Alan Wiseman

Fungal and other β-d-glucosidases — Their properties and applications

Abstract β- d -Glucosidase (β- d -glucoside glucohydrolase, EC 3.2.1.21) has been described in a variety of fungi and bacteria. Its function — to catalyse the hydrolysis of cellobiose, and aryl and alkyl β- d -glucosides — depends upon the nature of its source. Recent interest in this enzyme centres on its role in the enzymatic hydrolysis of cellulose. The rate and extent of cellulose hydrolysis can be increased by supplementing commercial cellulases with immobilized β- d -glucosidase, which has high stability and can be recovered and reused. The current state of β- d -glucosidase biotechnology is described.

Cytochrome P-450 and oxidative metabolism in molluscs

1. Cytochrome P-450 and the associated components and oxidative activities of a mixed-function oxidase (MFO) system are localized primarily in the microsomes of the digestive gland of molluscs. 2. Cytochrome P-450 and putative cytochrome P-450-catalysed oxidative activities, measured in vitro and/or in vivo, have variously been detected in 23 species of mollusc. 3. Cytochrome P-450 and other MFO components and activities may be increased by exposure to xenobiotics, but the results are variable and no correlation is obvious between changes in cytochrome P-450 content and measured MFO activities (benzo[a]pyrene hydroxylase (BPH) and 7-ethoxycoumarin O-deethylase (ECOD)). 4. Type II binding compounds (clotrimazole, miconazole, ketoconazole, metyrapone and pyridine) give type II difference spectra with mussel digestive gland microsomal P-450, whereas type I binding compounds (testosterone, 7-ethoxycoumarin, alpha-naphthoflavone, SKF525-A) give apparent reverse type I difference spectra. 5. The existence of multiple or particular forms (P450 IVA or LAw) of cytochrome P-450 is indicated from enzyme kinetics and inhibition studies, seasonality, purification studies and cDNA probes. 6. Microsomal MFO activities are observed even in the absence of added or generated NADPH, and the NADPH-independent BPH, ECOD and N,N-dimethylaniline N-demethylase activities are inhibited by reducing agents, including NADPH. 7. The major metabolites of microsomal benzo[a]pyrene metabolism are quinones. 8. One-electron oxidation is considered to be one possible mechanism of molluscan cytochrome P-450 catalytic action.

Evidence for the existence of cytochrome P450 gene families (CYP1A, 3A, 4A, 11A) and modulation of gene expression (CYP1A) in the mussel Mytilus spp.

The presence and expression of four gene families of the cytochrome P450 multi-gene family (CYP450) was investigated in the digestive gland of M. edulis using Southern and Northern blot analysis. The cDNA probes employed were for CYP1A1 (pfP1450-3′, 1.5kb from rainbow trout), CYP3A (phPCN12, human), CYP4A1 (2.1kb, rat) and CYP1A1 (1.9kb, human). Single hybridising mRNAs of approximately 2.1kb were seen for all probes, indicating presence and expression of genes from or similar to the four families. Hybridisation on Southern blots was demonstrated for CYP3A and CYP11A, confirming the presence of related CYP genes. The former gave a strong band at about 4.5kb and a weak one at 7.3kb, and the latter a single band at 13kb. The detection of CYP3A-like sequences is consistent with the reported presence of testosterone 6-β-hydroxylase activity in M. edulis. Marked variation in levels of CYP1A-like mRNA were seen in mussels from several sites in the Venice area (UNESCO-MURST Venice Lagoon Project), indicating modulation of gene expression. Some correlation with contaminant exposure was seen, indicating potential for use of this measurement as a biomarker for organic pollution.

Seasonal and sex variation in the mixed-function oxygenase system of digestive gland microsomes of the common mussel, Mytilus edulis L.

Abstract 1. Sex differences in the MFO system were minimal. 2. Coincident increases in contents of cytochrome P-450, the “416-peak” and total haemoprotein occurred in summer and early winter. 3. Changes in NADH-dependent reductase activities were similar, and distinct from those of NADPH-cytochrome c reductase and cytochrome b 5 . 4. NADPH-independent 7-ethoxycoumarin O -deethylase activity changed from undectable in summer to a maximum in early winter. The increase coincided with a peak in P-450 content, the appearance of a 51.5 K d protein (SDS-PAGE), and a blue shift in the λ max of P-450. 5. The results are interpreted in terms of isozymes of P-450 and a seasonal role in endogenous monooxygenation.bl]

Studies on the properties of highly purified cytochrome P-448 and its dependent activity benzo[a]pyrene hydroxylase, from Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae, produces a cytochrome P-450 enzyme with a Soret peak in the reduced-CO difference spectrum at 448 nm. The enzyme purified to homogeneity (88-97% pure on a specific content basis) has a molecular wt. of 55 500 as determined by SDS-PAGE. Amino acid analysis of yeast cytochrome P-448 revealed 407 amino acid residues per molecule with a 43% complement of hydrophobic residues. Although the number of residues is smaller than cytochrome P-448 enzymes from mammalian sources, the percentage of hydrophobic residues is almost identical. Estimation of the haem content of yeast cytochrome P-448 showed that one haem group was present per molecule. Phospholipid was present at very low levels. The molecular wt. of the polypeptide chain plus an estimated 5-6 units of hexose and of hexosamine is in good agreement with the molecular wt. value obtained from SDS-PAGE. A reconstituted system of purified cytochrome P-448, purified NADPH-cytochrome P-450 (c) reductase and phospholipid showed aryl hydrocarbon hydroxylase activity towards benzo[a]pyrene. Both protein components, NADPH and dilauroyl phosphatidylcholine (or emulgen 911) were necessary for full activity. The NADPH requirement could be replaced by cumene hydroperoxide or H2O2 generated in situ from a glucose oxidase system; in each case Vmax is increased, but the apparent affinity for benzo[a]pyrene, as measured by an increased Km, is lowered. The spin state of purified yeast cytochrome P-448 was 94% low spin (22 degrees C) as determined from the temperature-dependent spin-state equilibrium. The addition of benzo[a]pyrene to this enzyme resulted in a change to higher spin state (18% high spin at 22 degrees C). Equilibrium gel filtration analysis of the number of benzo[a]pyrene binding sites per mole of enzyme monomer showed a value of 1 for purified yeast cytochrome P-448 and 6 for this enzyme in microsomal form. The corresponding values for purified and microsomal cytochrome P-450 from phenobarbital-pretreated rats are 1 and 6, respectively. However, purified cytochrome P-448 from beta-naphthoflavone-induced rats gave a value of 6 benzo[a]pyrene binding sites. Type I binding spectra with purified yeast cytochrome P-448 were observed with benzo[a]pyrene, lanosterol, ethylmorphine, dimethylnitrosamine, sodium phenobarbitone and perhydrofluorene. Type II spectral changes were observed with imidazole, aniline and benzphetamine. Cytochrome P-448 from Saccharomyces cerevisiae is identified as a distinct enzyme of the P-450 family. This enzyme however has many properties in common with cytochrome P-448 from mammalian sources.(ABSTRACT TRUNCATED AT 400 WORDS)

The involvement of salt links in the stabilization of baker's yeast invertase. Evidence from immobilization and chemical modification studies.

Abstract Bakers yeast invertase was immobilized onto microcrystalline cellulose DEAE- and CM-Sephadex and insolubilized concanavalin A, with 46, 41, 70 and 73% recovery of activity, respectively. The concanavalin A and microcrystalline cellulose invertase complexes exhibited spreading of their pH optimum curves, possessed similar Km and V and were more thermally stable in the absence and presence of 0.12 M sucrose, than the soluble enzyme. DEAE- and CM-Sephadex · invertase complexes were extremely unstable towards heat, compared to the soluble enzyme, losing all activity within 1 min of heating to 65°C. However, normal thermal stability of these complexes was observed in the presence of 20 mM succinic acid and 20 mM ethylenediamine, respectively. The treatment of purified invertase with citraconic anhydride, which converts the amino group function to carboxyl groups, resulted in the loss of enzyme activity. The enzyme, with 50% activity after such tretments, was less thermally stable than the untreated enzyme. Similar results were obtained using ethylene diamine and a water-soluble carbodiimide that converts carboxyl group function to amino group function. Treatment of invertase with methylacetimidate (which modifies amino groups of proteins but retains the positive charge) resulted in no loss in activity and very little effect on thermal stability. It is suggested that salt linkages may be important in maintaining the conformational stability of invertase. Modification of either amin or carboxyl groups chemically or by immobilization on to charged supports, would result in the loss of conformational stability, unless these stabilizing salt linkages were preserved.

Selenium‐Dependent Glutathione Peroxidase and other Selenoproteins: Their Synthesis and Biochemical Roles

Selenium-dependent glutathione peroxidase (SeGPX, EC 1.11.1.9) acts as part of the antioxidant defence of the body. It functions as a highly efficient catalyst to reduce a wide variety of intracellular peroxides, including hydrogen and lipid peroxides, thereby detoxifying these potentially damaging molecules. Thus selenium has a critical role in normal cellular biochemistry and the proposal has been made that deficiency of selenium in the diet can lead to a number of clinical disorders. World-wide interest in selenoproteins is now evident. In general, the active site of SeGPX and of other selenoproteins is based upon a selenium moiety (generally a single covalently bound atom of selenium) present as the amino acid selenocysteine. There has been an extension of the genetic code to include the codon TGA (in the correct DNA context) as the 21st codon specifying the presence of selenocysteine in the polypeptide structure of selenoproteins. Recent discoveries in SeGPX biochemistry and molecular biology in relation to its protective function are reviewed here in comparison to other selenoproteins. The mechanisms of selenium incorporation into selenoproteins is also described. In the future, antioxidant enzymes such as selenium-dependent glutathione peroxidase may find a use in biotechnology for the protection of enzymes and cellular membranes. While it is recognised that in cost terms the use of enzyme mimics of SeGPX may be more advantageous, large-scale in-vitro production of the selenoproteins is now possible and their value can be readily assessed.

Purification and characterization of the cytochrome P-448 component of a benzo(a)pyrene hydroxylase from Saccharomyces cerevisiae

Abstract Cytochrome P-448, a type of cytochrome P-450, from brewers yeast (Saccharomyces cerevisiae) grown under conditions of glucose repression was isolated and purified. Triton X-100 in very low concentration proved to be very effective in stabilizing P-448 in the microsomal fraction and later prevented its conversion to cytochrome P-420 through solubilization with various ionic and nonionic detergents. Highest yields were obtained with 1% sodium cholate, in the presence of 0.1% Triton X-100 and reduced glutathione. A novel combination of hydrophobic adsorption and other chromatographic techniques was used for the purification of cytochrome P-448. These involve the use of amino octyl-Sepharose 4B, instead of the low-yielding aminohexyl derivative, followed by the fast-running hydroxyapatite-cellulose column. Finally, the use of DEAE-Sephacel was found to increase greatly the purity of the cytochrome P-448 obtained. The molecular weight of this preparation was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr, 55,500). Using the known molar extinction coefficient of the carbon monoxide-difference spectrum the estimate of degree of purity of cytochrome P-448 obtained by this purification procedure was between 88 and 97%. Electrophoresis also showed that this preparation was completely homogeneous and assays showed that it was also completely free of cytochrome bs, cytochrome c reductase and cytochrome P-420. Purified cytochrome P-448 reconstituted with cytochrome P-450 (cytochrome c) reductase, isolated from yeast, showed 10-fold higher aryl hydrocarbon hydroxylase activity with benzo[a]pyrene as a substrate than the corresponding microsomal fraction enzyme. Kinetics of benzo[a]pyrene hydroxylation were determined: Km (33 μ m ) was comparable with that reported for purified hepatic cytochrome P-448. The number of binding sites of microsomal and purified cytochromes P-450 (from liver of phenobarbital-induced rats) and yeast cytochrome P-448 with benzo[a]pyrene has been determined using and equilibrium gel filtration method. There is one binding site in each case (contrast with six sites for microsomal enzymes). The Scatchard plot gives number of binding sites, apparent association constants (K), and the equivalent dissociation constants (Ks). Comparison is made with spectral dissociation constants for these enzymes and benzo[a]pyrene. Thus the proportion bound, dissociation constant (Ks), and stoichiometry of rat liver (phenobarbital induced) and yeast cytochrome P-448 with benzo[a]pyrene were compared with corresponding values for microsomal fractions of both systems. Purified enzymes had higher Ks values in both cases, and the proportion of enzyme that bound benzo[a]pyrene was high (53%) for liver and this value is 100% for purified enzyme from yeast, which is the same as the value obtained for the microsomal enzyme from yeast.

Molecular Modelling of Lanosterol 14α-Demethylase (CYPSl) from Saccharomyces Cerevisiae via Homology with CYP102, a Unique Bacterial Cytochrome P450 Isoform: Quantitative Structure-Activity Relationships (QSARs) within two Related Series of Antifungal Azole Derivatives

The construction of a three-dimensional molecular model of the fungal form of cytochrome P450 (CYP51) from Saccharomyces cerevisiae, based on homology with the haemoprotein domain of CYP102 from Bacillus megaterium (a unique bacterial P450 of known crystal structure) is described. It is found that the endogenous substrate, lanosterol, can readily occupy the putative active site of the CYP51 model such that the known mono-oxygenation reaction, leading to C14-demethylation of lanosterol, is the preferred route of metabolism for this particular substrate. Key amino acid contacts within the CYP51 active site appear to orientate lanosterol for oxidative attack at the C14-methyl group, and the position of the substrate relative to the haem moiety is consistent with the phenyl-iron complexation studies reported by Tuck et al. [J. Biol. Chem., 267, 13175-13179 (1992)]. Typical azole inhibitors, such as ketoconazole, are able to fit the putative active site of CYP51 by a combination of haem ligation, hydrogen bonding, pi-pi stacking and hydrophobic interactions within the enzymes haem environment. The mode of action of azole antifungals, as described by the modelling studies, is supported by quantitative structure-activity relationship (QSAR) analyses on two groups of structurally related fungal inhibitors. Moreover, the results of molecular electrostatic isopotential (EIP) energy calculations are compatible with the proposed mode of binding between azole antifungal agents and the putative active site of CYP51, although membrane interactions may also have a role in the antifungal activity of azole derivatives.

Regulation of the biosynthesis of cytochrome P-450 in brewer's yeast. Role of cyclic AMP.

The drug metabolising enzyme cytochrome P-450 has been studied in great detail in mammalian systems and its presence in microorganisms is also well established. However, neither its function nor its means of control in brewers yeast, Saccharomyces cerevisiae, has been investigated. We demonstrate here using yeast protoplasts that it is the intracellular concentration of cyclic AMP which controls, by repression, the de novo synthesis of the enzyme, and also that cyclic AMP concentrations are in turn inversely related to the concentration of glucose in the yeast growth medium.