Directed evolution of squalene synthase for dehydrosqualene biosynthesis

Squalene synthase (SQS) catalyzes the first step of sterol/hopanoid biosynthesis in various organisms. It has been long recognized that SQSs share a common ancestor with carotenoid synthases, but it is not known how these enzymes selectively produce their own product. In this study, SQSs from yeast, human, and bacteria were independently subjected to directed evolution for the production of the C₃₀ carotenoid backbone, dehydrosqualene. This was accomplished via high-throughput screening with Pantoea ananatis phytoene desaturase, which can selectively convert dehydrosqualene into yellow carotenoid pigments. Genetic analysis of the resultant mutants revealed various mutations that could effectively convert SQS into a “dehydrosqualene synthase.” All of these mutations are clustered around the residues that have been proposed to be important for NADPH binding.     

Carotenoid biosynthesis and the evolution of carotenogenesis genes in rust fungi

Diseases caused by rust fungi pose a significant threat to global plant production. Although carotenoid pigments are produced in spores of nearly all rust species, the corresponding biosynthesis pathway(s) have not been investigated. Here, candidate genes for carotenoid biosynthesis in Puccinia graminis f. sp. tritici (Pgt) were identified, cloned and functionally complemented using specifically engineered strains of Escherichia coli. A part of the carotenoid biosynthesis pathway in rust fungi was elucidated, with only two genes, CrtYB and CrtI, catalysing the reactions from geranyl–geranyl diphosphate (GGPP) to γ-carotene. The CrtYB gene encodes a bi-functional lycopene cyclase/phytoene synthase, which catalyses the condensation of two GGPP into phytoene, as well as the cyclisation of the ψ-end of lycopene to form γ-carotene. The CrtI gene encodes a phytoene desaturase that carries out four successive desaturations of phytoene, through the intermediates phytofluene and neurosporene to lycopene. The evolution of carotenoid pigmentation in rust fungi, including Pgt, P. graminis avenae, P. graminis secalis (Pgs), P. graminis lolli, P. striiformis f. sp. tritici, P. striiformis f. sp. pseudohordei, P. striiformis f. sp. hordei, the “scabrum” rust (putative hybrids between Pgt and Pgs), P. triticina, and P. hordei, was investigated by phylogenetic analysis. Both CrtYB and CrtI were found to be closely related among rust fungi, other pathogenic fungi, and some aphids. Our results provide a springboard to increase the understanding of the physiological role(s) of carotenoid pigments in rust fungi, to better understand evolution within the Pucciniales, and to develop robust molecular diagnostics for rust fungi.     

Synthesis of dehydrosqualene in microsomal fraction of Saccharomyces cerevisiae.

When the microsomal fraction of Saccharomyces cerevisiae was incubated with farnesyl pyrophosphate or presqualene pyrophosphate in the presence of Mn²⁺, dehydrosqualene was formed. Incubation of the reaction mixture in the presence of NADPH gave squalene, not dehydrosqualene, as the product. Little dehydrosqualene was formed when Mn²⁺ was replaced with Mg²⁺. These observations suggest that dehydrosqualene formation is closely associated with squalene synthesis in yeast, which synthesizes neither carotenes nor related pigments.     

Near edge X-ray absorption fine structure spectroscopy (NEXAFS) of pigment-protein complexes: peridinin-chlorophyll a protein (PCP) of Amphidinium carterae

Peridinin-chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a “building block” approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving “optically dark” states of carotenoids in pigment-protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR).     

Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.)

A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar “White Winter Pearmain”. When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4 °C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.     

Functional properties of diapophytoene and related desaturases of C30 and C40 carotenoid biosynthetic pathways

The desaturation reactions of C₃₀ 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 C₄₀ phytoene to some extend but exhibited a high affinity to ζ-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C₄₀ carotenes revealed that this desaturase can also convert C₃₀ diapophytoene. Other homologous bacterial C₄₀ carotene desaturases could also utilize C₃₀ substrates, including one type of ζ-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C₃₀ carotenogenic pathway is determined by this initial enzyme which is highly homologous to C₄₀ phytoene synthases.     

Two reaction pathways for transformation of high potential cytochrome b559 of PS II into the intermediate potential form

This study describes an analysis of different treatments that influence the relative content and the midpoint potential of HP Cyt b559 in PS II membrane fragments from higher plants. Two basically different types of irreversible modification effects are distinguished: the HP form of Cyt b559 is either predominantly affected when the heme group is oxidized (“O-type” effects) or when it is reduced (“R-type” effects). Transformation of HP Cyt b559 to lower potential redox forms (IP and LP forms) by the “O-type” mechanism is induced by high pH and detergent treatments. In this case the effects consist of a gradual decrease in the relative content of HP Cyt b559 while its midpoint potential remains unaffected. Transformation of HP Cyt b559 via an “R-type” mechanism is caused by a number of exogenous compounds denoted L: herbicides, ADRY reagents and tetraphenylboron. These compounds are postulated to bind to the PS II complex at a quinone binding site designated as Q_C which interacts with Cyt b559 and is clearly not the Q_B site. Binding of compounds L to the Q_C site when HP Cyt b559 is oxidized gives rise to a gradual decrease in the E_m of HP Cyt b559 with increasing concentration of L (up to 10 K_ox(L) values) while the relative content of HP Cyt b559 is unaffected. Higher concentrations of compounds L required for their binding to Q_C site when HP Cyt b559 is reduced (described by K_red(L)) induce a conversion of HP Cyt b559 to lower potential redox forms (“R-type” transformation). Two reaction pathways for transitions of Cyt b559 between the different protein conformations that are responsible for the HP and IP/LP redox forms are proposed and new insights into the functional regulation of Cyt b559 via the Q_C site are discussed.     

Cloning and Functional Characterization of a Squalene Synthase from Paris polyphylla var. yunnanensis

Paris polyphylla Smith var. yunnanensis (Franch.) Hand. – Mazz. is a precious traditional Chinese medicine, and steroidal saponins are its major bioactive constituents possessing extensive biological activities. Squalene synthase (SQS) catalyzes the first dedicated step converting two molecular of farnesyl diphosphate (FDP) into squalene, a key intermediate in the biosynthetic pathway of steroidal saponins. In this study, a squalene synthase gene (PpSQS1) was cloned and functionally characterized from P. polyphylla var. yunnanensis, representing the first identified SQS from the genus Paris. The open reading frame of PpSQS1 is 1239 bp, which encodes a protein of 412 amino acids showing high similarity to those of other plant SQSs. Expression of PpSQS1 in Escherichia coli resulted in production of soluble recombinant proteins. Gas chromatography-mass spectrometry analysis showed that the purified recombinant PpSQS1 protein could produce squalene using FDP as a substrate in the in vitro enzymatic assay. qRT-PCR analysis indicated that PpSQS1 was highly expressed in rhizomes, consistent with the dominant accumulation of steroidal saponins there, suggesting that PpSQS1 is likely involved in the biosynthesis of steroidal saponins in the plant. The findings lay a foundation for further investigation on the biosynthesis and regulation of steroidal saponins, and also provide an alternative gene for manipulation of steroid production using synthetic biology.     

Binding modes of zaragozic acid A to human squalene synthase and staphylococcal dehydrosqualene synthase

Zaragozic acids (ZAs) belong to a family of fungal metabolites with nanomolar inhibitory activity toward squalene synthase (SQS). The enzyme catalyzes the committed step of sterol synthesis and has attracted attention as a potential target for antilipogenic and antiinfective therapies. Here, we have determined the structure of ZA-A complexed with human SQS. ZA-A binding induces a local conformational change in the substrate binding site, and its C-6 acyl group also extends over to the cofactor binding cavity. In addition, ZA-A effectively inhibits a homologous bacterial enzyme, dehydrosqualene synthase (CrtM), which synthesizes the precursor of staphyloxanthin in Staphylococcus aureus to cope with oxidative stress. Size reduction at Tyr(248) in CrtM further increases the ZA-A binding affinity, and it reveals a similar overall inhibitor binding mode to that of human SQS/ZA-A except for the C-6 acyl group. These structures pave the way for further improving selectivity and development of a new generation of anticholesterolemic and antimicrobial inhibitors.     

Mechanism of cis-prenyltransferase reaction probed by substrate analogues

Undecaprenyl pyrophosphate synthase (UPPS) is a cis-type prenyltransferases which catalyzes condensation reactions of farnesyl diphosphate (FPP) with eight isopentenyl pyrophosphate (IPP) units to generate C₅₅ product. In this study, we used two analogues of FPP, 2-fluoro-FPP and [1,1-²H₂]FPP, to probe the reaction mechanism of Escherichia coli UPPS. The reaction rate of 2-fluoro-FPP with IPP under single-turnover condition is similar to that of FPP, consistent with the mechanism without forming a farnesyl carbocation intermediate. Moreover, the deuterium secondary KIE of 0.985 ± 0.022 measured for UPPS reaction using [1,1-²H₂]FPP supports the associative transition state. Unlike the sequential mechanism used by trans-prenyltransferases, our data demonstrate E. coli UPPS utilizes the concerted mechanism.     

Biosynthesis of Carotenoids in the Chloroplasts of Algae and Higher Plants

Physiological, biochemical, and genetic aspects of carotenoid biosynthesis in the chloroplast membranes of green algae and higher plants are discussed starting from the earliest stages of biosynthesis of key C₅-isoprene units. The latter are synthesized either from acetate (C₂) to mevalonic acid (C₆) or from glucose (C₆) by forming glyceraldehyde 3-phosphate (C₃) and pyruvate decarboxylation product (C₂) through intermediate compounds to isopentenyl diphosphate (C₅). In all organisms, the further carotenoid synthesis from isopentenyl diphosphate and its isomer dimethylallyl diphosphate (C₅) proceeds through their transformation into geranyl diphosphate (C₁₀), farnesyl diphosphate (C₁₅), geranylgeranyl diphosphate (C₂₀) and phytoene (C₄₀). Phytoene desaturation (dehydrogenation) to carotene, neurosporene, and lycopene, and all steps of their cyclization to , and carotenes are discussed in detail. The synthesis of xanthophylls in chloroplasts is presented as the sequential formation of hydroxy-, epoxy- and oxo- groups. Genetic control of biosynthesis, as well as the localization and functional role of carotenoids in the chloroplast membranes of plants and algae are briefly discussed.     

甜瓜鲨烯合酶基因克隆及酶分子结构分析

葫芦素类是主要分布于葫芦科植物中具有多种医药活性的四环三萜类化合物,目前药用葫芦素原料主要从甜瓜蒂中提取。该研究从甜瓜中克隆葫芦素类合成关键酶——鲨烯合酶(SQS)的基因,并对其序列进行了生物信息学分析。结果表明:DNA测序和BLASTRefSeqGene分析表明,克隆的甜瓜SQS基因片段具有完整的该酶基因开放阅读框架(ORF)序列。ORF分析显示,甜瓜SQS由417氨基酸残基构成,等电点为7.56。对推衍的甜瓜SQS氨基酸序列分析结果提示,该酶二级结构以α螺旋为主。结构域预测结果表明,SQS属于异戊二烯合酶家族,具有法呢酰基二磷酸及镁离子的结合位点。三级结构预测提示,甜瓜SQS为单体酶,其活性中心主要由几个α螺旋围绕形成的穴状结构。磷酸化位点分析显示,S~(48)处于酶活性中心相关~(47)VSRSF~(52)的模体中,而S~(196)是正选择位点,提示这两处磷酸化位点可能是甜瓜SQS酶活性调节的关键部位。以甜瓜SQS基因ORF序列构建系统发生树的系统发生分类结果与形态学分类结果一致。该研究结果为葫芦素类的生物合成调控研究提供了新的线索和实验依据。     

Isolation and characterization of squalene synthase cDNA fromcentella asiatica (L) urban

We have cloned and characterized a gene for squalene synthase (SQS) fromCentella asiatica (L) Urban, a species that produces a large quantity of triterpene saponins such as asiaticoside and madecassoside. Its full-length cDNA clone was isolated by RACE PCR. The sequence ofpSQS contains an open reading frame of 1248 nucleotides, which code for 416 amino acids with a molecular mass of 47.3 kDa. Southern analysis revealed that one copy might exist in the C.asiatica genome. We also determined that 0.1 mM methyl jasmonate was sufficient to up-regulate those levels ofCaSQS mRNA.     

Arabidopsis thaliana isoprenyl diphosphate synthases produce the C25 intermediate geranylfarnesyl diphosphate

Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short‐chain all‐trans (E) prenyl diphosphates geranyl diphosphate (GDP, C₁₀), farnesyl diphosphate (FDP, C₁₅) or geranylgeranyl diphosphate (GGDP, C₂₀). In the genome of Arabidopsis thaliana, 15 trans‐product‐forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC‐MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C₂₅) instead of GGDP as their major product in enzyme assays performed in vitro. Over‐expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N‐terminal to the first aspartate‐rich motif is responsible for C₂₅ versus C₂₀ product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C₂₀) as a product and a larger R group (Met) resulting in GFDP (C₂₅).     

Different reaction mechanisms for cis- and trans-prenyltransferases

Octaprenyl diphosphate synthase (OPPs) and undecaprenyl diphosphate synthases (UPPs) catalyze consecutive condensation reactions of farnesyl diphosphate (FPP) with 5 and 8 isopentenyl diphosphate (IPP) to generate C₄₀ and C₅₅ products with trans- and cis-double bonds, respectively. In this study, we used IPP analogue, 3-bromo-3-butenyl diphosphate (Br-IPP), in conjunction with radiolabeled FPP, to probe the reaction mechanisms of the two prenyltransferases. Using this alternative substrate with electron-withdrawing bromo group at the C3 position to slow down the condensation step, trapping of farnesol in the OPPs reaction from radiolabeled FPP under basic condition was observed, consistent with a sequential mechanism. In contrast, UPPs reaction yielded no farnesyl carbocation intermediate under the same condition with radiolabeled FPP and Br-IPP, indicating a concerted mechanism. Our data demonstrate the different reaction mechanisms for cis- and tran-prenyltransferases although they share the same substrates.     

Towards elucidating the energy of the first excited singlet state of xanthophyll cycle pigments by X-ray absorption spectroscopy

The first excited singlet state (S₁) of carotenoids (also termed 2A_g⁻) plays a key role in photosynthetic excitation energy transfer due to its close proximity to the S₁ (Q_y) level of chlorophylls. The determination of carotenoid 2A_g⁻ energies by optical techniques is difficult; transitions from the ground state (S₀, 1A_g⁻) to the 2A_g⁻ state are forbidden (“optically dark”) due to parity (g ← //→ g) as well as pseudo-parity selection rules (− ← //→ −). Of particular interest are S₁ energies of the so-called xanthophyll-cycle pigments (violaxanthin, antheraxanthin and zeaxanthin) due to their involvement in photoprotection in plants. Previous determinations of S₁ energies of violaxanthin and zeaxanthin by different spectroscopic techniques vary considerably. Here we present an alternative approach towards elucidation of the optically dark states of xanthophylls by near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The indication of at least one π* energy level (about 0.5 eV below the lowest 1B_u⁺ vibronic sublevel) has been found for zeaxanthin. Present limitations and future improvements of NEXAFS to study optically dark states of carotenoids are discussed. NEXAFS combined with simultaneous optical pumping will further aid the investigation of these otherwise hardly accessible states.