Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450 cin , CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450 cin gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450 cin may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at ϳ2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450 cin , it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450 cin by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K D ‫؍‬ 0.7 M), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450 cin catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.The cytochrome P450s (P450s) 1 are a superfamily of oxidative hemoproteins (1, 2) that carry out an enormous variety of oxidative transformations. These range from simple alkene epoxidation and heteroatom oxidation all the way through oxygen insertion into unactivated C-H bonds and C-C bond cleavage (3). P450s are broadly categorized as either soluble, e.g. most bacterial P450s, or as membrane bound/microsomal monooxygenases, e.g. most eukaryotic P450s. All P450s, however, contain a prosthetic heme group that is ligated to the protein backbone by a cysteinyl sulfur coordinated to the heme iron. It is this heme-thiolate moiety that is responsible for much of the chemistry carried out by these enzymes. The typical reaction catalyzed by a P450 is given by Reaction...

Cytochrome P450cin catalyzes the monooxygenation of 1,8-cineole, which is structurally very similar to d-camphor, the substrate for the most thoroughly investigated cytochrome P450, cytochrome P450cam. Both 1,8-cineole and d-camphor are C(10) monoterpenes containing a single oxygen atom with very similar molecular volumes. The cytochrome P450cin-substrate complex crystal structure has been solved to 1.7 A resolution and compared with that of cytochrome P450cam. Despite the similarity in substrates, the active site of cytochrome P450cin is substantially different from that of cytochrome P450cam in that the B' helix, essential for substrate binding in many cytochrome P450s including cytochrome P450cam, is replaced by an ordered loop that results in substantial changes in active site topography. In addition, cytochrome P450cin does not have the conserved threonine, Thr252 in cytochrome P450cam, which is generally considered as an integral part of the proton shuttle machinery required for oxygen activation. Instead, the analogous residue in cytochrome P450cin is Asn242, which provides the only direct protein H-bonding interaction with the substrate. Cytochrome P450cin uses a flavodoxin-like redox partner to reduce the heme iron rather than the more traditional ferredoxin-like Fe(2)S(2) redox partner used by cytochrome P450cam and many other bacterial P450s. It thus might be expected that the redox partner docking site of cytochrome P450cin would resemble that of cytochrome P450BM3, which also uses a flavodoxin-like redox partner. Nevertheless, the putative docking site topography more closely resembles cytochrome P450cam than cytochrome P450BM3.

Background and Aims Pollen limitation is most prevalent among bee-pollinated plants, self-incompatible plants, and tropical plants. However, we have very little understanding of the extent to which pollen limitation affects fruit set in mass-flowering trees despite tree crops accounting for at least 600 million tons of the 9,200 million tons of annual global food production. Methods We determined the extent of pollen limitation in a bee-pollinated, partially self-incompatible, subtropical tree by hand cross-pollinating the majority of flowers on mass-flowering macadamia (Macadamia integrifolia) trees that produce about 200,000–400,000 flowers. We measured tree yield and kernel quality and estimated final fruit set. We genotyped individual kernels by MassARRAY to determine levels of outcrossing in orchards and assess paternity effects on nut quality. Key Results Macadamia trees were pollen limited. Supplementary cross-pollination increased nut-in-shell yield, kernel yield and fruit set by as much as 97%, 109% and 92%, respectively. The extent of pollen limitation depended upon the proximity of experimental trees to trees of another cultivar because macadamia trees were highly outcrossing. Between 84% and 100% of fruit arose from cross-pollination, even at 200 m (25 rows) from orchard blocks of another cultivar. Large variations in nut-in-shell mass, kernel mass, kernel recovery and kernel oil concentration were related to differences in fruit paternity, including between self-pollinated and cross-pollinated fruit, thus demonstrating pollen-parent effects on fruit quality, i.e. xenia. Conclusions This study is the first to demonstrate pollen limitation in a mass-flowering tree. Improved pollination led to increased kernel yield of 0.31–0.59 tons per hectare, which equates currently to higher farm-gate income of approximately US3,720–US7,080 per hectare. The heavy reliance of macadamia flowers on cross-pollination and the strong xenia effects on kernel mass demonstrate the high value that pollination services can provide to food production.