Biochemical characterization of the carotenoid 1,2-hydratases (CrtC) from Rubrivivax gelatinosus and Thiocapsa roseopersicina

Two carotenoid 1,2-hydratase (CrtC) genes from the photosynthetic bacteria Rubrivivax gelatinosus and Thiocapsa roseopersicina were cloned and expressed in Escherichia coli in an active form and purified by affinity chromatography. The biochemical properties of the recombinant enzymes and their substrate specificities were studied. The purified CrtCs catalyze cofactor independently the conversion of lycopene to 1-HO- and 1,1′-(HO)2-lycopene. The optimal pH and temperature for hydratase activity was 8.0 and 30°C, respectively. The apparent Km and Vmax values obtained for the hydration of lycopene were 24 μM and 0.31 nmol h−1 mg−1 for RgCrtC and 9.5 μM and 0.15 nmol h−1 mg−1 for TrCrtC, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed two protein bands of 44 and 38 kDa for TrCrtC, which indicate protein processing. Both hydratases are also able to convert the unnatural substrate geranylgeraniol (C20 substrate), which functionally resembles the natural substrate lycopene

Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX

Two Chlamydomonas reinhardtii mutants defective in CHLM encoding Mg-protoporphyrin IX methyltransferase (MgPMT) were identified. The mutants, one with a missense mutation (chlM-1) and a second mutant with a splicing defect (chlM-2), do not accumulate chlorophyll, are yellow in the dark and dim light, and their growth is inhibited at higher light intensities. They accumulate Mg-protoporphyrin IX (MgProto), the substrate of MgPMT and this may be the cause for their light sensitivity. In the dark, both mutants showed a drastic reduction in the amounts of core proteins of photosystems I and II and light-harvesting chlorophyll a/b-binding proteins. However, LHC mRNAs accumulated above wild-type levels. The accumulation of the transcripts of the LHC and other genes that were expressed at higher levels in the mutants during dark incubation was attenuated in the initial phase of light exposure. No regulatory effects of the constitutively 7- to 18-fold increased MgProto levels on gene expression were detected, supporting previous results in which MgProto and heme in Chlamydomonas were assigned roles as second messengers only in the transient activation of genes by light

Orientation of nitrous oxide on palladium(110) by STM

The adsorption structure of N2O on Pd(1 1 0) was analyzed below 14 K by scanning-tunneling microscopy. The N2O monomer was oriented along the [0 0 1] direction in the on-top form. Furthermore, the formation of small aggregates extending along the [1-1 0] direction was observed. The observed images were well-simulated for two types of cluster structures optimized by density-functional theory calculations. The components in the aggregates are proposed to be in a tilted form either on bridge sites or on-top sites

Xantha-l encodes a membrane subunit of the aerobic Mg-protoporphyrin IX monomethyl ester cyclase involved in chlorophyll biosynthesis

Chlorophyll biosynthesis is a process involving ≈20 different enzymatic steps. Half of these steps are common to the biosynthesis of other tetrapyrroles, such as heme. One of the least understood enzymatic steps is formation of the isocyclic ring, which is a characteristic feature of all (bacterio)chlorophyll molecules. In chloroplasts, formation of the isocyclic ring is an aerobic reaction catalyzed by Mg-protoporphyrin IX monomethyl ester cyclase. An in vitro assay for the aerobic cyclase reaction required membrane-bound and soluble components from the chloroplasts. Extracts from barley (Hordeum vulgare L.) mutants at the Xantha-l and Viridis-k loci showed no cyclase activity. Fractionation of isolated plastids by Percoll gradient centrifugation showed that xantha-l and viridis-k mutants are defective in components associated with chloroplast membranes. The Xantha-l gene, corresponding to Arabidopsis thaliana CHL27, Rubrivivax gelatinosus acsF, Chlamydomonas reinhardtii CRD1, and CTH1 and situated at the short arm of barley chromosome 3 (3H), was cloned, and the mutations in xantha-l(35), xantha-l(81), and xantha-l(82) were characterized. This finding connected biochemical and genetic data because it demonstrated that Xantha-l encodes a membrane-bound cyclase subunit. The evidence suggests that the aerobic cyclase requires at least one soluble and two membrane-bound components

Chloroplast envelope membranes: a dynamic interface between plastids and the cytosol.

Chloroplasts are bounded by a pair of outer membranes, the envelope, that is the only permanent membrane structure of the different types of plastids. Chloroplasts have had a long and complex evolutionary past and integration of the envelope membranes in cellular functions is the result of this evolution. Plastid envelope membranes contain a wide diversity of lipids and terpenoid compounds serving numerous biochemical functions and the flexibility of their biosynthetic pathways allow plants to adapt to fluctuating environmental conditions (for instance phosphate deprivation). A large body of knowledge has been generated by proteomic studies targeted to envelope membranes, thus revealing an unexpected complexity of this membrane system. For instance, new transport systems for metabolites and ions have been identified in envelope membranes and new routes for the import of chloroplast-specific proteins have been identified. The picture emerging from our present understanding of plastid envelope membranes is that of a key player in plastid biogenesis and the co-ordinated gene expression of plastid-specific protein (owing to chlorophyll precursors), of a major hub for integration of metabolic and ionic networks in cell metabolism, of a flexible system that can divide, produce dynamic extensions and interact with other cell constituents. Envelope membranes are indeed one of the most complex and dynamic system within a plant cell. In this review, we present an overview of envelope constituents together with recent insights into the major functions fulfilled by envelope membranes and their dynamics within plant cells