Liver prenylated methylated protein methyl esterase is an organophosphate-sensitive enzyme

Prenylation and subsequent methylation are essential modifications on a significant proportion of eucaryotic proteins. Proteins such as the G-gamma subunits of G-protein coupled receptors, nuclear lamins, and guanine nucleotide-binding proteins such as Ras are prenylated and undergo methylation. Prenylated methylated protein methyl esterase (PMPMEase) readily hydrolyses the prenylated protein methyl esters, thus making this step reversible and possibly regulatory. Benzoyl-glycyl-farnesyl-cysteine methyl ester (BzGFCM) was developed as a specific PMPMEase substrate and characterized by electron spray ionization mass spectrometry (ESI-MS) to be of the calculated molecular mass. Rat liver and brain PMPMEase hydrolyzed BzGFCM, forming benzoyl-glycyl-farnesyl-cysteine (BzGFC) in a time- and concentration-dependent manner. Both enzymes cleaved BzGFCM with K(m) values of 4.58 +/- 0.30 and 25.57 +/- 2.36 microM and V(max) values of 2.21 +/- 0.03 and 0.17 +/- 0.003 nmol/min/mg, respectively. The liver enzyme eluted from a gel-filtration column as a single peak of apparent size, 89 kDa. The brain enzyme eluted as two main peaks of 53 and 890 kDa. Organophosphorus pesticides (OPs), which are suspected to be involved in human disorders such as parkinsonism, neuronal, and retinal degeneration, inhibited the liver enzyme with IC(50) values from 4.77 muM for parathion to 0.04 microM for paraoxon, respectively. Only about 25% of the brain enzyme was inhibited by 0.5-1 mM solutions of mipafox, while 0.1 and 1 mM paraoxon inhibited over 50% and 95% of the enzyme, respectively. Paraoxon is thus about 2,250 times less potent against the brain than the liver PMPMEase. BzGFCM was not hydrolyzed by various cholinesterases, indicating its specificity for PMPMEase. Perturbations in prenylated protein metabolism might play a role in noncholinergic OPs-induced toxicity, since prenylated proteins play such important roles in cell signaling, proliferation, differentiation, and apoptosis.     

Insights into the physiological role of pig liver esterase: Isoenzymes show differences in the demethylation of prenylated proteins

The possible physiological role of PLE (E.C. 3.1.1.1) located in the endoplasmic reticulum (ER) of pig liver cells in the conversion of endogenous compounds was investigated as it was reported, that PLE acts as prenylated methylated protein methyl esterase (PMPMEase) hydrolysing methylesters of prenylated proteins. Using the specific PMPMEase substrate benzoyl–glycyl–farnesyl–cysteine methyl ester (BzGFCM), six different PLE isoenzymes expressed recombinantly in the yeast Pichia pastoris were found active. Activities ranged from 1.6–15.6 mU per mg protein and it is suggested that Pro285 has a major influence on high activity. In addition, the role of the C-terminal HAEL retention signal for translocation of pig liver esterase (PLE) in the endoplasmic reticulum (ER) of eukaryotic cells was studied using the γ-isoenzyme of PLE expressed in Pichia pastoris. Using truncated versions (HAE, HA, H and without retention signal) of the enzyme it was found that in contrast to earlier reports no influence of the signal peptide on the expression rate of PLE was found. However, higher enzyme activities were obtained in the periplasmatic fraction compared to the supernatant irrespective of the presence or absence of HAEL and the trimeric formation seems to occur in the supernatant of P. pastoris X33 enabling an easier transition of monomeric forms through cell membranes.     

A novel post-translational modification of yeast elongation factor 1A. Methylesterification at the C terminus

Protein methylation reactions can play important roles in cell physiology. After labeling intact Saccharomyces cerevisiae cells with S-adenosyl-l-[methyl-(3)H]methionine, we identified a major methylated 49-kDa polypeptide containing [(3)H]methyl groups in two distinct types of linkages. Peptide sequence analysis of the purified methylated protein revealed that it is eukaryotic elongation factor 1A (eEF1A, formerly EF-1alpha), the protein that forms a complex with GTP and aminoacyl-tRNAs for binding to the ribosomal A site during protein translation. Previous studies have shown that eEF1A is methylated on several internal lysine residues to give mono-, di-, and tri-N-epsilon-methyl-lysine derivatives. We confirm this finding but also detect methylation that is released as volatile methyl groups after base hydrolysis, characteristic of ester linkages. In cycloheximide-treated cells, methyl esterified eEF1A was detected largely in the ribosome and polysome fractions; little or no methylated protein was found in the soluble fraction. Because the base-labile, volatile [methyl-(3)H]radioactivity of eEF1A could be released by trypsin treatment but not by carboxypeptidase Y or chymotrypsin treatment, we suggest that the methyl ester is present on the alpha-carboxyl group of its C-terminal lysine residue. From the results of pulse-chase experiments using radiolabeled intact yeast cells, we find that the N-methylated lysine residues of eEF1A are stable over 4 h, whereas the eEF1A carboxyl methyl ester has a half-life of less than 10 min. The rapid turnover of the methyl ester suggests that the methylation/demethylation of eEF1A at the C-terminal carboxyl group may represent a novel mode of regulation of the activity of this protein in yeast.     

Evidence for an S-farnesylcysteine methyl ester at the carboxyl terminus of the Saccharomyces cerevisiae RAS2 protein

The protein products of yeast and mammalian ras genes are posttranslationally modified to give mature forms that are localized to the inner surface of the plasma membrane. We have previously demonstrated that the mature form of the Saccharomyces cerevisiae RAS2 gene product is methyl esterified at a modified C-terminal cysteine residue. Here we provide evidence that this residue is an S-farnesylcysteine alpha-carboxyl methyl ester. This result establishes common posttranslational modifications for RAS proteins and fungal sex factors. These polypeptides exhibit sequence similarities at their C-termini that appear to be the critical recognition elements for a common set of modification enzymes. In mammalian cells, proteins with analogous C-terminal sequences appear to be prenylated and carboxyl methylated by a similar mechanism.     

Inhibitors of sterol synthesis. Oleate ester of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one as a substrate for pancreatic cholesterol esterase

5 alpha-Cholest-8(14)-en-3 beta-yl-15-one oleate (15-ketosteryl oleate), the oleate ester of a compound with the capacity to lower serum cholesterol, was effectively hydrolyzed by partially purified porcine pancreatic cholesterol esterase with an apparent Km of 0.28 +/- 0.01 mM and a Vmax of 0.62 +/- 0.01 mumol/min per mg protein compared to an apparent Km of 0.19 +/- 0.02 mM and a Vmax of 0.37 +/- 0.02 mumol/min per mg protein for cholesteryl oleate. The 15-ketosteryl oleate was also hydrolyzed by highly purified rat pancreatic cholesterol esterase with an apparent Km of 0.20 +/- 0.01 mM and a Vmax of 86.7 +/- 3.0 mumol/min per mg protein compared to an apparent Km of 0.43 +/- 0.01 mM and a Vmax of 119.8 +/- 2.6 mumol/min per mg protein for cholesteryl oleate. 15-Ketosteryl oleate is, therefore, a good substrate for pancreatic cholesterol esterase from either source. The 15-ketosterol is a weak competitive inhibitor of partially purified porcine pancreatic cholesterol esterase when cholesteryl oleate is the substrate.     

Binding of the delta subunit to rod phosphodiesterase catalytic subunits requires methylated, prenylated C-termini of the catalytic subunits

PDE6 (type 6 phosphodiesterase) from rod outer segments consists of two types of catalytic subunits, alpha and beta; two inhibitory gamma subunits; and one or more delta subunits found only on the soluble form of the enzyme. About 70% of the phosphodiesterase activity found in rod outer segments is membrane-bound, and is thought to be anchored to the membrane through C-terminal prenyl groups. The recombinant delta subunit has been shown to solubilize the membrane-bound form of the enzyme. This paper describes the site and mechanism of this interaction in more detail. In isolated rod outer segments, the delta subunit was found exclusively in the soluble fraction, and about 30% of it did not coimmunoprecipitate with the catalytic subunits. The delta subunit that was bound to the catalytic subunits dissociated slowly, with a half-life of about 3.5 h. To determine whether the site of this strong binding was the C-termini of the phosphodiesterase catalytic subunits, peptides corresponding to the C-terminal ends of the alpha and beta subunits were synthesized. Micromolar concentrations of these peptides blocked the phosphodiesterase/delta subunit interaction. Interestingly, this blockade only occurred if the peptides were both prenylated and methylated. These results suggested that a major site of interaction of the delta subunit is the methylated, prenylated C-terminus of the phosphodiesterase catalytic subunits. To determine whether the catalytic subunits of the full-length enzyme are methylated in situ when bound to the delta subunit, we labeled rod outer segments with a tritiated methyl donor. Soluble phosphodiesterase from these rod outer segments was more highly methylated (4.5 +/- 0.3-fold) than the membrane-bound phosphodiesterase, suggesting that the delta subunit bound preferentially to the methylated enzyme in the outer segment. Together these results suggest that the delta subunit/phosphodiesterase catalytic subunit interaction may be regulated by the C-terminal methylation of the catalytic subunits.     

Hydrolysis of 13-sophorosyloxydocosanoic acid esters by acetyl- and carboxylesterases isolated from Candida bororiensis.

Two enzymes have been isolated from Candida bogoriensis which catalyze the hydrolysis of 13-sophorosyloxydocosanoic acid (Glc2HDA) esters obtained from this organism. The 6',6"-diacetyl derivative of Glc2HDA (Ac2Glc2HDA) is hydrolyzed by an acetylesterase (EC 3.1.1.6) which has been purified 1300-fold. The acetylesterase has a molecular weight of 35,000 estimated from gel filtration, and is much more active with p-nitrophenyl acetate than with the acetylated glycolipid. The rate of hydrolysis increases with Ac2Glc2HDA concentration until a plateau is reached at a concentration of about 40 muM, near the critical micelle concentration of this glycolipid. These kinetic data are interpreted as an enzyme specificity for the monomeric, but not the micellar form of the glycolipid. The acetylesterase is inhibited by 0.1 to 10 mM diisopropyl fluorophosphate, 5 mM p-hydroxymercuribenzoate, and 5 mM N-ethylmaleimide, but only slightly by 5 mM iodoacetamide. The methyl ester of Ac2Glc2HDA is hydrolyzed by at least two carboxylesterases (EC 3.1.1) which differ in size according to gel filtration. Their molecular weights are estimated as 140,000 for carboxyesterase A and 40,000 for carboxyesterase B. Both carboxylesterases were purified over 20-fold, and carboxylesterase A was characterized further. Carboxylesterase A activity was inhibited completely by 0.1 to 10 mM diisopropyl fluorophosphate and by 10 mM p-hydroxymercuribenzoate, but only slightly by lower concentrations of p-hydroxymercuribenzoate or by N-ethylmaleimide or iodoacetamide. The carboxylesterase A preparation also acted as a thioesterase with palmityl-CoA (palmityl-CoA hydrolase, EC 3.1.2.2), showing the following approximate relative activities: palmityl-CoA, 100; octanoyl-CoA, 90; methyl Glc2HD, 22; butyryl-CoA, 18; methyl AcGlc2HD, 15; methyl Ac2Glc2HD, 10; and acetyl-CoA, O. Methyl Ac2Glc2HD showed some substrate inhibition at higher concentrations, but neither methyl Ac2Glc2HD nor palmityl-CoA approached enzyme saturation until well above their critical micelle concentrations, indicating hydrolysis of the micellar substrate was occurring. The carboxylesterase and palmityl-CoA hydrolase activities were destroyed in a parallel fashion by heat denaturation, and each substrate inhibited the action of the preparation on the other substrate, but the preparation has not been purified sufficiently to establish with certainty that both activities reside in the same protein.     

Effects of phospholipids on hydrolysis of trioleoylglycerol by human serum carboxylesterase

Human serum carboxylesterase (EC 3.1.1.1), purified by affinity chromatography on trimethylammonium anilinium-Sepharose, hydrolyzed the short-chain fatty acid ester tributyrin (40 mumol/mg protein per h), but scarcely hydrolyzed the long-chain fatty acid ester triolein (less than 0.2 mumol/mg protein per h). Phospholipids enhanced triolein hydrolysis by carboxylesterase to various extents, cardiolipin causing the most enhancement (2.5 mumol/mg protein per h). Phosphatidylserine and phosphatidylinositol also enhanced carboxylesterase-catalyzed hydrolysis of triolein (450-980 nmol/mg protein per h). The optimal pH for tributyrin hydrolysis was pH 8.0, but the pH range for triolein hydrolysis was broad, being pH 4.5-7.5. The rates of hydrolyses of monoolein, diolein and triolein by carboxylesterase in the absence and presence of 100 micrograms/ml cardiolipin were 3.9, 0.5 and 0.2 mumol/mg esterase per h and 2.0, 0.6 and 4.0 mumol/mg protein per h, respectively. Thus, on addition of cardiolipin, triolein hydrolysis was enhanced, but tributyrin hydrolysis was reciprocally decreased. Triton X-100 (0.1%) and NaCl (1.0 M) decreased triolein hydrolysis, but did not decrease tributyrin hydrolysis. Mercaptoethanol decreased triolein hydrolysis, but not tributyrin hydrolysis. These results suggest that cardiolipin modifies the interaction of carboxylesterase with substrates in such a way as to facilitate its interaction with a hydrophobic substrate, and that disulfide bonding might be involved in the substrate recognition site.     

Hydrolysis of substance p and neurotensin by converting enzyme and neutral endopeptidase

Angiotensin I converting enzyme (ACE) and neutral endopeptidase ("enkephalinase"; NEP), were purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln6-Phe7,-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. NEP hydrolyzed 0.1 mM SP, NT and their C-terminal fragments at the following rates (mumol/min/mg): SP1-11 = 7.8, SP4-11 = 11.7, SP5-11 = 15.4, SP6-11 = 15.6, SP8-11 = 6.7, NT1-13 = 2.9, and NT8-13 = 4.0. Purified ACE rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl- dependent and inhibited by captopril. ACE released mainly C-terminal tripeptide from SP methyl ester, but only dipeptide from SP free acid. Modification of arginine residues in ACE with cyclohexanedione or butanedione similarly inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80-93%) indicating an active site arginine is required for hydrolysis of SP. ACE hydrolyzed NT at Tyr11-Ile12 to release Ile12-Leu13. SP, NT and their derivatives (0.1 mM) were cleaved by ACE at the following rates (mumol/min/mg): SP1-11 = 1.2, SP methyl ester = 0.7, SP free acid = 8.5, SP4-11 = 2.4, SP5-11 = 0.9, SP6-11 = 1.4, SP8-11 = 0, NT1-13 = 0.2, and NT8-13 = 1.3. Peptide substrates were used as inhibitors of ACE (substrate = FA-Phe-Gly-Gly) and NEP (substrate = Leu5-enkephalin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification, expression, and purification of a pyrethroid-hydrolyzing carboxylesterase from mouse liver microsomes

Carboxylesterases are enzymes that catalyze the hydrolysis of a wide range of ester-containing endogenous and xenobiotic compounds. Although the use of pyrethroids is increasing, the specific enzymes involved in the hydrolysis of these insecticides have yet to be identified. A pyrethroid-hydrolyzing enzyme was partially purified from mouse liver microsomes using a fluorescent reporter similar in structure to cypermethrin (Shan, G., and Hammock, B. D. (2001) Anal. Biochem. 299, 54-62 and Wheelock, C. E., Wheelock, A. M., Zhang, R., Stok, J. E., Morisseau, C., Le Valley, S. E., Green, C. E., and Hammock, B. D. (2003) Anal. Biochem. 315, 208-222) and subsequently identified as a carboxylesterase (NCBI accession number BAC36707). The expressed sequence tag was then cloned, expressed in baculovirus, and purified to homogeneity. Kinetic constants for a large number of both type I and type II pyrethroid or pyrethroid-like substrates were determined. This esterase possesses similar kinetic constants for cypermethrin and its fluorescent-surrogate (k(cat) = 0.12 +/- 0.03 versus 0.11 +/- 0.01 s(-1)). Compared with their cis- counterparts, trans-permethrin and cypermethrin were hydrolyzed 22- and 4-fold faster, respectively. Of the four fenvalerate isomers the (2R)(alphaR)-isomer was hydrolyzed at least 1 order of magnitude faster than any other isomer. However, it is unlikely that this enzyme accounts for the total pyrethroid hydrolysis in the microsomes because both isoelectrofocusing and native PAGE indicate the presence of a second region of cypermethrin-metabolizing enzymes. A second carboxylesterase gene (NCBI accession number NM_133960), isolated during a cDNA mouse liver library screening, was also found to hydrolyze pyrethroids. Both these enzymes could be used as preliminary tools in establishing the relative toxicity of new pyrethroids.     

Methylation suppresses the proteasome-inhibitory function of green tea polyphenols

Under physiological conditions, biotransformation reactions, such as methylation, can modify green tea polyphenols (GTPs) and therefore limit their in vivo cancer-preventive activity. Although a recent study suggested that methylated polyphenols are less cancer-protective, the molecular basis is unknown. We previously reported that ester bond-containing GTPs, for example (-)-epigallocatechin-3-gallate [(-)-EGCG] or (-)-epicatechin-3-gallate [(-)-ECG], potently and specifically inhibit the proteasomal chymotrypsin-like activity. In this study, we hypothesize that methylated GTPs have decreased proteasome-inhibitory abilities. To test this hypothesis, methylated (-)-EGCG and (-)-ECG analogs that can be found in vivo were synthesized and studied for their structure-activity relationships (SARs) using a purified 20S proteasome. The addition of a single methyl group on (-)-EGCG or (-)-ECG led to decreased proteasome inhibition and, as the number of methyl groups increased, the inhibitory potencies further decreased. These SARs were supported by our findings from in silico docking analysis published recently. Previously, we synthesized a peracetate-protected (-)-EGCG molecule, Pro-EGCG (1), to enhance its cellular permeability and stability, and current HPLC analysis confirms conversion of Pro-EGCG (1) to (-)-EGCG in cultured human leukemic Jurkat T cells. Furthermore, in this study, peracetate-protected forms of methylated GTPs were added in intact Jurkat T cells to observe the intracellular effects of methylation. Peracetate-protected, monomethylated (-)-EGCG induced greater cellular proteasome inhibition and apoptosis than did peracetate-protected, trimethylated (-)-EGCG, consistent with the potencies of the parent methylated analogs against a purified 20S proteasome. Therefore, methylation on GTPs, under physiological conditions, could decrease their proteasome-inhibitory activity, contributing to decreased cancer-preventive effects of tea consumption.     

Paralogous gene analysis reveals a highly enantioselective 1,2-O-isopropylideneglycerol caprylate esterase of Bacillus subtilis.

Carboxylesterase NP of Bacillus subtilis Thai I-8, characterized in 1992 as a very enantioselective (S)-naproxen esterase, was found to show no enantiopreference towards (S)-1,2-O-isopropylideneglycerol (IPG) esters. The ybfK gene was identified by the B. subtilis genome project as an unknown gene with homology to carboxylesterase NP. The purpose of the present study was to characterize the ybfK gene product in order to determine whether this paralogue of carboxylesterase NP had an altered or enhanced stereospecificity. The ybfK gene was cloned and expressed in B. subtilis using a combination of two strong promoters in a multicopy vector. The enzyme was purified from the cytoplasm of B. subtilis by means of anion exchange and hydrophobic interaction chromatography. The purified YbfK is an enzyme of 296 amino acids and shows an apparent molecular mass of 32 kDa (SDS/PAGE). Comparison of the activities of YbfK and carboxylesterase NP towards caprylate esters of IPG revealed that YbfK produces (S)-IPG with 99.9% enantioselectivity. Therefore, we conclude that we have isolated a paralogue of carboxylesterase NP that can be used for the enantioselective production of (S)-IPG.     

Identification of a gamma-glutamyl methyl ester in bacterial membrane protein involved in chemotaxis.

Evidence is presented that a methyltransferase enzyme, previously shown to be necessary for chemotaxis and identified as the cheR gene product, catalyzes the formation of a gamma-glutamyl methyl ester in one or more membrane proteins of Salmonella typhimurium. The rates of release of methyl label from the methylated protein in acid, base, and hydroxylamine are consistent with a methyl ester and not with a methylated imidazole, guanidino, or amino group. A gamma-glutamyl methyl ester was isolated from a proteolytic digest of the modified protein.     

Purification of cat submaxillary kallikrein.

The cat submaxillary gland contains 1,000--2,400 kallikrein units (KU)/g of tissue. The submaxillary kallikrein was purified to homogeneity by acetone fractionation, DEAE-Sephadex A-50 chromatography, Sephadex G-75 gel filtration, and Ampholine isoelectric focusing. The kallikrein was separated by isoelectric focusing into 6--7 forms with pI's between 4.2 and 5.1. One mg of the purified kallikrein contained 930--1,260 KU in the dog vasodilator assay, and hydrolyzed 15--25 and 9--12 mumol of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and N-alpha-toluenesulfonyl-L-arginine methyl ester (TAME), respectively, in 1 min at 25 degrees C and pH 8.0. The Km's of the purified kallikrein with BAEE and TAME were 0.67 and 0.34 mM, respectively. Hydrolysis of N-alpha-benzoyl-L-tyrosine ethyl ester (BTEE), N-alpha-benzoylarginine-p-nitroanilide (BApNA), and casein was small or negligible. The apparent molecular weight of the kallikrein was estimated to be 5 X 10(4) by Sephadex G-100 gel filtration and 4.7 X 10(4) by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). The kallikrein was found to contain 18.5% carbohydrate by weight. Trasylol and soybean trypsin inhibitor were not specific inhibitors of this kallikrein.     

Isolation and characterization of p-coumaroyl esterase from the anaerobic fungus Neocallimastix strain MC-2.

An extracellular p-coumaroyl esterase produced by the anaerobic fungus Neocallimastix strain MC-2 released p-coumaroyl groups from 0-[5-0-((E)-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-0-beta -D-xylopyranosyl-(1----4)-D-xylopyranose (PAXX). The esterase was purified 121-fold from culture medium in successive steps involving ultrafiltration column chromatography on S-sepharose and hydroxylapatite, isoelectric focusing, and gel filtration. The native enzyme had an apparent mass of 11 kDa under nondenaturing conditions and a mass of 5.8 kDa under denaturing conditions, suggesting that the enzyme may exist as a dimer. The isoelectric point was 4.7, and the pH optimum was 7.2. The purified esterase had 100 times more activity towards PAXX than towards the analogous feruloyl ester (FAXX). The apparent Km and Vmax of the purified p-coumaroyl esterase for PAXX at pH 7.2 and 40 degrees C were 19.4 microM and 5.1 microM min(-1), respectively. p-Coumaroyl tetrasaccharides isolated from plant cell walls were hydrolyzed at rates similar to that for PAXX, whereas a dimer of PAXX was hydrolyzed at a rate 20-fold lower, yielding 4,4'-dihydroxy-alpha-truxillic acid as an end product. Ethyl and methyl p-coumarates were hydrolyzed at very slow rates, if at all. The purified esterase released p-coumaroyl groups from finely, but not coarsely, ground plant cell walls, and this activity was enhanced by the addition of xylanase and other cell wall-degrading enzymes.     

Screening and its potential application of lipolytic activity from a marine environment: characterization of a novel esterase from <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> CL180

To develop an enantioselective lipase/esterase hydrolyzing racemic ofloxacin ester to levofloxacin, samples were collected from a variety of marine environments such as cold sea, hydrothermal vent area, sediment, tidal flat area, arctic sea, marine organisms, and so on. Microorganisms were isolated by plating on an enrichment medium with simultaneous detection of lipolytic activities and screened for the hydrolysis of ofloxacin ester. Three candidates among isolates were selected, and one of them, identified as Yarrowia lipolytica CL180, hydrolyzed preferentially S-enantiomer of racemic ofloxacin ester. The lipase/esterase gene (yli180) was cloned by screening a genomic library. The sequence analysis revealed an open reading frame consisting of 1,431 bp that encoded a protein of 476 amino acids with a molecular mass of 53 kDa. The yli180 gene was expressed in Escherichia coli and purified to homogeneity. The optimum activity of the recombinant protein (rYli180) occurred at pH 7.5 and 35°C, respectively. rYli180 preferentially hydrolyzed p-nitrophenyl esters of fatty acids with short chain lengths of ≤10 carbon atoms. This study represents a novel esterase of type B1 carboxylesterase/lipase family from a marine isolate, showing a potential usage as a biocatalyst because of enantioselectivity toward racemic ofloxacin ester.     

An NAD:cysteine ADP-ribosyltransferase is present in human erythrocytes

A novel enzymatic activity, i.e., the catalysis of the formation of ADP-ribosylcysteine, was found in the cytosol of human erythrocytes. The NAD:cysteine ADP-ribosyltransferase was partially purified by sequential chromatographic steps on phenyl-Sepharose, phosphocellulose, and Sepharose CL-6B. The enzyme has an apparent molecular weight of 27,000 +/- 3,000, as determined by gel permeation. The formation of ADP-ribosylcysteine was associated with the stoichiometric release of nicotinamide from NAD. The enzyme was found to be highly specific toward cysteine and cysteine methyl ester as ADP-ribose acceptors. S-Benzoyl-L-cysteine, cystine, histidine, glutamic acid, arginine, arginine methyl ester, and agmatine were ineffective as acceptors for this enzyme.     

Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. strain MB1

A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M(r) of approximately 65,000. The apparent K(m) of the enzyme (mean +/- standard deviation) for cocaine was measured as 1.33 +/- 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.     

A metabolite of aspartame inhibits angiotensin converting enzyme

Aspartame (L-aspartyl-L-phenylalanine methyl ester, is a widely used artificIal sweetener. In humans and other animals aspartame is initially hydrolyzed to L-aspartyl-L-phenylalanine by intestinal esterases. L-Aspartyl-L-phenylalanine inhibits angiotensin converting enzyme purified from rabbit lungs with a Ki of 11 +/- 2 microM, equipotent to the IC50 of 12 microM for 2-D-methyl-succinyl-L-proline which has been reported to be an orally active antihypertensive agent in rats. Thus the possibility exists that L-aspartyl-L-phenylalanine inhibits angiotensin converting enzyme in humans consuming large quantities of aspartame. Both aspartame itself and the diketopiperazine formed from it, 3-carboxymethyl-6-benzyl-2,5-diketopiperazine, are weak inhibitors with Ki's greater than 1 mM.     

红纹黄单胞菌α-氨基酸酯水解酶的分离纯化及酶学性质

【目的】从红纹黄单胞菌中分离纯化了胞内α-氨基酸酯水解酶(AEH),并进行了酶学性质研究。【方法】采用乙酸丁酯破碎细胞,并相继用磷酸钙凝胶沉淀、硫酸铵分级沉淀、DEAE Sephadex A-50阴离子交换处理、CM Cellulose 52离子交换层析和Sephadex G-200凝胶过滤层析纯化得到了电泳纯α-氨基酸酯水解酶,并研究了此酶的酶学性质。【结果】SDS-PAGE显示α-氨基酸酯水解酶的亚基分子量为70 kDa。酶促合成头孢克洛的最适pH为6.8,最适温度为42℃,在pH5.0-8.0和35℃以下,酶保持了良好的稳定性。Mn2+和Ca2+对酶活有一定的促进作用,Cu2+、Fe2+及高浓度的丙酮对酶活有强的抑制作用。AEH催化D-苯甘氨酸甲酯、D-对羟基苯甘氨酸甲酯和头孢克洛水解反应的kcat/Km分别为123.7±3.7 mmol-1.s-1.L、2.9±0.6 mmol-1.s-1.L和101.3±2.1 mmol-1.s-1.L,AEH对D-苯甘氨酸甲酯的催化效率最高。AEH催化双底物反应的机制为乒乓机制,催化合成头孢克洛的kcat为547.3±38.2 s-1。【结论】有关红纹黄单胞菌α-氨基酸酯水解酶的酶学性质研究相对较少,本文的研究将为该酶催化合成β-内酰胺类抗生素的工业化应用提供重要参数。