Algal carotenoids with novel end groups

The structures of three previously unidentified carotenoids from Eutreptiella gymnastica are reported. These include siphonein with defined n-2-trans-2-dodecenoic esterifying acid and assigned 3R(?), 3′R,6′R chirality, (3R)-3′,4′-anhydrodiatoxanthin and eutreptiellanone (3,6-epoxy-3′,4′,7′,8′-tetradehydro-5,6-dihydro-β,β-caroten-4-one) with probable 3S,5R,6S chirality.     

A carotenogenic gene cluster from Brevibacterium linens with novel lycopene cyclase genes involved in the synthesis of aromatic carotenoids

The carotenogenic (crt) gene cluster from Brevibacterium linens, a member of the commercially important group of coryneform bacteria, was cloned and identified. An expression library of B. linens genes was constructed and a fragment of the crt cluster was obtained by functional complementation of a colourless B. flavum mutant, screening transformed cells for production of a yellow pigment. Subsequent screening of a cosmid library resulted in the cloning of the wholecrt cluster from B. linens. All genes necessary for the synthesis of the aromatic carotenoid isorenieratene were identified on the basis of sequence homologies. In addition a novel type of lycopene cyclase was identified by complementation of a lycopene-accumulating B. flavum mutant. Two genes, named crtYc and crtYd, which code for polypeptides of 125 and 107 amino acids, respectively, are necessary to convert lycopene to β-carotene. The amino acid sequences of these polypeptides show no similarity to any of the known lycopene cyclases. This is the first example of a carotenoid biosynthetic conversion in which two different gene products are involved, probably forming a heterodimer. Received: 17 July 1999 / Accepted: 7 December 1999     

Molecular evolution of proteins involved in vertebrate phototransduction

Vision is one of the most important senses for vertebrates. As a result, vertebrates have evolved a highly organized system of retinal photoreceptors. Light triggers an enzymatic cascade, called the phototransduction cascade, that leads to the hyperpolarization of photoreceptors. It is expected that a systematic comparison of phototransduction cascades of various vertebrates can provide insights into the diversity of vertebrate photoreceptors and into the evolution of vertebrate vision. However, only a few attempts have been made to compare each phototransduction protein participating in this cascade. Here, we determine phylogenetic trees of the vertebrate phototransduction proteins and compare them. It is demonstrated that vertebrate opsin sequences fall into five fundamental subfamilies. It is speculated that this is crucial for the diversity of the spectral sensitivity observed in vertebrate photoreceptors and provides the vertebrates with the molecular tools to discriminate the color of incident light. Other phototransduction proteins can be classified into only a few subfamilies. Cones generally share isoforms of phototransduction proteins that are different from those found in rods. The difference in sensitivity to light between rods and cones is likely due to the difference in the molecular properties of these isoforms. The phototransduction proteins seem to have co-evolved as a system. Switching the expression of these isoforms may characterize individual vertebrate photoreceptors.     

Molecular evolution of enzymes involved in the arachidonic acid cascade

The metabolic pathway called the arachidonic acid cascade produces a wide range of eicosanoids, such as prostaglandins, thromboxanes and leukotrienes with potent biological activities. Recombinant DNA techniques have made it possible to determine the nucleotide sequences of cDNAs and/or genomic structures for the enzymes involved in the pathway. Sequence comparison analyses of the accumulated sequence data have brought great insights into the structure, function and molecular evolution of the enzymes. This paper reviews the sequence comparison analyses of the enzymes involved in the arachidonic acid cascade.     

Molecular modeling of the non-covalent binding of the dietary tomato carotenoids lycopene and lycophyll, and selected oxidative metabolites with 5-lipoxygenase

Numerous studies on human prostate cancer cell lines indicate a role for arachidonic acid (AA) and its oxidative metabolites in prostate cancer proliferation. The metabolism of AA by either the cyclooxygenase (COX) or the lipoxygenase (LOX) pathways generates eicosanoids involved in tumor promotion, progression, and metastasis. In particular, products of the 5-LOX pathway (including 5-HETE and 5-oxo-EET) have been implicated as potential 'survival factors' that may confer escape after androgen withdrawal therapy through fatty-acid (i.e., AA) drive. Potent natural dietary antioxidant compounds such as lycopene and lycophyll, with tissue tropism for human prostate, have been shown to be effective in ameliorating generalized oxidative stress at the DNA level. Suppressing the 5-LOX axis pharmacologically is also a promising avenue for intervention in human patients. The recently recognized direct interaction of the astaxanthin-based soft-drug Cardax to human 5-LOX with molecular modeling, and the downregulation of both 5-HETE and 5-oxo-EET in vivo in a murine peritonitis model, suggest that other important dietary carotenoids may share this enzyme regulatory feature. In the current study, the acyclic tomato carotene lycopene (in all-trans and 5-cis isomeric configurations) and its natural dihydroxy analog lycophyll (also present in tomato fruit) were subjected to molecular modeling calculations in order to investigate their predicted binding interaction(s) with human 5-LOX. Two bioactive oxidative metabolites of lycopene (4-methyl-8-oxo-2,4,6-nonatrienal and 2,7,11-trimethyl-tetradecahexaene-1,14-dial) were also investigated. A homology model of 5-LOX was constructed using 8-LOX and 15-LOX structures as templates. The model was validated by calculating the binding energy of Cardax to 5-LOX, which was demonstrated to be in good agreement with the published experimental data. Blind docking calculations were carried out in order to explore the possible binding sites of the carotenoids on 5-LOX, followed by focused docking to more accurately calculate the predicted energy of binding. Lycopene and lycophyll were predicted to bind with high affinity in the superficial cleft at the interface of the beta-barrel and the catalytic domain of 5-LOX (the 'cleavage site'). Carotenoid binding at this cleavage site provides the structural rationale by which polyenic compounds could modify the 5-LOX enzymatic function via an allosteric mechanism, or by radical scavenging in proximity to the active center. In addition, the two bioactive metabolites of lycopene were predicted to bind to the catalytic site with high affinity--therefore suggesting potential direct competitive inhibition of 5-LOX activity that should be shared by both lycopene and lycophyll after in vivo supplementation, particularly in the case of the dial metabolite.     

Divergent evolution of oxidosqualene cyclases in plants

Triterpenes are one of the largest classes of plant metabolites and have important functions. A diverse array of triterpenoid skeletons are synthesized via the isoprenoid pathway by enzymatic cyclization of 2,3-oxidosqualene. The genomes of the lower plants Chlamydomonas reinhardtii and moss (Physcomitrella patens) contain just one oxidosqualene cyclase (OSC) gene (for sterol biosynthesis), whereas the genomes of higher plants contain nine to 16 OSC genes. Here we carry out functional analysis of rice OSCs and rigorous phylogenetic analysis of 96 OSCs from higher plants, including Arabidopsis thaliana, Oryza sativa, Sorghum bicolor and Brachypodium distachyon. The functional analysis identified an amino acid sequence for isoarborinol synthase (OsIAS) (encoded by Os11g35710/OsOSC11) in rice. Our phylogenetic analysis suggests that expansion of OSC members in higher plants has occurred mainly through tandem duplication followed by positive selection and diversifying evolution, and consolidated the previous suggestion that dicot triterpene synthases have been derived from an ancestral lanosterol synthase instead of directly from their cycloartenol synthases. The phylogenetic trees are consistent with the reaction mechanisms of the protosteryl and dammarenyl cations which parent a wide variety of triterpene skeletal types, allowing us to predict the functions of the uncharacterized OSCs.     

The formation of end groups in cellulose during alkali cooking

Cotton that had been subjected to alkali cooking at 170° was hydrolysed to determine the car?ylic acid end-groups. Large proportions of 3-deoxy-ribo-hexonic, 3-deoxy-arabino-hexonic, and 2-C-methylglyceric acids, together with a minor proportion of 2-C-methylribonic acid, were isolated and identified. Reduction of the cellulose end-groups and subsequent analysis of the hydrolysate revealed 3-deoxy-ribo-hexitol, 3-deoxy-arabino-hexitol, 2-C-methylglycerol, and a small proportion of 2-C-methylribitol. It is concluded from these results that, in addition to 3-deoxyhexonic acid end-groups, significant quantities of terminal 2-C-methylglyceric and minor amounts of 2-C-methylribonic acid groups are formed during the alkali cooking. No alditol end-groups were detected in the unreduced cellulose.     

Molecular mechanisms involved in Bacillus subtilis biofilm formation

Biofilms are the predominant lifestyle of bacteria in natural environments, and they severely impact our societies in many different fashions. Therefore, biofilm formation is a topic of growing interest in microbiology, and different bacterial models are currently studied to better understand the molecular strategies that bacteria undergo to build biofilms. Among those, biofilms of the soil‐dwelling bacterium Bacillus subtilis are commonly used for this purpose. Bacillus subtilis biofilms show remarkable architectural features that are a consequence of sophisticated programmes of cellular specialization and cell–cell communication within the community. Many laboratories are trying to unravel the biological role of the morphological features of biofilms, as well as exploring the molecular basis underlying cellular differentiation. In this review, we present a general perspective of the current state of knowledge of biofilm formation in B. subtilis and thereby placing a special emphasis on summarizing the most recent discoveries in the field.     

Hypotheses for the origin and early evolution of triterpenoid cyclases

Hopanes and steranes are found almost universally in the sedimentary rock record where they often are used as proxies for aerobic organisms, metabolisms, and environments. In order to interpret ancient lipid signatures confidently we require a complementary understanding of how these modern biochemical pathways evolved since their conception. For example, generally it has been assumed that hopanoid biosynthesis was an evolutionary predecessor to steroid biosynthesis. Here we re‐evaluate this assumption. Using a combined phylogenetic and biochemical perspective, we address the evolution of polycyclic triterpenoid biosynthesis and suggest several constraints on using these molecules as aerobic biomarkers. Amino acid sequence data show that the enzymes responsible for polycyclic triterpenoid biosynthesis (i.e. squalene and 2,3‐oxidosqualene cyclases) are homologous. Numerous conserved domains correspond to active sites in the enzymes that are required to complete the complex cyclization reaction. From these sites we develop an evolutionary analysis of three independent characters to explain the evolution of the major classes of polycyclic triterpenoids. These characters are: (i) the number of unfavourable anti‐Markovnikov ring closures, (ii) all‐chair (CCC) or chair‐boat‐chair (CBC) substrate conformation, and (iii) the choice between squalene and 2,3‐oxidosqualene as the substrate. We use these characters to construct four competing phylogenies to describe the evolution of polycyclic triterpenoid biosynthesis. The analysis suggests that malabaricanoids would be the most ancient polycyclic triterpenoids. The two most parsimonious evolutionary trees are the ones in which hopanoid and steroid cyclases diverged from a common ancestor. The transition from a CCC‐ to CBC‐fold marks the major divergence in the evolution of these pathways, and it is diagnosable in the geological record. However, this transition does not require the simultaneous adoption of the aerobic substrate, 2,3‐oxidosqualene, because these characters are controlled by independent parts of the enzyme.     

Significant correlations of dermal total carotenoids and dermal lycopene with their respective plasma levels in healthy adults

Carotenoids in skin have been known to play a role in photoprotection against UV radiation. We performed dermal biopsies of healthy humans (N = 27) and collected blood samples for pair-wise correlation analyses of total and individual carotenoid content by high performance liquid chromatography (HPLC). The hydrocarbon carotenoids (lycopene and beta-carotene) made up the majority of carotenoids in both skin and plasma, and skin was somewhat enriched in these carotenoids relative to plasma. Beta-cryptoxanthin, a monohydroxycarotenoid, was found in similar proportions in skin as in plasma. In contrast, the dihydroxycarotenoids, lutein and zeaxanthin, were relatively lacking in human skin in absolute and relative levels as compared to plasma. Total carotenoids were significantly correlated in skin and plasma (r = 0.53, p < 0.01). Our findings suggest that human skin is relatively enriched in lycopene and beta-carotene, compared to lutein and zeaxanthin, possibly reflecting a specific function of hydrocarbon carotenoids in human skin photoprotection.     

Molecular evolution of plant P5CS gene involved in proline biosynthesis

The P5CS ({Delta} 1-Pyrroline–5-Carboxylate Synthetase) gene encodes for a bifunctional enzyme that catalyzes the rate limiting reaction in proline biosynthesis in living organisms. A wide range of multifunctional roles of proline have now been shown in stress defense. The proline biosynthetic genes, especially, P5CS is commonly used in metabolic engineering for proline overproduction conferring stress tolerance in plants. The gene is functionally well characterized at the molecular level, but there is more to learn about its evolutionary path in the plant kingdom, particularly the drive behind functional (osmoprotective and developmental) divergence of duplication of P5CS genes. In this review, we present the current understanding of the evolutionary trail of plant P5CS gene which plays a key role in stress tolerance.     

Origins and evolution of the formin multigene family that is involved in the formation of actin filaments

In eukaryotes, the assembly and elongation of unbranched actin filaments is controlled by formins, which are long, multidomain proteins. These proteins are important for dynamic cellular processes such as determination of cell shape, cell division, and cellular interaction. Yet, no comprehensive study has been done about the origins and evolution of this gene family. We therefore performed extensive phylogenetic and motif analyses of the formin genes by examining 597 prokaryotic and 53 eukaryotic genomes. Additionally, we used three-dimensional protein structure data in an effort to uncover distantly related sequences. Our results suggest that the formin homology 2 (FH2) domain, which promotes the formation of actin filaments, is a eukaryotic innovation and apparently originated only once in eukaryotic evolution. Despite the high degree of FH2 domain sequence divergence, the FH2 domains of most eukaryotic formins are predicted to assume the same fold and thus have similar functions. The formin genes have experienced multiple taxon-specific duplications and followed the birth-and-death model of evolution. Additionally, the formin genes experienced taxon-specific genomic rearrangements that led to the acquisition of unrelated protein domains. The evolutionary diversification of formin genes apparently increased the number of formin's interacting molecules and consequently contributed to the development of a complex and precise actin assembly mechanism. The diversity of formin types is probably related to the range of actin-based cellular processes that different cells or organisms require. Our results indicate the importance of gene duplication and domain acquisition in the evolution of the eukaryotic cell and offer insights into how a complex system, such as the cytoskeleton, evolved.     

Molecular basis of maillard amide-advanced glycation end product (AGE) formation in vivo

The Maillard reaction in vivo entails alteration of proteins or free amino acids by non-enzymatic glycation or glycoxidation. The resulting modifications are called advanced glycation end products (AGEs) and play a prominent role in various pathologies, including normoglycemic uremia. Recently, we established a new class of lysine amide modifications in vitro. Now, human plasma levels of the novel amide-AGEs N(6)-acetyl lysine, N(6)-formyl lysine, N(6)-lactoyl lysine, and N(6)-glycerinyl lysine were determined by means of LC-MS/MS. They were significantly higher in uremic patients undergoing hemodialysis than in healthy subjects. Model reactions with N(1)-t-butoxycarbonyl-lysine under physiological conditions confirmed 1-deoxy-d-erythro-hexo-2,3-diulose as an immediate precursor. Because formation of N(6)-formyl lysine from glucose responded considerably to the presence of oxygen, glucosone was identified as another precursor. Comparison of the in vivo results with the model experiments enabled us to elucidate possible formation pathways linked to Maillard chemistry. The results strongly suggest a major participation of non-enzymatic Maillard mechanisms on amide-AGE formation pathways in vivo, which, in the case of N(6)-acetyl lysine, parallels enzymatic processes.     

Structure and function of the genes involved in the biosynthesis of carotenoids in the mucorales

Carotenoids are widely distributed natural pigments which are in an increasing demand by the market, due to their applications in the human food, animal feed, cosmetics, and pharmaceutical industries. Although more than 600 carotenoids have been identified in nature, only a few are industrially important (β-carotene, astaxanthin, lutein or lycopene). To date chemical processes manufacture most of the carotenoid production, but the interest for carotenoids of biological origin is growing since there is an increased public concern over the safety of artificial food colorants. Although much interest and effort has been devoted to the use of biological sources for industrially important carotenoids, only the production of biological β-carotene and astaxanthin has been reported. Among fungi, several Mucorales strains, particularlyBlakeslea trispora, have been used to develop fermentation process for the production of β-carotene on almost competitive cost-price levels. Similarly, the basidiomycetous yeastXanthophyllomyces dendrorhous (the perfect state ofPhaffia rhodozyma), has been proposed as a promising source of astaxanthin. This paper focuses on recent findings on the fungal pathways for carotenoid production, especially the structure and function of the genes involved in the biosynthesis of carotenoids in the Mucorales. An outlook of the possibilities of an increased industrial production of carotenoids, based on metabolic engineering of fungi for carotenoid content and composition, is also discussed.