Selective targeting of a laccase from Stachybotrys chartarum covalently linked to a carotenoid-binding peptide.

Atwo-step targeting strategy was used to identify improved laccases for bleaching carotenoid-containing stains on fabric. We first applied a modified phage display technique to identify peptide sequences capable of binding specifically to carotenoid stains and not to fabric. Prior deselection on the support on which the carotenoid was localized, increased stringency during the biopanning target selection process, and analysis of the phage peptides' binding to the target after acid elution and polymerase chain reaction (PCR) postacid elution, were used to isolate phage peptide libraries with increased binding selectivity and affinity. Peptide sequences were selected based on identified consensus motifs. We verified the enhanced carotenoid-binding properties of the peptide YGYLPSR and subsequently cloned and expressed C-terminal variants of laccase from Stachybotrys chartarum containing carotenoid-binding peptides YGYLPSR, IERSAPATAPPP, KASAPAL, CKASAPALC, and SLLNATK. These targeted peptide-laccase fusions demonstrate enhanced catalytic properties on stained fabrics.

The structural requirements of sterols for membrane function in Saccharomyces cerevisiae.

Cultures of Saccharomyces cerevisiae strain GL7 auxotrophic for sterol were incubated with a series of sterols and sterol-like molecules (tetracyclic and pentacyclic triterpenoids) in order to determine the structural requirements of sterols for bulk membrane function. For growth support, the 3 beta-OH group could not be replaced by H, OMe, OBu, NH2, NHOH, OAc, keto, or 3 alpha-OH. A methyl group at C-14 was neither deleterious nor essential for activity. Removal of the C-4 geminal methyl group was obligatory for activity. Thus, no sterol-like triterpenoid supported growth (e.g., tetrahymanol, lanosterol, and cycloartenol). Growth support required a sterol with the longest methylene segment extending from C-20 not to exceed six contiguous C-atoms and the stereochemistry must be C-20 R. No significance could be attributed to branching at C-20 (i.e., to C-21), C-24 (when alkylated), or C-25 (regarding the isopropyl group). Double bonds in the nucleus were not essential for activity since cholestanol supported growth. In several incubations, the addition of trace levels of dietary ergosterol (0.5 microgram/ml) to the medium was necessary to promote growth and transformation of the bulk sterol to a membrane competent sterol(s).

Structural requirements for transformation of substrates by the S-adenosyl-L-methionine:delta 24(25)-sterol methyltransferase. Inhibition by analogs of the transition state coordinate.

Microsomes from sunflower seedlings were used to investigate the transition state coordinate for the C-24 methylation reaction that mediates phytosterol biosynthesis. They were then used to study structurally related cationic and uncharged compounds of the natural sterol substrate, which were designed to interfere with the reaction progress. The hypothetical reaction course is described to proceed through an Sn2 formation of an activated complex involving the initial production of a covalent structure with a dative bond (methyl from AdoMet attacks si-face of the 24,25-double bond of the sterol) and the secondary production of a series of high energy intermediates, the stabilization of which determines the final C-24 methylated product. Derivatives of lanosterol and cholesterol with a methyl, hydrogen, oxygen, or bromine atom introduced into the side chain and/or at C-3 in place of the natural nucleophile were studied as inhibitors that interfere with the formation of the hypothetical tertiary isopropylcarbinyl cation intermediate in the conversion of cycloartenal to 24(28)-methylene cycloartanol. The data indicate the most potent inhibitor is a sterol with an aziridine group attached to C-24(25), which mimics the bridged C-24(25) carbenium ion generated in the transition state, and the methyltransferase possesses two strategic sites: one that recognizes the proximal end of the sterol acting as a proton donor and the other that recognizes the distal end that acts as a proton acceptor. The best fit (binding/catalysis) involves a flat sterol (including substrate and inhibitor) with intact unsubstituted nucleophilic centers at C-3 and C-24 and a freely rotating side chain that can assume the pseudocyclic conformation.

Structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase.

The membrane-bound enzyme of microsomes obtained from sunflower embryos that catalyzes the bi-substrate transfer reaction whereby the methyl group of (S)-adenosyl-L-methionine is transferred to C-24 of the sterol side chain has been investigated. Optimal incubation conditions for assay of the microsomal (S)-adenosyl-L-methionine:sterol delta 24-methyl transferase (SMT) have been established for the first time. The microsomal preparation was found to catalyze the formation of a delta 24(28)-sterol and to be free of contaminating methyl transferase enzymes, e.g. those which form delta 23-24 methyl sterols (cyclosadol) and delta 25-24 beta-methyl sterols (cyclolaudenol) and other sterolic enzymes which might transform the acceptor molecule to metabolites which could compete in the assay with the test substrate. From a series of incubations with 27 sterol and sterol-like (triterpenoids) substrates of which 23 compounds possessed a 24,25-double bond, we observed a marked dependence on precise structural features and three-dimensional shape of the acceptor molecule in its ability to be transformed by the SMT. In contrast to the yeast SMT where cycloartenol fails to bind to the SMT and zymosterol is the best substrate for methylation, the sunflower SMT studied here utilizes cycloartenol preferentially to zymosterol and the other substrates. Of the chemical groups which distinguishes cycloartenol, a free 3 beta-OH,9 beta,19-cyclopropyl group, trimethylated saturated nucleus, and delta 24-double bond, only the nucleophilic centers at C-3 and C-24 were obligatory for substrate binding and methylation. Of the bent or flat conformations which cycloartenol may orient in the enzyme-substrate complex, our results indicate a selection for acceptor molecules which possess the shape that closely resembles the crystal state and solution orientation of cycloartenol which is now known to be flat rather than bent (Nes, W. D., Benson, M., Lundin, R. E., and Le, P. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5759-5763).

Regulation of sterol biosynthesis in sunflower by 24(R,S),25-epiminolanosterol, a novel C-24 methyl transferase inhibitor.

Whereas sitosterol and 24(28)-methylene cycloartanol were competitive inhibitors (with Ki = 26 microM and 14 microM, respectively), 24(R,S)-25-epiminolanosterol was found to be a potent non-competitive inhibitor (Ki = 3.0 nM) of the S-adenosyl-L-methionine-C-24 methyl transferase from sunflower embryos. Because the ground state analog, 24(R,S)-oxidolanosterol, failed to inhibit the catalysis and 25-azalanosterol inhibited the catalysis with a Ki of 30 nM we conclude that the aziridine functions in a manner similar to the azasteriod (Rahier, A., et al., J. Biol. Chem. (1984) 259, 15215) as a transition state analog mimicking the carbonium intermediate found in the normal transmethylation reaction. Additionally, we observed that the aziridine inhibited cycloartenol metabolism (the preferred substrate for transmethylation) in cultured sunflower cells and cell growth.