Plant-Derived Enzymes Producing Chiral Aroma Compounds and Potential Application

Aroma (volatile) compounds play important ecological functions in plants, and also contribute to the quality of plant-derived foods. Moreover, chiral aroma compounds affect their functions in plants and lead to different flavor quality properties. Formations of chiral aroma compounds are due to the presence of enzymes producing these compounds in plants, which are generally involved in the final biosynthetic step of the aroma compounds. Here, we review recent progress in research on the plant-derived enzymes producing chiral aroma compounds, and their changes in response to environmental factors. The chiral aroma enzymes that have been reported produce (R)-linalool, (S)-linalool, (R)-limonene, and (S)-limonene, etc., and these enzymes are found in various plant species. We also discuss the origins of enantioselectivity in the plant-derived enzymes producing chiral aroma compounds and summarize the potential use of plants containing enzymes producing chiral aroma compounds for producing chiral flavors/fragrances.     

Prostatic trypsin-like kallikrein-related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs)

The prostate is a site of high expression of serine proteinases including members of the kallikrein-related peptidase (KLK) family, as well as other secreted and membrane-anchored serine proteinases. It has been known for some time that members of this enzyme family elicit cellular responses by acting directly on cells. More recently, it has been recognised that for serine proteinases with specificity for cleavage after arginine and lysine residues (trypsin-like or tryptic enzymes) these cellular responses are often mediated by cleavage of members of the proteinase-activated receptor (PAR) family--a four member sub-family of G protein-coupled receptors. Here, we review the expression of PARs in prostate, the ability of prostatic trypsin-like KLKs and other prostate-expressed tryptic enzymes to cleave PARs, as well as the prostate cancer-associated consequences of PAR activation. In addition, we explore the dysregulation of trypsin-like serine proteinase activity through the loss of normal inhibitory mechanisms and potential interactions between these dysregulated enzymes leading to aberrant PAR activation, intracellular signalling and cancer-promoting cellular changes.     

Distinction between mouse DNA polymerases alpha and beta by tryptic peptide mapping.

Results presented here and in a previous paper (Tanabe et al. (1979) Biochemistry 18, 3401--3406) indicate that mouse beta-polymerase is a single polypeptide with an apparent molecular weight of 40,000. This polypeptide has now been analyzed by tryptic peptide mapping. Comparison of the results with identical analysis of mouse alpha-polymerase reveals that the tryptic peptides derived from the two enzymes are different. These results indicate that beta-polymerase is neither a subunit of alpha-polymerase nor a proteolytic degradation product of alpha-polymerase.     

Digestive processes during the development of the roach, Rutilus rutilus L.

During development of the roach from larvae to adults relative gut length increases from 44 to 104% of the body length. Gut passage rate correlates with gut length, ranging from 2.25 h in larvae to 6.2 h in adults (at 20 ± 1°C). In controlled experiments (20°C, feeding on carp diet) tryptic activity of the gut content increases with the age of the fish. In larvae artificial diet leads to considerable increase of tryptic activity, but to low growth rate and finally to total mortality. The reabsorption of digestive enzymes in the hindgut is efficient only in adults in which tryptic activity in the second half of the intestine is reduced to about 12% of total activity.     

An approach to the antigenic structure of arginine kinase and creatine kinase. Physical, chemical and immunological study of some modified derivatives.

The antigenic structure of arginine kinase and creatine kinase has been approached using chemical modifications and enzymatic cleavage. Mild performic oxidation that, with a restricted number of oxidized amino acid residues, results for both enzymes in a severe decrease of the helical structure and in a large increase of the protein-solvent interactions, affects differently their antigenic reactivity: when compared with antisera to the homologous native enzymes, arginine kinase and its oxidized derivative cross-react fully, while creatine kinase and its oxidized derivative cross-react only about 30%. The persistence of the antigenic reactivity of arginine kinase through drastic structural alterations is confirmed by the high inhibitory capacity (about 80%) of crude tryptic hydrolyzates towards the combination of argining kinase with its specific antibodies. Tryptic peptides of creatine kinase, obtained in the same conditions, inhibit weakly (about 12%) the homologous antigen-antibody interaction. The participation of the lysines in the antigenicity of arginine kinase and creatine kinase is suggested by the enhanced inhibitory capacity of the tryptic hydrolyzates when the cleavage is restricted to the arginyl peptide bounds, and was verified for arginine kinase through assays with lysine-modified derivatives.     

METHODOLOGIES FOR THE STEREOSELECTIVE SYNTHESIS OF ADRENERGIC β-BLOCKERS: AN OVERVIEW

The importance of the adrenergic β-blockers with structure of (S) 1-aryloxy-3-amino-2-propanol in the treatment of different diseases has led the development of a variety of stereoselective synthetic methodologies for this stereoisomer. In this review we present several methodologies to obtain this compound using (i) chiral substrates, (ii) chiral catalysts (organometallic or enzymes) and (iii) preparative chiral chromatography, showing the advantages and disadvantages of each methodology.     

Tryptic and chymotryptic proteases released by larvae of the blowfly, Lucilia cuprina

Proteases released by larvae of the sheep blowfly have been suggested to have a primary role in wound formation and larval nutrition. Assays were carried out on two larval products to analyse the substrate specificity of these proteases, their abundance and approximate molecular weights. Tryptic and chymotryptic activities were found in both products though there were more chymotrypsin-like enzymes in products from 48 h cultures (CESP) than in product collected direct from 48 h larvae (LESP). Sodium dodecyi sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels incubated with azocasein showed plaques of major enzyme activity at molecular weights of 20,000 and 26,000 in LESP and at 20,000 in CESP. SDS-PAGE gels, when reacted with peptide substrates showed tryptic activity at 20,000 and 26,000 in LESP, whereas CESP showed only chymotryptic activity at 20,000 and higher molecular weights. The results suggest at least three enzymes, a trypsin and chymotrypsin in LESP, a chymotrypsin in CESP and a tryptic enzyme which is not stable to SDS-PAGE probably in both LESP and CESP. In addition, reactivity with elastase and plasmin substrates suggests the presence of enzymes with general effects on skin substrates and inflammatory pathways.     

Tryptic and chymotryptic proteases released by larvae of the blowfly, lucilia cuprina

Proteases released by larvae of the sheep blowfly have been suggested to have a primary role in wound formation and larval nutrition. Assays were carried out on two larval products to analyse the substrate specificity of these proteases, their abundance and approximate molecular weights. Tryptic and chymotryptic activities were found in both products though there were more chymotrypsin-like enzymes in products from 48 h cultures (CESP) than in product collected direct from 48 h larvae (LESP). Sodium dodecyi sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels incubated with azocasein showed plaques of major enzyme activity at molecular weights of 20,000 and 26,000 in LESP and at 20,000 in CESP. SDS-PAGE gels, when reacted with peptide substrates showed tryptic activity at 20,000 and 26,000 in LESP, whereas CESP showed only chymotryptic activity at 20,000 and higher molecular weights. The results suggest at least three enzymes, a trypsin and chymotrypsin in LESP, a chymotrypsin in CESP and a tryptic enzyme which is not stable to SDS-PAGE probably in both LESP and CESP. In addition, reactivity with elastase and plasmin substrates suggests the presence of enzymes with general effects on skin substrates and inflammatory pathways.     

The active sites of cellulases are involved in chiral recognition: a comparison of cellobiohydrolase 1 and endoglucanase 1

The cellulases cellobiohydrolase 1 (CBH 1) and endoglucanase 1 (EG 1) from the fungus Trichoderma reesei are closely related with 40% sequence identity and very similar in structure. In CBH 1 the active site is enclosed by long loops and some antiparallel β-strands forming a 40 Å long tunnel, whereas in EG 1 part of those loops are missing so that the enzyme has a more common active site groove. Both enzymes were immobilized on silica and these materials were used as chiral stationary phases for chromatographic separation of the enantiomers of two chiral drugs, propranolol and alprenolol. The CBH 1 phase showed much better resolution than did the EG 1 phase, suggesting that the tunnel structure of the protein may play an important role in the chiral separation. The chiral compounds were found to be competitive inhibitors of both enzymes when p-nitrophenyl lactoside (pNPL) was used as substrate. (S)-enantiomers showed stronger inhibitory effects and also longer retention time on the stationary phases than the (R)-enantiomers. The consistency between kinetic data and retention on the stationary phases clearly shows that the enzymatically active sites of CBH 1 and EG 1 are involved in chiral recognition.     

Stereochemical course of biotin-independent malonate decarboxylase catalysis.

Malonate decarboxylases, which catalyze the conversion of malonate to acetate, can be classified into biotin-dependent and biotin-independent enzymes. In order to reveal the stereochemical course of the reactions catalyzed by the biotin-independent enzymes from Acinetobacter calcoaceticus and Pseudomonas fluorescens, a chiral substrate, malonate carrying (13)C in one carboxyl group and (3)H at one of the methylene positions, was prepared and used in the reactions catalyzed by these two enzymes. The decarboxylation of (R)-[1-(13)C(1), 2-(3)H]malonate in (2)H(2)O gave a pseudo-racemate of chiral acetate which was converted via acetyl-CoA into malate with malate synthase. From the relative proportions of the isotopomers of malate present, determined by (3)H NMR analysis, it was concluded that in the decarboxylation of malonate by these two biotin-independent enzymes COOH is replaced by H with retention of configuration. The same stereochemical outcome had been previously observed for the reaction catalyzed by the biotin-dependent malonate decarboxylase from Malonomonas rubra (J. Micklefield et al. J. Am. Chem. Soc. 117, 1153-1154, 1995).     

Purification of homologous protein carboxyl methyltransferase isozymes from human and bovine erythrocytes

We have purified the two major isozymes of the L-isoaspartyl/D-aspartyl protein methyltransferase from both human and bovine erythrocytes. These four enzymes all have polypeptide molecular weights of approximately 26,500 and appear to be monomers in solution. Each of these enzymes cross-reacts with antibodies directed against protein carboxyl methyltransferase I from bovine brain. Their structures also appear to be similar when analyzed by dodecyl sulfate gel electrophoresis for the large fragments produced by digestion with Staphylococcus aureus protease V8 or when analyzed by high-performance liquid chromatography (HPLC) for tryptic peptides. The structural relatedness of these enzymes was confirmed by sequence analysis of a total of 433 residues in 32 tryptic fragments of the human erythrocyte isozymes I and II and of the bovine erythrocyte isozyme II. We found sequence identify or probable identity in 111 out of 112 residues when we compared the human isozymes I and II and identities in 127 out of 134 residues when the human and bovine isozymes II were compared. These results suggest that the erythrocyte isozymes from both organisms may have nearly identical structures and confirm the similarities in the function of these methyltransferases that have been previously demonstrated.     

The precursor to B-type natriuretic peptide is an O-linked glycoprotein

Human pro-B-type natriuretic peptide (proBNP), the precursor for B-type natriuretic peptide (BNP), was expressed in Chinese hamster ovary cells (CHO) and compared by Western blot analysis to BNP cross-reacting material immunoprecipitated from the plasma of heart failure patients. Both recombinant and native forms co-migrated as a diffuse band centered around 25 kDa and were reduced to a 12 kDa species by treatment with a mixture of O-link deglycosylation enzymes. The 108-amino acid CHO-expressed protein was examined by tryptic mapping and LC-MS and found to be an O-linked glycoprotein. Determination of the sites of O-glycosyl addition by blank cycle sequencing of tryptic and Glu-C (Staphylococcus aureus V8 protease) peptides showed that there are seven sites of glycosylation confined to a 36-amino acid residue stretch within the center of the propeptide region. This data is consistent with previous observations of higher molecular weight isoforms of BNP.     

Rapid screening of enzymes for the enzymatic hydrolysis of chiral esters in drug discovery

Thirty-five enzymes were rapidly screened for their ability to selectively hydrolyze chiral esters to their corresponding carboxylic acids for the efficient generation of chiral intermediates in drug discovery. Optimization of the enzymatic reactions at various incubation times was performed using a robotic liquid handler. Enantiomeric pairs of chiral esters and carboxylic acids were then analyzed simultaneously by chiral GC/MS in a single analysis. This analytical approach is particularly useful for compounds that do not possess a conjugated chromophore or are volatile and difficult to analyze by chiral HPLC/UV or HPLC/MS. The resulting data was used to determine enantiomeric excesses and percent conversions to the desired enantiomer of the carboxylic acid for the selection of efficient enzymes for bioconversions in drug discovery in a pharmaceutical company.     

Protease-activated kinase II as the potential mediator of insulin-stimulated phosphorylation of ribosomal protein S6

One of the earliest responses to insulin in target cells is stimulation of the phosphorylation of ribosomal protein S6. When exponentially growing 3T3-L1 cells are serum-starved, little phosphorylation of S6 is observed; however, following addition of insulin (10(-7) M), up to 5 phosphoryl groups are incorporated into S6. An enzyme mediating the insulin-stimulated phosphorylation of S6 has been identified as protease-activated kinase II. Two-dimensional peptide maps of tryptic digests of S6 from insulin-treated 3T3-L1 cells contain 5 phosphopeptides; the same 5 phosphopeptides are observed with tryptic digests of 40 S ribosomal subunits phosphorylated in vitro by protease-activated kinase II from rabbit reticulocytes. Protease-activated kinase II has also been identified and partially purified from the postribosomal supernatant of serum-starved and insulin-treated 3T3-L1 cells. The enzyme is present in the inactive proenzyme form in serum-starved cells; following insulin treatment, approximately 50% of the enzyme is in an activated form. Identical tryptic phosphopeptide maps are observed with these enzymes.     

Different sensitivity of the sarcoplasmic reticulum Ca(2+)-ATPase enzyme to fluorescein-isothiocyanate in rabbit and carp muscles.

1. Carp and rabbit sarcoplasmatic reticulum Ca(2+)-ATPase enzymes were compared with respect to their sensitivity to FITC labelling. 2. The carp enzyme showed much lower sensitivity to FITC in the Ca(2+)-Mg2+ activated ATPase activity. Fifty percent inhibition was observed at 20 microM labelling FITC concentration; in rabbit enzyme this inhibition was already achieved at 2 microM FITC. 3. The tryptic cleavage products of the carp enzyme identified with immunoblot analysis as well as with FITC fluorescence, suggest multiple cleavage, yielding different fragments from the ones well known in rabbit and in rat enzyme. 4. The present results indicates major structural differences with respect to the FITC binding, and tryptic cleavage between the SR Ca(2+)-ATPase enzymes from carp and rabbit, despite the cross-reactivity with polyclonal antibodies.     

Structural comparison of bovine erythrocyte, brain, and liver NADH-cytochrome b5 reductase by HPLC mapping

NADH-cytochrome b5 reductases purified from bovine erythrocytes and from bovine brain and liver microsomes solubilized with lysosomal protease were subjected to structural analysis by using HPLC mapping, amino acid analysis of the resulting peptides, and NH2-terminal sequence analysis of apoproteins. HPLC maps of the tryptic peptides derived from these enzymes were very similar to each other, and amino acid analysis of the HPLC-separated peptides indicated that the structures of these enzymes are identical except for the NH2-terminal region. The NH2-terminal sequence of the brain enzyme determined by automated Edman degradation was as follows: NH2-Phe-Gln-Arg-Ser-Thr-Pro-Ala-Ile-Thr-Leu-Glu-Asn-Pro-Asp- Ile-Lys-Tyr-Pro-Leu-Arg-Leu-Ile-Asp-Lys-Glu-Val-Ile- This sequence is identical to that of liver enzyme except that the liver enzyme started at the 3rd Arg or 4th Ser. The NH2-terminal amino acid residue of the soluble erythrocyte enzyme was not detected by automated Edman degradation. The sequence analysis of a tryptic peptide from the erythrocyte enzyme indicated that Leu is present before the NH2-terminal Phe of the brain enzyme. The recently reported sequence of the apparently identical protein (Ozols et al. (1985) J. Biol. Chem. 260, 11953-11961) differs in two amino acid assignments from our sequence.