Preparation, characterization, and optimization of an in vitro C30 carotenoid pathway

The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4'-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoid-based pathways, could lead to the synthesis of novel carotenoids and their derivatives.     

Functional properties of diapophytoene and related desaturases of C(30) and C(40) carotenoid biosynthetic pathways

The desaturation reactions of C(30) carotenoids from diapophytoene to diaponeurosporene was investigated in vitro and by complementation in Escherichia coli. The expressed diapophytoene desaturase from Staphylococcus aureus inserts three double bonds in an FAD-dependent reaction. The enzyme is inhibited by diphenylamine. In the complementation experiment diapophytoene desaturase was able to convert C(40) phytoene to some extend but exhibited a high affinity to zeta-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C(40) carotenes revealed that this desaturase can also convert C(30) diapophytoene. Other homologous bacterial C(40) carotene desaturases could also utilize C(30) substrates, including one type of zeta-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C(30) carotenogenic pathway is determined by this initial enzyme which is highly homologous to C(40) phytoene synthases.     

The major carotenoid in all known species of heliobacteria is the C30 carotenoid 4,4′-diaponeurosporene, not neurosporene

The carotenoids of five species of heliobacteria (Heliobacillus mobilis, Heliophilum fasciatum, Heliobacterium chlorum, Heliobacterium modesticaldum, and Heliobacterium gestii) were examined by spectroscopic methods, and the C₃₀ carotene 4,4′-diaponeurosporene was found to be the dominant pigment; heliobacteria were previously thought to contain the C₄₀ carotenoid neurosporene. In addition, trace amounts of the C₃₀ diapocarotenes diapolycopene, diapo-ζ-carotene, diapophytofluene, and diapophytoene were also found. Up to now, diapocarotenes have been found in only three species of chemoorganotrophic bacteria, but not in phototropic organisms. Furthermore, the esterifying alcohol of bacteriochlorophyll g from all known species of heliobacteria was determined to be farnesol (C₁₅) instead of the usual phytol (C₂₀). Heliobacteria may be unable to produce geranylgeranyol (C₂₀). Received: 10 March 1997 / Accepted: 3 June 1997     

A unique reaction in a common pathway: mechanism and function of chorismate synthase in the shikimate pathway

Chorismate synthase, the seventh enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate which is the last common precursor in the biosynthesis of numerous aromatic compounds in bacteria, fungi and plants. The enzyme has an absolute requirement for reduced FMN as a cofactor, although the 1,4-anti elimination of phosphate and the C(6proR)-hydrogen does not involve a net redox change. The role of the reduced FMN in catalysis has long been elusive. However, recent detailed kinetic and bioorganic approaches have fundamentally advanced our understanding of the mechanism of action, suggesting an initial electron transfer from tightly bound reduced flavin to the substrate, a process which results in C—O bond cleavage. Studies on chorismate synthases from bacteria, fungi and plants revealed that in these organisms the reduced FMN cofactor is made available in different ways to chorismate synthase: chorismate synthases in fungi – in contrast to those in bacteria and plants – carry a second enzymatic activity which enables them to reduce FMN at the expense of NADPH. Yet, as shown by the analysis of the corresponding genes, all chorismate synthases are derived from a common ancestor. However, several issues revolving around the origin of reduced FMN, as well as the possible regulation of the enzyme activity by means of the availability of reduced FMN, remain poorly understood. This review summarizes recent developments in the biochemical and genetic arena and identifies future aims in this field. Received: 22 June 1998 / Accepted: 7 August 1998     

Identification of neoxanthin synthase as a carotenoid cyclase paralog.

Neoxanthin, a precursor of the plant hormone abscisic acid, is an allenic xanthophyll recognized as the last product of carotenoid synthesis in green plants. A cDNA for neoxanthin synthase (NSY) was isolated from tomato using a molecular approach based on the mechanistic and structural similarities of NSY to two other closely related carotenogenic enzymes, lycopene cyclase (LCY) and capsanthin-capsorubin synthase (CCS). The identified tomato NSY cDNA (T.NSY) encodes a 56-kDa plastid-targeted protein that when expressed in Escherichia coli, catalyzes the conversion of violaxanthin to neoxanthin. In tobacco leaves that transiently express T.NSY, an increase in neoxanthin content with a concomitant decrease in violaxanthin is observed. NSY is structurally similar to LCY and CCS. However, in Cyanobacteria, the generally accepted progenitor of plastids, both CCS and NSY are absent while LCY is present. LCY catalyzes a simplified version of the reaction catalyzed by NSY and CCS suggesting that these two enzymes were remodeled from LCY during higher plant evolution to create new forms of oxidized carotenoids.     

Identification of the gene responsible for torulene cleavage in the Neurospora carotenoid pathway

Torulene, a C₄₀ carotene, is the precursor of the end product of the Neurospora carotenoid pathway, the C₃₅ xanthophyll neurosporaxanthin. Torulene is synthesized by the enzymes AL-2 and AL-1 from the precursor geranylgeranyl diphosphate and then cleaved by an unknown enzyme into the C₃₅ apocarotenoid. In general, carotenoid cleavage reactions are catalyzed by carotenoid oxygenases. Using protein data bases, we identified two putative carotenoid oxygenases in Neurospora, named here CAO-1 and CAO-2. A search for novel mutants of the carotenoid pathway in this fungus allowed the identification of two torulene-accumulating strains, lacking neurosporaxanthin. Sequencing of the cao-2 gene in these strains revealed severe mutations, pointing to a role of CAO-2 in torulene cleavage. This was further supported by the identical phenotype found upon targeted disruption of cao-2. The biological function was confirmed by in vitro assays using the purified enzyme, which cleaved torulene to produce β-apo-4′-carotenal, the corresponding aldehyde of neurosporaxanthin. The specificity of CAO-2 was shown by the lack of γ-carotene-cleaving activity in vitro. As predicted for a structural gene of the carotenoid pathway, cao-2 mRNA was induced by light in a WC-1 and WC-2 dependent manner. Our data demonstrate that CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the neurosporaxanthin biosynthetic pathway.     

Use of transposon promoter-probe vectors in the metabolic engineering of the obligate methanotroph Methylomonas sp. strain 16a for enhanced C40 carotenoid synthesis

The recent expansion of genetic and genomic tools for metabolic engineering has accelerated the development of microorganisms for the industrial production of desired compounds. We have used transposable elements to identify chromosomal locations in the obligate methanotroph Methylomonas sp. strain 16a that support high-level expression of genes involved in the synthesis of the C(40) carotenoids canthaxanthin and astaxanthin. with three promoterless carotenoid transposons, five chromosomal locations-the fliCS, hsdM, ccp-3, cysH, and nirS regions-were identified. Total carotenoid synthesis increased 10- to 20-fold when the carotenoid gene clusters were inserted at these chromosomal locations compared to when the same carotenoid gene clusters were integrated at neutral locations under the control of the promoter for the gene conferring resistance to chloramphenicol. A chromosomal integration system based on sucrose lethality was used to make targeted gene deletions or site-specific integration of the carotenoid gene cluster into the Methylomonas genome without leaving genetic scars in the chromosome from the antibiotic resistance genes that are present on the integration vector. The genetic approaches described in this work demonstrate how metabolic engineering of microorganisms, including the less-studied environmental isolates, can be greatly enhanced by identifying integration sites within the chromosome of the host that permit optimal expression of the target genes.     

BCDO2 acts as a carotenoid scavenger and gatekeeper for the mitochondrial apoptotic pathway

Carotenoids and their metabolites are widespread and exert key biological functions in living organisms. In vertebrates, the carotenoid oxygenase BCMO1 converts carotenoids such as β,β-carotene to retinoids, which are required for embryonic pattern formation and cell differentiation. Vertebrate genomes encode a structurally related protein named BCDO2 but its physiological function remains undefined. Here, we show that BCDO2 is expressed as an oxidative stress-regulated protein during zebrafish development. Targeted knockdown of this mitochondrial enzyme resulted in anemia at larval stages. Marker gene analysis and staining for hemoglobin revealed that erythropoiesis was not impaired but that erythrocytes underwent apoptosis in BCDO2-deficient larvae. To define the mechanism of this defect, we have analyzed the role of BCDO2 in human cell lines. We found that carotenoids caused oxidative stress in mitochondria that eventually led to cytochrome c release, proteolytic activation of caspase 3 and PARP1, and execution of the apoptotic pathway. Moreover, BCDO2 prevented this induction of the apoptotic pathway by carotenoids. Thus, our study identifying BCDO2 as a crucial protective component against oxidative stress establishes this enzyme as mitochondrial carotenoid scavenger and a gatekeeper of the intrinsic apoptotic pathway.     

Structural basis for the carotenoid binding and transport function of a START domain

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Construction of carotenoid biosynthetic pathways using squalene synthase

The first committed steps of steroid/hopanoid pathways involve squalene synthase (SQS). Here, we report the Escherichia coli production of diaponeurosporene and diapolycopene, yellow C₃₀ carotenoid pigments, by expressing human SQS and Staphylococcus aureus dehydrosqualene (C₃₀ carotenoid) desaturase (CrtN). We suggest that the carotenoid pigments are synthesized mainly via the desaturation of squalene rather than the direct synthesis of dehydrosqualene through the non-reductive condensation of prenyl diphosphate precursors, indicating the possible existence of a “squalene route” and a “lycopersene route” for C₃₀ and C₄₀ carotenoids, respectively. Additionally, this finding yields a new method of colorimetric screening for the cellular activity of squalene synthases, which are major targets for cholesterol-lowering drugs.     

Light induction of the carotenoid biosynthesis pathway in Blakeslea trispora

A gene of Blakeslea trispora has been cloned by heterologous hybridization with the Mucor circinelloides crgA gene, a repressor of light-inducible carotenogenesis. This gene is the ortholog of the M. circinelloides crgA, since it was able to restore the wild-type phenotype of a null crgA mutant of M. circinelloides. The expression of B. trispora crgA gene is light-induced and photoadapted, as occurs for M. circinelloides crgA. Light induction and photoadaptation of B. trispora crgA was also observed in M. circinelloides, which suggests that the mechanisms involved in light regulation are basically conserved between these filamentous fungi. Conservation of the regulatory pathway that controls carotene biosynthesis was supported by the light-induced and photoadapted expression of all structural carotenogenic genes of B. trispora. Consequently, the beta-carotene content of dark grown mycelia of B. trispora increased upon illumination with white light.     

Evolution and function of the sucrose-phosphate synthase gene families in wheat and other grasses

Suc-phosphate synthase (SPS) is a key regulatory enzyme in the pathway of Suc biosynthesis and has been linked to quantitative trait loci controlling plant growth and yield. In dicotyledonous plants there are three SPS gene families: A, B, and C. Here we report the finding of five families of SPS genes in wheat (Triticum aestivum) and other monocotyledonous plants from the family Poaceae (grasses). Three of these form separate subfamilies within the previously described A, B, and C gene families, but the other two form a novel and distinctive D family, which on present evidence is only found in the Poaceae. The D-type SPS proteins lack the phosphorylation sites associated with 14-3-3 protein binding and osmotic stress activation, and the linker region between the N-terminal catalytic glucosyltransferase domain and the C-terminal Suc-phosphatase-like domain is 80 to 90 amino acid residues shorter than in the A, B, or C types. The D family appears to have arisen after the divergence of mono- and dicotyledonous plants, with a later duplication event resulting in the two D-type subfamilies. Each of the SPS gene families in wheat showed different, but overlapping, spatial and temporal expression patterns, and in most organs at least two different SPS genes are expressed. Analysis of expressed sequence tags indicated similar expression patterns to wheat for each SPS gene family in barley (Hordeum vulgare) but not in more distantly related grasses. We identified an expressed sequence tag from rice (Oryza sativa) that appears to be derived from an endogenous antisense SPS gene, and this might account for the apparently low level of expression of the related OsSPS11 sense gene, adding to the already extensive list of mechanisms for regulating the activity of SPS in plants.     

Structural dynamics in the C terminal domain homolog of orange carotenoid Protein reveals residues critical for carotenoid uptake

The structural features enabling carotenoid translocation between molecular entities in nature is poorly understood. Here, we present the three-dimensional X-ray structure of an expanded oligomeric state of the C-terminal domain homolog (CTDH) of the orange carotenoid protein, a key water-soluble protein in cyanobacterial photosynthetic photo-protection, at 2.9 Å resolution. This protein binds a canthaxanthin carotenoid ligand and undergoes structural reorganization at the dimeric level, which facilitates cargo uptake and delivery. The structure displays heterogeneity revealing the dynamic nature of its C-terminal tail (CTT). Molecular dynamics (MD) simulations based on the CTDH structures identified specific residues that govern the dimeric transition mechanism. Mutagenesis based on the crystal structure and these MD simulations then confirmed that these specific residues within the CTT are critical for carotenoid uptake, encapsulation and delivery processes. We present a mechanism that can be applied to other systems that require cargo uptake.