Characterization of the structure and composition of gecko adhesive setae.

The ability of certain reptiles to adhere to vertical (and hang from horizontal) surfaces has been attributed to the presence of specialized adhesive setae on their feet. Structural and compositional studies of such adhesive setae will contribute significantly towards the design of biomimetic fibrillar adhesive materials. The results of electron microscopy analyses of the structure of such setae are presented, indicating their formation from aggregates of proteinaceous fibrils held together by a matrix and potentially surrounded by a limiting proteinaceous sheath. Microbeam X-ray diffraction analysis has shown conclusively that the only ordered protein constituent in these structures exhibits a diffraction pattern characteristic of beta-keratin. Raman microscopy of individual setae, however, clearly shows the presence of additional protein constituents, some of which may be identified as alpha-keratins. Electrophoretic analysis of solubilized setal proteins supports these conclusions, indicating the presence of a group of low-molecular-weight beta-keratins (14-20 kDa), together with alpha-keratins, and this interpretation is supported by immunological analyses.

Cloning and characterisation of a novel P-glycoprotein homologue from barley.

P-glycoproteins are members of a large superfamily of transport proteins (the 'traffic ATPases') that utilize ATP to translocate a wide range of substrates across biological membranes. Using a PCR-based approach, and degenerate oligonucleotides corresponding to conserved motifs, two 300-bp cDNA fragments (pBMDR1 and pBMDR2) with a significant sequence similarity to mammalian P-glycoproteins were amplified from barley (Hordeum vulgare) root poly A+ RNA and used as probes to screen a barley root cDNA library. A single full-length clone pHVMDR2 coding for a polypeptide of 1232 residues (c. 134 kDa) was isolated. Comparison of this barley sequence with Arabidopsis ATPGP1 and human MDR1 and MDR3 P-glycoprotein sequences showed that the barley cDNA has 44%, 37% and 38% amino acid (aa) identity, respectively, with these sequences, and conserved structural features. RNase protection analysis showed that HVMDR2 mRNA is expressed at low levels in both barley roots and leaves. Southern blot analyses indicated that there is a small multigene family related to P-glycoproteins in barley. Possible functions for these barley P-glycoproteins are discussed.

Expression of CYP71B7, a cytochrome P450 expressed sequence Tag from Arabidopsis thaliana.

The systematic sequencing of anonymous cDNA clones (expressed sequence tags or ESTs) from the plant Arabidopsis thaliana has identified a number of cDNAs with similarity to known cytochrome P450 sequences. The partial sequence of one of these cDNAs, 5G6, indicated that it was likely to encode a full-length cytochrome P450 monooxygenase (cyt P450) sequence. In this paper we describe the complete sequence of this clone, which has been designated CYP71B7 in accordance with the nomenclature for the cyt P450 gene superfamily. The cDNA was used to determine the pattern of expression of the corresponding gene in A. thaliana. Northern hybridization analysis indicated that maximal expression of CYP71B7 occurred in rosette leaves. Weaker hybridizing bands were also detected by Northern analysis of RNA from roots, leaves, flowers, and siliques. No expression could be detected in stem tissue. Southern analysis indicated that the CYP71B7 gene was likely to exist as a single copy in the genome of A. thaliana. CYP71B7 was expressed episomally in yeast, and microsomes prepared from transgenic yeast exhibited a carbon monoxide difference spectrum characteristic of cyt P450. Microsomes from yeast expressing CYP71B7 were assayed for enzymatic activity with synthetic model cyt P450 substrates. Microsomes from yeast cells expressing CYP71B7 or those from control cells exhibited no detectable NADPH-supported 7-ethoxycoumarin or 7-ethoxyresorufin deethylase activities. However, in the presence of cumene hydroperoxide, activity was observed with microsomes from cells expressing CYP71B7 with 7-ethoxycoumarin as substrate. Organic hydroperoxides are well known to support cyt P450 catalysis in the absence of electrons from NADPH. The yeast microsomes contained high levels of endogenous NADPH-ferricytochrome P450 reductase (CPR) activity. The data suggest that this A. thaliana cyt P450, although expressed in an active form, is incapable of accepting electrons from the endogenous yeast CPR protein.

Spatially distinct expression of two new cytochrome P450s in leaves of Nepeta racemosa: identification of a trichome-specific isoform.

Using a PCR-based approach, two novel cytochrome P450 cDNAs were isolated from a catmint (Nepeta racemosa) leaf cDNA library. The cDNAs (pBSK3C7 and pBSK4C3) were 76.9% identical in their nucleotide sequences, indicating that they are the products of two closely-related genes. A comparison of the sequence of these cDNAs with database sequences indicated that they represent new members of the CYP71 gene family of plant cytochrome P450s. Clone pBSK3C7 contains the full-length coding sequence of a cytochrome P450, whilst pBSK4C3 lacks ca. 6 codons at the 5' end. The cytochromes P450 encoded by these clones were designated CYP71A5 and CYP71A6 (pBSK3C7 and pBSK4C3, respectively). Southern blot analysis indicated that the corresponding genes were present as single copies in the genome of N. racemosa. Northern blot analysis showed that a gene homologous with CYP71A5 was expressed in the related species N. cataria, but no homologue of CYP71A6 was detected in this species. Expression of CYP71A5 in N. racemosa was maximal in flowers, tissues within the apical bud, and young expanded leaves. That of CYP71A6 was maximal in older leaves. Expression of CYP71A5 occurred exclusively in trichomes present on the leaf surfaces, in contrast to that of CYP71A6, which occurred predominantly within the leaf blade tissues.

Purification and characterization of an acyclic monoterpene primary alcohol:NADP+ oxidoreductase from catmint (Nepeta racemosa).

A soluble monoterpene primary alcohol:NADP+ oxidoreductase has been purified to apparent homogeneity from leaves of the catmint, Nepeta racemosa. The purified enzyme consisted of two polypeptides, with molecular masses of 42,000 and 40,000 Da, and contained zinc ions. A number of monoterpene alcohols (geraniol, nerol, citronellol, and their hydroxylated derivatives) were substrates, but the enzyme was inactive toward ethanol. The enzyme required NADP(H) as cofactor, with NAD(H) ineffective. Gas chromatographic and coupled mass spectrometric analysis of the reaction products showed that 10-hydroxygeraniol and 10-hydroxynerol were oxidized by the enzyme in the presence of NADP+, at both C-1 and C-10. These results are consistent with a role for this enzyme in the biosynthesis of iridoid monoterpenes.

Cytochrome P-450 in plant/insect interactions: geraniol 10-hydroxylase and the biosynthesis of iridoid monoterpenoids.

The interactions between plant secondary metabolites (particularly monoterpenes) and insects are discussed. Such metabolites are likely to have influenced the evolution of cyt P450-linked detoxification systems in animals, through animal/plant coevolution. The biosynthesis of many classes of plant secondary metabolites involves cyt P450 enzymes. Of these, one of the best characterised is the geraniol/nerol 10-hydroxylase which catalyses a key step in the biosynthesis of the iridoid class of plant terpenes. It would appear that these monoterpenoids are synthesised (via cyt P450 hydroxylation) from different precursors in different plant species, namely geraniol, its isomer nerol, or the related monoterpenoid, citronellol. We show that cyt P450 from the plants Catharanthus roseus and Nepeta racemosa are capable of hydroxylating geraniol, nerol and citronellol, and thus do not impose precursor specificity on iridoid biosynthesis in plants.

Cytochrome P-450-catalysed monoterpenoid oxidation in catmint (Nepeta racemosa) and avocado (Persea americana); evidence for related enzymes with different activities.

A cytochrome P-450 present in ripening avocado (Persea americana) fruit mesocarp (CYP71A1) had previously been shown to metabolize the monoterpenoids nerol and geraniol (Hallahan et al. (1992) Plant Physiol. 98, 1290-1297). Using DNA encoding CYP71A1 as a hybridization probe, we have shown by Southern analysis that a related gene is present in the catmint, Nepeta racemosa. RNA blot analysis, together with Western analysis of catmint leaf polypeptides using avocado cyt P-450 antiserum, showed that a closely related gene is expressed in catmint leaves. Cytochrome P-450 in catmint microsomes catalysed the specific hydroxylation of nerol and geraniol at C-10, whereas avocado CYP71A1, in either avocado microsomes or heterologously expressed in yeast, catalysed 2,3- or 6,7-epoxidation of these substrates. These results suggest that orthologous genes of the CYP71 family are expressed in these two plant species, but catalyse dissimilar reactions with monoterpenoid substrates.

Interactions of Avocado (Persea americana) Cytochrome P-450 with Monoterpenoids.

The microsomal fraction of avocado (Persea americana) mesocarp is a rich source of cytochrome P-450 active in the demethylation of xenobiotics. Cytochrome P-450 from this tissue has been purified and well characterized at the molecular level (DP O'Keefe, KJ Leto [1989] Plant Physiol 89: 1141-1149; KR Bozak, H Yu, R Sirevag, RE Christoffersen [1990] Proc Natl Acad Sci USA 87: 3904-3908). Despite this extensive characterization, the role of the enzyme in vivo was not established. Optical and electron paramagnetic resonance binding studies described here suggest that the monoterpenoids, nerol and geraniol, are substrates of avocado cytochrome P-450 (spectral dissociation constant of 7.2 and 35 micromolar, respectively). Avocado microsomes have been shown to catalyze the hydroxylation of these monoterpenoids, and both nerol and geraniol have been shown to inhibit the activity of avocado cytochrome P-450 toward the artificial substrate 7-ethoxycoumarin, with nerol a competitive inhibitor of this activity.

Synthesis and biological activity of an azido derivative of paclobutrazol, an inhibitor of gibberellin biosynthesis.

A photolabile azido derivative of the kaurene oxidase inhibitor 1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-l-yl) pentan-3-ol (paclobutrazol) has been synthesized for use as a photoaffinity labeling agent. The compound was tested as an inhibitor of the oxidation of ent-kaurene catalyzed by cell-free preparations from endosperm of Cucurbita maxima. The I(50) of the azido derivative was 9.5 nanomolar, which compares well with that of paclobutrazol (6.3 nanomolar in our measurements). The azido compound bound to Cytochrome P-450 in microsomes from Cucurbita maxima, and induced a Type II spectral change, with an apparent binding constant of 0.24+/-0.04 micromolar.

Reversible activation of hydrogenase from Escherichia coli.

Hydrogenase from Escherichia coli exhibited low activity when assayed for hydrogen:methyl viologen reductase activity and no activity when assayed for hydrogen-uptake activity with acceptors of high redox potential (dichloroindophenol, methylene blue). Nor did the enzyme as isolated catalyse proton-tritium exchange activity. Incubation under hydrogen resulted in an increase in hydrogen-uptake activity with methyl viologen and the appearance of hydrogen-uptake activity with dichloroindophenol and methylene blue. Following such treatment, the enzyme also readily catalysed isotope exchange. This process is interpreted as the conversion of the hydrogenase from an inactive 'unready' state to an 'active' state. Oxidation of active hydrogenase with dichloroindophenol caused conversion to a state resembling that of the enzyme as isolated but capable of more rapid activation under reducing conditions. This form is termed the 'ready' state. Such interconversions have been reported for hydrogenases from Desulfovibrio gigas and D. desulfuricans, and the possibility that they constitute a regulatory mechanism suggested.

Differential inhibition of catalytic sites in Desulfovibrio gigas hydrogenase.

The hydrogenase of Desulfovibrio gigas has been shown to contain one nickel atom, a cluster with three irons and two clusters of the [4Fe-4S] type in an 89 kDa molecule. Though evidence that the nickel ion is involved in the site of hydrogen activation has been presented for this and other hydrogenases, the role of nickel and of the other redox centres in the protein remains to be firmly identified. We have examined the effects of inhibitors of hydrogenase activity in an attempt to identify the functions of the prosthetic redox centres. We have shown carbon monoxide to inhibit at the site of hydrogen activation. The dye, procion red, was found to compete with electron acceptors at a different site, and partial denaturation with the detergent lithium dodecyl sulphate resulted in the differential inhibition of hydrogen activation and substrate reduction. These results imply the presence of distinct domains within the protein with different catalytic activities.