Stereochemical studies of chiral resolving agents, M9PP and H9PP acids

The stereoselective Grignard reaction of (1R,2S,5R)-(-)-2-isopropyl-5-methylcyclohexyl pyruvate (menthyl pyruvate) with 9-phenanthrylmagnesium bromide yielded diastereomeric hydroxy-esters, where intramolecular OH em leader O=C hydrogen bond was observed in IR and (1)H NMR spectra. The alkaline hydrolysis of the major product gave (+)-2-hydroxy-2-(9-phenanthryl)propionic acid (H9PP acid (3)), whose absolute configuration was assigned as S based on the chemical correlation with (1R,2S,5R)-2-isopropyl-5-methylcyclohexyl ester of (S)-2-methoxy-2-(9-phenanthryl)propionic acid (M9PP acid (2)); the absolute configuration of 2 had been previously established by X-ray crystallography. The enantioresolution of (+/-)-6-methyl-5-hepten-2-ol, sulcatol, an insect pheromone, was carried out using (S)-(+)-M9PP acid 2.     

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Solvent isotope effects for lipoprotein lipase catalyzed hydrolysis of water-soluble p-nitrophenyl esters

Solvent deuterium isotope effects on the rates of lipoprotein lipase (LpL) catalyzed hydrolysis of the water-soluble esters p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB) have been measured and fall in the range 1.5-2.2. The isotope effects are independent of substrate concentration, LpL stability, and reaction temperature and hence are effects on chemical catalysis and not due to a medium effect of D2O on LpL stability and/or conformation. pL (L = H or D) vs. rate profiles for the Vmax/Km of LpL-catalyzed hydrolysis of PNPB increase sigmoidally with increasing pL. Least-squares analysis of the profiles gives pKaH2O = 7.10 +/- 0.01, pKaD2O = 7.795 +/- 0.007, and a solvent isotope effect on limiting velocity at high pL of 1.97 +/- 0.03. Because the pL-rate profiles are for the Vmax/Km of hydrolysis of a water-soluble substrate, the measured pKa's are intrinsic acid-base ionization constants for a catalytically involved LpL active-site amino acid side chain. Benzeneboronic acid, a potent inhibitor of LpL-catalyzed hydrolysis of triacylglycerols [Vainio, P., Virtanen, J. A., & Kinnunen, P. K. J. (1982) Biochim. Biophys. Acta 711, 386-390], inhibits LpL-catalyzed hydrolysis of PNPB, with Ki = 6.9 microM at pH 7.36, 25 degrees C. This result and the solvent isotope effects for LpL-catalyzed hydrolysis of water-soluble esters are interpreted in terms of a proton transfer mechanism that is similar in many respects to that of the serine proteases.     

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

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

Interfacial reaction dynamics and acyl-enzyme mechanism for lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters

The fatty acyl (lipid) p-nitrophenyl esters p-nitrophenyl caprylate, p-nitrophenyl laurate and p-nitrophenyl palmitate that are incorporated at a few mol % into mixed micelles with Triton X-100 are substrates for bovine milk lipoprotein lipase. When the concentration of components of the mixed micelles is approximately equal to or greater than the critical micelle concentration, time courses for lipoprotein lipase-catalyzed hydrolysis of the esters are described by the integrated form of the Michaelis-Menten equation. Least square fitting to the integrated equation therefore allows calculation of the interfacial kinetic parameters Km and Vmax from single runs. The computational methodology used to determine the interfacial kinetic parameters is described in this paper and is used to determine the intrinsic substrate fatty acyl specificity of lipoprotein lipase catalysis, which is reflected in the magnitude of kcat/Km and kcat. The results for interfacial lipoprotein lipase catalysis, along with previously determined kinetic parameters for the water-soluble esters p-nitrophenyl acetate and p-nitrophenyl butyrate, indicate that lipoprotein lipase has highest specificity for the substrates that have fatty acyl chains of intermediate length (i.e. p-nitrophenyl butyrate and p-nitrophenyl caprylate). The fatty acid products do not cause product inhibition during lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles. The effects of the nucleophiles hydroxylamine, hydrazine, and ethylenediamine on Km and Vmax for lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl laurate are consistent with trapping of a lauryl-lipoprotein lipase intermediate. This mechanism is confirmed by analysis of the product lauryl hydroxamate when hydroxylamine is the nucleophile. Hence, lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles occurs via an interfacial acyl-lipoprotein lipase mechanism that is rate-limited by hydrolysis of the acyl-enzyme intermediate.     

Studies on the catalytic behaviour of a cholinesterase-like abzyme in an AOT microemulsion system

The hydrolytic activity of a monoclonal catalytic antibody (9A8) (abzyme) with acetylcholinesterase-like activity was investigated in water-in-oil (w/o) microemulsions (reverse micelles) based on sodium bis-2-(ethylhexyl)sulfosuccinate (AOT) in isooctane, using p- and o-nitrophenylacetate (p-and o-NPA) as substrates. The dependence of the abzyme hydrolytic activity on the molar ratio of water to surfactant (w(o)) showed a bell-shaped curve, presenting a maximum at w(o)=11.1. An increase of the AOT concentration at constant w(o), resulted in a decrease of the catalytic activity suggesting a possible inhibition effect of the surfactant. The incorporation of the abzyme into the reverse micelle system caused a blue shift of the fluorescence emission maximum by a magnitude of 7-10 nm depending on the w(o) value. This result indicates that the antibody molecule, or a large part of it, is located in the aqueous microphase of the system. Kinetic studies showed that the hydrolysis of p-and o-NPA in microemulsion system as well as in aqueous solution follows Michaelis-Menten kinetics. The catalytic efficiency (k(cat)/K(m)) in w/o microemulsion was significant lower than in aqueous solution.     

Biotransformation of β-amino nitriles: the role of the N-protecting group

N-Tolylsulfonyl- and N-butyloxycarbonyl-protected β-amino nitriles were prepared to study the effect of the N-protecting group on the biotransformation of the β-amino nitriles to the corresponding β-amino amides and acids. The bioconversions were carried out by using whole cells of Rhodococcus sp. R312 and Rhodococcus erythropolis NCIMB 11540. The bioconversion products of five-membered carbocyclic nitriles were mainly the respective acids whereas the carbocyclic six-membered nitriles were accumulated at the stage of the amide. Benefits of the enzymatic compared with the chemical hydrolysis of β-amino nitriles are the mild reaction conditions for the transformation of the nitrile group in the presence of acid or base labile N-protecting groups. In the present work we concentrated on this chemoselectivity of the biotransformation rather than its potential enantioselectivity, which will be subject of future investigations. Thus, some new compounds were prepared: (±)-(2-cyano-cyclohexyl) carbamic acid tert-butyl ester (4a), (±)-(2-carbamoyl-cyclopentyl) carbamic acid tert-butyl ester (3b) and (±)-(2-carbamoyl-cyclohexyl) carbamic acid tert-butyl ester (4b).     

Water-in-oil macroemulsions sustain long-term viability of microbial cells in organic solvents

Extremely stable water-in-oil macroemulsions have been obtained by dispersing water in isooctane in the presence of lecithin. Either prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and Rhodotorula minuta) cells hosted in these water-in-oil macroemulsions are viable for weeks despite the consistent excess of organic solvent (ranging from 70 to 84%, v/v) in these ternary systems. Conjugation occurs upon mixing macroemulsions containing F(+) or F(-) Escherichia coli strains, indicating consistent mass transfer between the water droplets. Populations of yeasts hosted in water-in-oil macroemulsion feature a higher frequency of cells aggregation when compared with the corresponding populations suspended in homogeneous aqueous media.     

Multiple effects of isoproterenol on horse plasma cholinesterase

Isoproterenol inhibits the hydrolysis of butyrylthiocholine by horse plasma cholinesterase, while it stimulates the hydrolysis of p-nitrophenyl butyrate. The inhibition pattern obtained for butyrylthiocholine is consistent with a dimeric model for the enzyme showing negative cooperativity. The kinetics of inhibition point to a dissociative effect of isoproterenol, superimposed on its competitive inhibitory action. The hydrolysis of p-nitrophenyl butyrate is not sensitive to changes in the subunit composition of the enzyme.     

Stereospecific hydrolysis of 3-(4-methoxyphenyl)glycidic ester in supercritical carbon dioxide by immobilized lipase

In the hydrolysis of racemic 3-(4-methoxyphenyl)glycidic acid methyl ester by immobilized Mucor miehel lipase in supercritical CO₂ the initial hydrolysis rate of the (2S,3R)-form was faster than the rate of the (2R,3S) -form. The stereoisomeric excess of the (2R,3S)-form reached 87 % at 53 % total conversion level. The water content of the reaction mixture and the initial concentration of the 3-(4-methoxyphenyl) glycidic acid methyl ester had no effect on isomeric purity. The reaction rate in supercritical CO₂ was considerably faster than in toluene/water -mixture.     

High level expression and characterization of a thermostable lysophospholipase from Thermococcus kodakarensis KOD1

Phospholipases can catalyze the hydrolysis of one or more ester and phosphodiester bonds and have a considerable interest in the food, oil leather and pharmaceutical industries. In this report, a lysophospholipase gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (LysoPL-tk) was cloned. The gene of 783?bp encodes a 260-amino acid protein with a molecular mass of 29?kDa. LysoPL-tk has a consensus motif (GxSxG) and a catalytic triad (S, D, H) of esterases in the deduced amino acid sequence. LysoPL-tk was expressed in Escherichia coli and purified to homogeneity. The enzyme can degrade substrates with both short and long acyl chain lengths. The apparent K (m) value for p-nitrophenyl butyrate was 607.1?μM with V (max) values of 95.5?U/mg. The enzyme was active at a broad range of pH (5-8) and temperatures (70-95?°C) with the optimum pH and temperature being 8.0 and 85?°C, respectively. The high yield, broad substrate range along with its thermo-stability indicates that LysoPL-tk is a potential enzyme in industrial application.     

Soluble NTPDase: An additional system of nucleotide hydrolysis in rat blood serum

The participation of a nucleoside triphosphate diphosphohydrolase in the nucleotide hydrolysis by rat blood serum was evaluated. Nucleoside triphosphate diphosphohydrolase and phosphodiesterase are enzymes possibly involved in ATP and ADP hydrolysis. The specific activity of the phosphodiesterase activity (using thymidine 5'-monophosphate p-nitrophenyl ester as substrate) was 4.92 +/- 0.73 (mean +/- SD, n = 10) nmol p-nitrophenol.min(-1).mg(-1) protein and the specific activities for ATP and ADP were 1.31 +/- 0.37 (mean +/- SD, n = 7) and 1.36 +/- 0.25 (mean +/- SD, n = 5) nmol Pi.min(-1).mg(-1) protein, respectively. A competition plot demonstrated that ATP and ADP hydrolysis occurs at the same active site. The effect of suramin and phenylalanine on ATP, ADP and thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis was investigated. The results were opposite considering the hydrolysis of ATP and ADP and that of the substrate marker for the enzyme phosphodiesterase. These results are indicative of the presence of, at least, two enzymes participating in the serum nucleotide hydrolysis. The presence of cAMP did not affect the hydrolysis velocity of ATP and ADP, while thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis was inhibited by cAMP by approximately 47%, suggesting that the hydrolysis of the ATP and ADP, under our assay conditions, occurs at a different site from the phosphodiesterase site. Both enzyme activities, in the rat blood serum, may be involved in the modulation of the nucleotide/nucleoside ratio in the circulation, serving an in vivo homeostatic and antithrombotic function. In addition, the phosphodiesterase may act on DNA or RNA liberated upon tissue injury and/or cell death.     

Development of a serum-free system for the in vitro cultivation of Brugia malayi infective-stage larvae

Over the past several years, numerous attempts have been made to culture the infective-stage (L3) larvae of the human filarial parasite Brugia malayi in an in vitro system that promotes molting to the fourth larval stage (L4). Although there have been reports in the literature of successful L3 to L4 development in vitro, all of these systems have required serum supplementation. The complexity of serum as a culture supplement has made reproducibility of results and identification of specific factors necessary for L3 development problematic. We have developed a serum-free in vitro system consisting of RPMI 1640 supplemented with one of three fatty acids (arachidonic, linoleic, or linolenic) that supports consistent and reproducible molting to the fourth larval stage in the presence of a basidiomycetous yeast, Rhodotorula minuta. Coculture with this yeast, initially isolated as a culture contaminant, is required for successful molting. In serum-free cultures lacking R. minuta, L3 larvae survive for upward of 2 weeks, but do not molt successfully. The L4 larvae generated in cultures containing R. minuta are well formed, as seen by light and electron microscopy, and are capable of further development upon transfer to a permissive host. This culture system is inexpensive and easily reproducible, thus making it a useful tool for studying the requirements for the development of B. malayi L3.     

Expression and characterization of a novel enantioselective lipase from Acinetobacter species SY-01

A novel lipase gene, lipase A, of Acinetobacter species SY-01 (A. species SY-01) was cloned, sequenced, and expressed in Bacillus subtilis 168. The deduced amino acid (aa) sequences for the lipase A and its chaperone, lipase-specific chaperone, were found to encode mature proteins of 339 aa (37.2 kDa) and 347 aa (38.1 kDa), respectively. The aa sequence of lipase A and lipase-specific chaperone shared high homology 82 and 67% identity with the lipase A and the lipase B of A. species RAG-1. This new lipase was defined as a group I Proteobacterial lipase family. The expressed lipase A was purified through sequential treatment with Q-Sepharose, Resource Q, and Superdex-S75 columns. The maximal activity was observed at 50 degrees C for hydrolysis of p-nitrophenyl monoesters and found to be stable at pH 9-11, with optimal activity at pH 10. Lipase A hydrolyzed wide range of fatty acid esters of p-nitrophenyl, but preferentially hydrolyzed short length acyl chains (C2 and C4). Moreover, lipase A from A. species SY-01 catalyzed hydrolysis of the two acetate isomers of cis-(+/-)-2-(bromomethyl)-2-(2,4-dichloro phenyl)-1,3-dioxolane-4-methyl acetate, an intermediate required for the synthesis of Itraconazole which was an anti-fungal drug, at different rate and yielded cis-(-)-isomer in 81.5% conversion with 91.9% enantiomeric excess.     

Rhodotorula pinicola sp. nov., a basidiomycetous yeast species isolated from xylem of pine twigs

Three pink-colored yeast strains 3-1-3, 10-3-3 and 19-3-3 were isolated from xylem of surface-sterilized twigs of Pinus tabulaeformis collected from Dongling Mountain, Beijing, in different seasons. These strains were identified as Rhodotorula minuta (Saito) F.C. Harrison by conventional taxonomic characterization. However, molecular phylogenetic analysis based on internal transcribed spacer region (including 5.8S rDNA) and large-subunit rDNA D1/D2 domain sequences indicated that they represent a novel basidiomycetous yeast species, for which Rhodotorula pinicola is proposed (type strain: AS 2.2193(T)=CBS 9130(T)). The new species was most closely related to Rhodotorula laryngis Reiers?l in the R. minuta complex.     

Arginine 127 stabilizes the transition state in carboxypeptidase

Crystallographic studies suggest that Arg-127 is a key amino acid in the hydrolysis of peptides and esters by carboxypeptidase A. The guanidinium group of Arg-127 is hypothesized to stabilize the oxyanion of the tetrahedral intermediate formed by the attack of water on the scissile carbonyl bond. We have replaced this amino acid in rat carboxypeptidase A1 with lysine (R127K), methionine (R127M), and alanine (R127A), in order to define the role of Arg-127 in carboxypeptidase catalyzed hydrolysis. The wild-type and mutant enzymes were expressed in yeast and purified. Kinetic studies show that Arg-127 substitution decreases kcat for both ester and amide substrates, whereas Km is relatively unchanged; for R127M and R127A this corresponds to a 6 kcal/mol decrease in transition state stabilization of the rate-limiting step. The binding affinity for the phosphonate transition state analog, Cbz-Phe-Ala(P)-OAla, was decreased by 5.4 kcal/mol, whereas binding affinity for the ground state inhibitor, DL-benzylsuccinic acid, was decreased by only 1.7 kcal/mol for R127M. Electrostatic calculations employing a finite difference solution to the Poisson-Boltzmann equation predict that the positive charge of Arg-127 should stabilize the transition state by 6-8 kcal/mol. Therefore, the experimental and theoretical data suggest that the primary role of Arg-127 is stabilization of the transition state through electrostatic interaction with the oxyanion.     

High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates

The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.     

Cholesterol esterase catalyzed hydrolysis of mixed micellar thiophosphatidylcholines: a possible charge-relay mechanism

Mechanistic features of cholesterol esterase catalyzed hydrolysis of two thiophospholipids, rac-1-(hexanoylthio)-2-hexanoyl-3-glycerophosphorylcholine (6TPC) and rac-1-(decanoylthio)-2-decano-yl-3-glycerophosphorylcholine (10TPC), have been characterized. The hydrolysis of 10TPC that is contained in mixed micelles with Triton X-100 occurs strictly at the micellar interface, since the reaction rate is independent of the micelle concentration but depends hyperbolically on the mole fraction of the substrate in the micelles. This latter observation allows one to calculate the interfacial kinetic parameters V*max and K*m. The hydrolyses of 10TPC and p-nitrophenyl butyrate are similarly inhibited by the transition state analogue inhibitor phenyl-n-butylborinic acid, and therefore, physiological and nonphysiological substrates are processed at the same active site. The similarity of k*cat values for the acyl-similar substrates 10TPC and p-nitrophenyl decanoate indicates that the phospholipase A1 activity of cholesterol esterase is partially rate limited by turnover of a decanoyl-enzyme intermediate. Solvent isotope effects on V*max and V*max/K*m (which monitors acylation only) are approximately 2-3 and are consistent with transition states that are stabilized by general acid-base proton transfers. Proton inventories of V*max/K*m indicate that simultaneous proton transfers stabilize the acylation transition state, which requires a multifunctional acid-base machinery (perhaps a charge-relay system) in the cholesterol esterase active site. Similar results are obtained for the 6TPC reaction, both in the presence and absence of Triton X-100 micelles.