline polymer. However, purified PDI had a high hydrolytic activity if native (amorphous) PHB (nPHB) was used as a substrate. N-terminal sequencing of PDI revealed that it was identical to recently described extracellular PHB depolymerase PhaZ7 which is specific for nPHB and which cannot hydrolyze dPHB. To confirm that the inhibition of hydrolysis of dPHB by PhaZ7 is an indirect surface competition effect at high depolymerase concentration, the activity of PHB depolymerases PhaZ2 and PhaZ5 in the presence of different amounts of protein mixtures was determined. The components of NB or LB medium inhibited hydrolysis of the polymer in a concentration-dependent manner but had no effect on the hydrolysis of p-nitrophenylbutyrate by PHB depolymerases. In combination with PHB depolymerases PhaZ2 and PhaZ5 the protein PhaZ7 ("PDI") enables the bacteria to hydrolyze dPHB and nPHB simultaneously.

The catalytic domain of human neuropathy target esterase mediates an organophosphate-sensitive ionic conductance across liposome membranes.

In humans and other vertebrates, reaction of organophosphates with a neuronal membrane protein, neuropathy target esterase (NTE), initiates events which culminate in axonal degeneration. The initiation process appears to involve modification of a property of the protein distinct from its esterase activity, subsequent to formation of a negatively charged adduct with the active site serine residue. Here, we show that membrane patches from liposomes containing NEST, a recombinant hydrophobic polypeptide comprising the esterase domain of human NTE, display a transmembrane ionic conductance with both stable and high-frequency flickering components. An asymmetric current-voltage relationship suggested that ion flow was favoured in one direction relative to the membrane and its associated NEST molecules. Flow of anions was slightly favoured compared with cations. The flickering current formed a much larger proportion of the overall conductance in patches containing wild-type NEST compared with the catalytically inactive S966A mutant form of the protein. The conductance across patches containing NEST, but not those with the S966A mutant, was significantly reduced after adding neuropathic organophosphates to the bathing medium. By contrast, non-neuropathic covalent inhibitors of the catalytic activity of NEST did not reduce NEST-mediated conductance. Future work may establish whether NTE itself mediates an organophosphate-sensitive ion flux across intracellular membranes within intact cells.

The gene pvaB encodes oxidized polyvinyl alcohol hydrolase of Pseudomonas sp. strain VM15C and forms an operon with the polyvinyl alcohol dehydrogenase gene pvaA.

A 5.7 kbp SphI fragment containing the polyvinyl alcohol (PVA) dehydrogenase gene pvaA and its 1.9 kbp 5'-flanking region was cloned from the PVA-degrading bacterium Pseudomonas sp. VM15C. The pvaB gene, encoding oxidized PVA hydrolase, was found in the region upstream of pvaA. Sequence data and expression studies indicated that pvaA and B constitute an operon in the order pvaBA. The pvaB gene encoded a protein of 379 amino acid residues (40610 Da), and a lipoprotein signal sequence and the lipase consensus sequence, Gly-X-Ser-X-Gly, characteristic of the active-site serine region in serine hydrolases, were detected in the deduced amino acid sequence. The pvaB product with the pvaA product constituted an enzyme system for the cleavage of PVA molecules. The pvaA product introduced beta-diketone groups into the PVA molecule, and the pvaB product hydrolysed these beta-diketone groups in oxidized PVA. The pvaB product also hydrolysed 4,6-nonanedione at a low rate, but not acetylacetone or 5-nonanone. It was completely inhibited by PMSF and was concluded to be a serine hydrolase. There were no proteins showing high similarity to the pvaB product in the databases, but minor similarity to a number of serine hydrolases including polyhydroxyalkanoate depolymerases was apparent.

Inactivation of Staphylococcus hyicus lipase by hexadecylsulfonyl fluoride: evidence for an active site serine.

The Staphylococcus hyicus lipase is an acyl hydrolase with broad substrate specificity including neutral glycerides and phospholipids. To obtain further insight into the mechanism of action of this enzyme, we tested several sulfonyl fluorides as active site-directed inhibitors. The enzyme is resistant to the well-known serine protease/esterase inhibitor phenylmethanesulfonyl fluoride (PMSF), but is rapidly inactivated by hexadecylsulfonyl fluoride. The kinetics of inactivation were studied in Triton X-100 micelles. Inactivation is fast and the rate of inactivation is constant over the pH range where this lipase is active. Metal ions like Ca2+ and Sr2+ do not appreciably influence the rate of inactivation, although the enzymatic activity is significantly increased, suggesting a structural role for these ions. The S. hyicus lipase contains a consensus sequence G-H/Y-S-X-G. Substitution by site-directed mutagenesis of this serine (Ser369) by a cysteine resulted in a mutant with only 0.2% residual activity. The activity of this mutant could not be inhibited with water-soluble sulfhydryl reagents either in the presence or absence of Triton X-100 micelles. In the presence of Triton X-100 micelles, inactivation of the mutant occurred with 4-nitrophenylhexadecyl disulfide (t1/2 = 125 min) while the wild-type enzyme does not react at all. We conclude that Ser369 is the active site residue and that in water this residue is inaccessible. Only after interfacial activation Ser369 (or Cys369) becomes exposed and reacts with irreversible inhibitors.

The "intracellular" poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases.

Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). In this study we reinvestigated the soluble R. rubrum PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (phaZ1) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser(42), Asp(138), and His(178) were identified as catalytic-triad amino acids, with Ser(42) as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R. rubrum.