NADPH-binding Motif Research Articles

Genome-Wide Identification and Characterization of the Medium-Chain Dehydrogenase/Reductase Superfamily of Trichosporon asahii and Its Involvement in the Regulation of Fluconazole Resistance.

The medium-chain dehydrogenase/reductase (MDR) superfamily contains many members that are widely present in organisms and play important roles in growth, metabolism, and stress resistance but have not been studied in Trichosporon asahii. In this study, bioinformatics and RNA sequencing methods were used to analyze the MDR superfamily of T. asahii and its regulatory effect on fluconazole resistance. A phylogenetic tree was constructed using Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, and T. asahii, and 73 MDRs were identified, all of which contained NADPH-binding motifs. T. asahii contained 20 MDRs that were unevenly distributed across six chromosomes. T. asahii MDRs (TaMDRs) had similar 3D structures but varied greatly in their genetic evolution at different phylum levels. RNA-seq and gene expression analyses revealed that the fluconazole-resistant T. asahii strain upregulates xylitol dehydrogenase, and downregulated alcohol dehydrogenase and sorbitol dehydrogenase concluded that the fluconazole-resistant T. asahii strain was less selective toward carbon sources and had higher adaptability to the environment. Overall, our study contributes to our understanding of TaMDRs, providing a basis for further analysis of the genes associated with drug resistance in T. asahii.

Flavin-Containing Monooxygenases (<i>FMO</i>) Genes in Garlic <i>Allium sativum</i> L.: Genome-Wide Identification, Characterization, and Expression Analysis in Response to <i>Fusarium proliferatum</i>

In this study, 39 flavin-containing monooxygenase genes were identified in the garlic (Allium sativum L.) genome. The distribution of AsFMOs into three phylogenetic clades associated with N-oxygenation (22 proteins), auxin biosynthesis (13 proteins), and S-oxygenation (4 proteins) has been shown. FAD and NADPH binding, FMO-identifying, and FATGY motifs were found in the AsFMO sequences. AsFMOs transcripts were present in all garlic organs with a maximum in roots, leaves, peduncle, and aerial bulbs. In response to infection with the pathogenic fungus Fusarium proliferatum, differential expression of clade I genes (AsFMO4, AsFMO11, AsFMO12, and AsFMO35) was detected in the roots of cv. Sarmat and Strelets, contrasting in Fusarium rot resistance. At the same time, the expression response of the clade III AsFMO18 gene involved in the alliin biosynthesis was similar for both cultivars, regardless of their resistance/susceptibility to Fusarium. This suggests the clades I and III genes redundancy in plant responses to infection. The AsFMO35 coding and regulatory sequences were analyzed in the Sarmat and Strelets cultivars. It was shown that the AsFMO35 promoter differs in the presence of the ABA-associated cis-regulatory element ABRE in cv. Strelets susceptible to Fusarium rot.

Elucidating the function of hypothetical PE_PGRS45 protein of Mycobacterium tuberculosis as an oxido-reductase: a potential target for drug repurposing for the treatment of tuberculosis

Mycobacterium tuberculosis (Mtb) encodes a total of 67 PE_PGRS proteins and definite functions of many of them are still unknown. This study reports PE_PGRS45 (Rv2615c) protein from Mtb as NADPH dependent oxido-reductase having substrate specificity for fatty acyl Coenzyme A. Computational studies predicted PE_PGRS45 to be an integral membrane protein of Mtb. Expression of PE_PGRS45 in non-pathogenic Mycobacterium smegmatis, which does not possess PE_PGRS genes, confirmed its membrane localization. This protein was observed to have NADPH binding motif. Experimental validation confirmed its NADPH dependent oxido-reductase activity (Km value = 34.85 ± 9.478 μM, Vmax = 96.77 ± 7.184 nmol/min/mg of protein). Therefore, its potential to be targeted by first line anti-tubercular drug Isoniazid (INH) was investigated. INH was predicted to bind within the active site of PE_PGRS45 protein and experiments validated its inhibitory effect on the oxido-reductase activity of PE_PGRS45 with IC50/Ki values of 5.66 μM. Mtb is resistant to first line drugs including INH. Therefore, to address the problem of drug resistant TB, docking and Molecular Dynamics (MD) simulation studies between PE_PGRS45 and three drugs (Entacapone, Tolcapone and Verapamil) which are being used in Parkinson’s and hypertension treatment were performed. PE_PGRS45 bound the three drugs with similar or better affinity in comparison to INH. Additionally, INH and these drugs bound within the same active site of PE_PGRS45. This study discovered Mtb’s PE_PGRS45 protein to have an oxido-reductase activity and could be targeted by drugs that can be repurposed for TB treatment. Furthermore, in-vitro and in-vivo validation will aid in drug-resistant TB treatment. HIGHLIGHTS In-silico and in-vitro studies of hypothetical protein PE_PGRS45 (Rv2615c) of Mycobacterium tuberculosis (Mtb) reveals it to be an integral membrane protein PE_PGRS45 protein has substrate specificity for fatty acyl Coenzyme A (fatty acyl CoA) and possess NADPH dependent oxido-reductase activity Docking and simulation studies revealed that first line anti-tubercular drug Isoniazid (INH) and other drugs with anti-TB property have strong affinity for PE_PGRS45 protein Oxido-reductase activity of PE_PGRS45 protein is inhibited by INH PE_PGRS45 protein could be targeted by drugs that can be repurposed for TB treatment Communicated by Ramaswamy H. Sarma

Structurally distinct unfolding intermediates formed from a staphylococcal capsule-producing enzyme retained NADPH binding activity

CapF, a capsule-producing enzyme expressed by Staphylococcus aureus, binds NADPH and exists as a dimer in the aqueous solution. Many other capsule-producing virulent bacteria also express CapF orthologs. To understand the folding-unfolding mechanism of S. aureus CapF, herein a recombinant CapF (rCapF) was individually investigated using urea and guanidine hydrochloride (GdnCl). Unfolding of rCapF by both the denaturants was reversible but proceeded via the synthesis of a different number of intermediates. While two dimeric intermediates (rCapF4 and rCapF5) were formed at 0.5 M and 1.5 M GdnCl, three dimeric intermediates (rCapF1, rCapF2, and rCapF3) were produced at 1 M, 2 M, and 3 M urea, respectively. rCapF5 showed 3.6 fold less NADPH binding activity, whereas other intermediates retained full NADPH binding activity. Compared to rCapF, all of the intermediates (except rCapF3) had a compressed shape. Conversely, rCapF3 possessed a native protein-like shape. The maximum shape loss was in rCapF4 though its secondary structure remained unperturbed. Additionally, the tertiary structure and hydrophobic surface area of the intermediates neither matched with each other nor with those of the native rCapF. Of the four Trp residues in rCapF, one or more Trp residues in the intermediates may have higher solvent accessibility. Using sequence alignment and a tertiary structural model of CapF, we have demonstrated that the region around Trp 137 of CapF may be most sensitive to unfolding, whereas the NADPH binding motif carrying region at the N-terminal end of this protein may be resistant to unfolding, particularly at the low denaturant concentrations. Communicated by Ramaswamy H. Sarma

Stimulated Biosynthesis of an C10-Deoxy Heptaene NPP B2 via Regulatory Genes Overexpression in Pseudonocardia autotrophica.

Polyene macrolides, such as nystatin A1, amphotericin B, and NPP A1, belong to a large family of valuable antifungal polyketide compounds that are typically produced by soil actinomycetes. Previously, NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, was generated by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase in Pseudonocardia autotrophica. This derivative showed superior antifungal activity to NPP A1. In this study, another novel derivative called NPP B2 was developed, which lacks a hydroxyl group at the C10 position by site-specific gene disruption of the P450 hydroxylase NppL. To stimulate the extremely low expression of the NPP B2 biosynthetic pathway genes, the 32-kb NPP-specific regulatory gene cluster was overexpressed via site-specific chromosomal integration. The extra copy of the six NPP-specific regulatory genes led to a significant increase in the NPP B2 yield from 0.19 to 7.67 mg/L, which is the highest level of NPP B2 production ever achieved by the P. autotrophica strain. Subsequent in vitro antifungal activity and toxicity studies indicated that NPP B2 exhibited similar antifungal activity but significantly lower hemolytic toxicity than NPP B1. These results suggest that an NPP biosynthetic pathway refactoring and overexpression of its pathway-specific regulatory genes is an efficient approach to stimulating the production of an extremely low-level metabolite, such as NPP B2 in a pathway-engineered rare actinomycete strain.

Two classes of cytochrome P450 reductase genes and their divergent functions in Camptotheca acuminata Decne

Cytochrome P450 monooxygenases (CYP450s) and their auxiliary cytochrome P450 reductases (CPRs) are important and commonly involved in the biosynthesis of camptothecin (CPT). To better understand the possible functions of CPRs in planta, we first isolated two CaCPRs genes from Camptotheca acuminata. Sequence analysis revealed the presence of common conserved FMN-, FAD- and NADPH-binding motifs in putative CaCPR1/CaCPR2 proteins. The two CaCPR paralogs were assigned to the Class I and Class II of CPRs, respectively, according to phylogenetic tree. The recombinant CrCYP72-CaCPR1 and CrCYP72-CaCPR2 enzymes and their substrate bioconversion rates of 23.09% and 35.23% demonstrated that both CaCPRs could support the enzyme activities of CrSLS1. Gene silencing of CaCPRs by VIGS led to downregulation of CaCPR1/CaCPR2 expression by 50–67%, accompanied with 10–15% slight decrease and 57–63% dramatic reduction for CaCPR1 and CaCPR2 individually in CPT accumulations. Moreover, CaCPR1/CaCPR2 displayed almost omnipresent expression patterns across Camptotheca tissues. While in comparison to constitutive expression of CaCPR1 gene, CaCPR2 and CYP450 genes were all dramatically phytohormone-induced expressed in leaves which were main tissues for isoprenoid and CPT biosynthesis. Our results suggested that, in Camptotheca seedlings, CaCPR2 had a distinct function from CaCPR1 that was clearly involved in the inducible specialized metabolism for CPT biosynthesis.

Biochemical Characterization of the Rice Cinnamyl Alcohol Dehydrogenase Gene Family.

Cinnamyl alcohol dehydrogenase (CAD) is involved in the final step of the phenylpropanod pathway, catalyzing the NADPH-dependent reduction of hydroxy-cinnamaldehydes into the corresponding alcohols. The rice genome contains twelve CAD and CAD-like genes, collectively called OsCADs. To elucidate the biochemical function of the OsCADs, OsCAD1, 2, 6, and 7, which are highly expressed in rice, were cloned from rice tissues. The cloned OsCADs were heterologously expressed in Escherichia coli as His-tag fusion proteins. The activity assay of the recombinant OsCADs showed that OsCAD2, 6, and 7 have CAD activity toward hydroxycinnamaldehydes, but OsCAD1 has no detectable catalytic activity. The kinetic parameters of the enzyme reactions demonstrated that OsCAD2 has the highest catalytic activity among the examined enzymes. This result agrees well with the finding that the Zn binding and NADPH binding motifs and the residues constituting the substrate binding pocket in bona fide plant CADs were fully conserved in OsCAD2. Although they have large variations in the residue for the substrate binding pocket, OsCAD6 and 7 catalyzed the reduction of hydroxycinnamaldehydes with a similar efficiency. Alignment of amino acid sequences showed that OsCAD1 lacks the GxxxxP motif for NADPH binding and has mismatches in residues important in the reduction process, which could be responsible for the loss of catalytic activity. OsCAD2 belongs to CAD Class I with bona fide CADs from other plant species and is constitutively expressed throughout the developmental stages of rice, with preferential expression in actively lignifying tissues such as the root, stem, and panicle, suggesting that it is mainly involved in developmental lignification in rice. The expression of OsCAD2 was also induced by biotic and abiotic stresses such as Xanthomonas oryzae pv. oryzae (Xoo) infection and UV-irradiation, suggesting that it plays a role in the defense response of rice, in addition to a bona fide role in developmental lignification. OsCAD6 and 7 belong in CAD Class II. Their expression is relatively lower than that of OsCAD2 and is confined to certain tissues, such as the leaf sheath, stem, and panicle. The expression of OsCAD6 was stimulated by Xoo infection and UV-irradiation. Thus OsCAD6 appears to be an inducible OsCAD that is likely involved in the defense response of rice against biotic and abiotic stresses.

Nystatin-like Pseudonocardia polyene B1, a novel disaccharide-containing antifungal heptaene antibiotic

Polyene macrolides such as nystatin A1 and amphotericin B belong to a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Recently, nystatin-like Pseudonocardia polyene (NPP) A1 has been identified as a unique disaccharide-containing tetraene antifungal macrolide produced by Pseudonocardia autotrophica. Despite its significantly increased water solubility and decreased hemolytic activity, its antifungal activity remains limited compared with that of nystatin A1. In this study, we developed NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase NppC. The low level NPP B1 production yield was successfully improved by eliminating the native plasmid encoding a polyketide biosynthetic gene cluster present in P. autotrophica. In vitro and in vivo antifungal activity and toxicity studies indicated that NPP B1 exhibited comparable antifungal activity against Candida albicans and was less toxic than the most potent heptaene antifungal, amphotericin B. Moreover, NPP B1 showed improved pharmacokinetic parameters compared to those of amphotericin B, suggesting that NPP B1 could be a promising candidate for development into a pharmacokinetically improved and less-toxic polyene antifungal antibiotic.

Two distinct cinnamoyl-CoA reductases in Selaginella moellendorffii offer insight into the divergence of CCRs in plants.

Two distinct cinnamoyl-coenzyme A reductases (CCRs) from Selaginella moellendorffii were evaluated, and of these, SmCCR2-1, which has both distinct sequence motifs and catalytic properties, was clustered into a new CCR subgroup. Cinnamoyl-coenzyme A reductases (CCRs) have been reported in many land plants to have critical functions in monolignol biosynthesis. In this study, we performed a genome-wide screen and obtained two distinct SmCCRs from S. moellendorffii. Phylogenetic analysis indicated that SmCCR2 (both SmCCR2-1 and 2-2) and SmCCR3 together with PpaCCR belong to a distinct subgroup of genuine CCRs with variations in the NAD(P)H-binding motif. Enzymatic assays showed detectable activity by both SmCCR1 and SmCCR2-1 toward four hydroxycinnamoyl-CoA esters. SmCCR1, which clustered with reported CCRs from angiosperms and gymnosperms, exhibited specificity toward feruloyl-CoA, while SmCCR2-1 showed a preference for sinapoyl-CoA. Interestingly, the reaction temperature profiles for SmCCR1 and SmCCR2-1 are complementary. Homology models and molecular simulations suggest that the variations in NADPH-binding motifs, especially R(X)6K instead of R(X)5K, affect the NADP+ conformation. Notably, the signature motif NWYCY was replaced with NGYCL in SmCCR1 and with EWYCL in SmCCR2-1, while the signature residues H202 and R253, reported in a previous study, were conserved in SmCCR1 and SmCCR2-1 but varied in SmCCR-like genes. It is likely that NWYCY is not a reliable signature for CCRs in plants. The detectable activity of site-direct mutant S123T of SmCCR1 suggested that S123 which consists of catalytic triad is changeable. Possible evolution process for the emergence of two subgroups of genuine CCRs was also revealed. Altogether, these findings revise our understanding of CCRs with regard to divergence and active sites.

A Root-Preferential DFR-Like Gene Encoding Dihydrokaempferol Reductase Involved in Anthocyanin Biosynthesis of Purple-Fleshed Sweet Potato.

Purple-fleshed sweet potato is good for health due to rich anthocyanins in tubers. Although the anthocyanin biosynthetic pathway is well understood in up-ground organs of plants, the knowledge on anthocyanin biosynthesis in underground tubers is limited. In the present study, we isolated and functionally characterized a root-preferential gene encoding dihydrokaempferol reductase (IbDHKR) from purple-fleshed sweet potato. IbDHKR showed highly similarity with the reported dihydroflavonol reductases in other plant species at the sequence levels and the NADPH-binding motif and the substrate-binding domain were also found in IbDHKR. The tissue profile showed that IbDHKR was expressed in all the tested organs, but with much higher level in tuber roots. The expression level of IbDHKR was consistent with the anthocyanin content in sweet potato organs, suggesting that tuber roots were the main organs to synthesize anthocyanins. The recombinant 44 kD IbDHKR was purified and fed by three different dihydroflavonol substrates including dihydrokaempferol (DHK), dihydroquerctin, and dihydromyrecetin. The substrate feeding assay indicated that only DHK could be accepted as substrate by IbDHKR, which was reduced to leucopelargonidin confirmed by LC-MS. Finally, IbDHKR was overexpressed in transgenic tobacco. The IbDHKR-overexpression tobacco corolla was more highly pigmented and contained higher level of anthocyanins than the wild-type tobacco corolla. In summary, IbDHKR was a root-preferential gene involved in anthocyanin biosynthesis and its encoding protein, specifically catalyzing DHK reduction to yield leucopelargonidin, was a candidate gene for engineering anthocyanin biosynthetic pathway.

Molecular identification and functional delineation of a glutathione reductase homolog from disk abalone (Haliotis discus discus): Insights as a potent player in host antioxidant defense

Glutathione reductase (GSR) is an enzyme that catalyzes the biochemical conversion of oxidized glutathione (GSSG) into the reduced form (GSH). Since the ratio between the two forms of glutathione (GSH/GSSG) is important for the optimal function of GSH to act as an antioxidant against H2O2, the contribution of GSR as an enzymatic regulatory agent to maintain the proper ratio is essential. Abalones are marine mollusks that frequently encounter environmental factors that can trigger the overproduction of reactive oxygen species (ROS) such as H2O2. Therefore, we conducted the current study to reveal the molecular and functional properties of a GSR homolog in the disk abalone, Haliotis discus discus. The identified cDNA sequence (2325 bp) has a 1356 bp long open reading frame (ORF), coding for a 909 bp long amino acid sequence, which harbors a pyridine nucleotide-disulfide oxidoreductase domain (171–246 aa), a pyridine nucleotide-disulfide oxidoreductase dimerization domain, and a NAD(P)(+)-binding Rossmann fold superfamily signature domain. Four functional residues: the FAD binding site, glutathione binding site, NADPH binding motif, and assembly domain were identified to be conserved among the other species. The recombinant abalone GSR (rAbGSR) exhibited detectable activity in a standard glutathione reductase activity assay. The optimum pH and optimal temperature for the reaction were found to be 7.0 and 50 °C, respectively, while the ionic strength of the medium had no effect. The enzymatic reaction was vastly inhibited by Cu+2 and Cd+2 ions. A considerable effect of cellular protection was detected with a disk diffusion assay conducted with rAbGSR. Moreover, an MTT assay and flow cytometry confirmed the significance of the protective role of rAbGSR in cell function. Furthermore, AbGSR was found to be ubiquitously distributed in different types of abalone tissues. AbGSR mRNA expression was significantly upregulated in response to three immune challenges: Vibrio parahaemolyticus, Listeria monocytogenes, and lipopolysaccharide (LPS), thus indicating its possible involvement in host defense mechanisms during pathogenic infections. Taken together, the results of the current study suggest that AbGSR plays an important role in antioxidant-mediated host defense mechanisms and also provide insights into the immunological contribution of AbGSR.

A novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase

Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions.

A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase

Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions.

Characterization of recombinant glutathione reductase from the psychrophilic Antarctic bacterium Colwellia psychrerythraea.

Glutathione reductases catalyze the reduction of oxidized glutathione (glutathione disulfide, GSSG) using NADPH as the substrate to produce reduced glutathione (GSH), which is an important antioxidant molecule that helps maintain the proper reducing environment of the cell. A recombinant form of glutathione reductase from Colwellia psychrerythraea, a marine psychrophilic bacterium, has been biochemically characterized to determine its molecular and enzymatic properties. C. psychrerythraea glutathione reductase was shown to be a homodimer with a molecular weight of 48.7 kDa using SDS-PAGE, MALDI-TOF mass spectrometry and gel filtration. The C. psychrerythraea glutathione reductase sequence shows significant homology to that of Escherichia coli glutathione reductase (66 % identity), and it possesses the FAD and NADPH binding motifs, as well as absorption spectrum features which are characteristic of flavoenzymes such as glutathione reductase. The psychrophilic C. psychrerythraea glutathione reductase exhibits higher k cat and k cat/K m at lower temperatures (4 °C) compared to mesophilic Baker's yeast glutathione reductase. However, C. psychrerythraea glutathione reductase was able to complement an E. coli glutathione reductase deletion strain in oxidative stress growth assays, demonstrating the functionality of C. psychrerythraea glutathione reductase over a broad temperature range, which suggests its potential utility as an antioxidant enzyme in heterologous systems.

Molecular cloning, characterization and expression analysis of the gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Aquilaria sinensis (Lour.) Gilg.

The major constituents of agarwood oils are sesquiterpenes that are obtained from isoprenoid precursors through the plastidial methylerythritol phosphate (MEP) pathway and the cytosolic mevalonate pathway. In this study, a novel full-length cDNA of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), which was the second key enzyme in the plastid MEP pathway of sesquiterpenes biosynthesis was isolated from the stem of Aquilaria sinensis (Lour.) Gilg by the methods of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique for the first time, and named as AsDXR. The full-length cDNA of AsDXR was 1768 bp, containing a 1437 bp open reading frame (ORF) encoding a polypeptide of 478 amino acids with a molecular weight of 51.859 kD and the theoretical isoelectric point of 6.29. Comparative and bioinformatic analysis of the deduced AsDXR protein showed extensive homology with DXRs from other plant species, especially Theobroma cacao and Gossypium barbadense, and contained a conserved transit peptide for plastids, and extended pro-rich region and a highly conserved NADPH-binding motif owned by all plant DXRs. Southern blot analysis indicated that AsDXR belonged to a small gene family. Tissue expression pattern analysis revealed that AsDXR expressed strongly in root and stem, but weakly in leaf. Additionally, AsDXR expression was found to be activated by exogenous elicitor of MeJA (methyl jasmonate). The contents of three sesquiterpenes (α-guaiene, α-humulene and Δ-guaiene) were significantly induced by MeJA. This study enables us to further elucidate the role of AsDXR in the biosynthesis of agarwood sesquiterpenes in A. sinensis at the molecular level.

The crystal structure of human GDP-l-fucose synthase

Human GDP-l-fucose synthase, also known as FX protein, synthesizes GDP-l-fucose from its substrate GDP-4-keto-6-deoxy-d-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. In this paper, the first crystal structure of human FX protein was determined at 2.37 Å resolution. The asymmetric unit of the crystal structure contains four molecules which form two homodimers. Each molecule consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain. Compared with the Escherichia coli GDP-l-fucose synthase, the overall structures of these two enzymes have four major differences. There are four loops in the structure of human FX protein corresponding to two α-helices and two β-sheets in that of the E. coli enzyme. Besides, there are seven different amino acid residues binding with NAPDH comparing human FX protein with that from E. coli. The structure of human FX reveals the key catalytic residues and could be useful for the design of drugs for the treatment of inflammation, auto-immune diseases, and possibly certain types of cancer.

Cloning and expression regulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase cDNA from Alpinia officinarum

The rhizome of Alpinia officinarum is a widely used Chinese herbal medicine. The essential oil in A. officinarum rhizome is mainly composed of 1, 8-cineole and other monoterpenes, as the major bioactive ingredients. In plants, monoterpenes are synthesized through the methylerythritol phosphate (MEP) pathway in the plastids, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an enzyme catalyzing a committed step of the MEP pathway. In the present study, the full-length cDNA encoding DXR was cloned from the rhizome of A. officinarum, using homology-based RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The new cDNA was designated as AoDXR and submitted to GenBank to be assigned with an accession number HQ874658. The full-length cDNA of AoDXR was 1 670 bp containing a 1 419 bp open reading frame encoding a polypeptide of 472 amino acids with a calculated molecular mass of 51.48 kDa and an isoelectric point of 6.15. Bioinformatic analyses revealed that AoDXR showed extensive homology with DXRs from other plant species and contained a conserved plastids transit peptide, a Pro-rich region and two highly conserved NADPH-binding motifs in its N-terminal region characterized by all plant DXRs. The phylogenetic analysis revealed that AoDXR belonged to angiosperm DXRs. The structural modeling of AoDXR showed that AoDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that AoDXR expressed strongly in leaves, weak in rhizomes of A. officinarum. Exogenous methyl jasmonate (MeJA) could enhance the expression of AoDXR and the production of 1, 8-cineole in A. officinarum rhizomes. The cloning and characterization of AoDXR will be helpful to reveal the molecular regulation mechanism of monoterpene biosynthesis in A. officinarum and provides a candidate gene for metabolic engineering in improving the medicinal quality of A. officinarum rhizome.

Identification and differential expression of two isogenes encoding 1-deoxy-d-xylulose 5-phosphate reductoisomerase in Glycine max

Plant 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) were considered to be encoded by single copy. In this study, we successfully isolated two DXR isogenes, designated GmDXR1 and GmDXR2, from soybean. The multicopy nature of DXRs in soybean was supported by Southern blot. Alignment of the two GmDXRs showed the presence of the N-terminal transit peptide for plastids, the conserved cleavage site, proline-rich region, and the NADPH-binding motif. Phylogenetic analysis revealed GmDXR1 and GmDXR2 belonged to different branches of angiosperm DXRs clusters. Expression pattern analysis indicated that GmDXR1 was expressed in all analyzed organs except the pod walls, with the highest level in seeds, whereas no message was detected for GmDXR2. In response to heat stress, GmDXR1 showed declined transcript levels in the experiments; in contrast, GmDXR2 was highly responsive, with mRNA accumulation peaking at 6 h after treatment. We also demonstrated that both GmDXRs were localized in chloroplasts. Overexpression of GmDXRs induced increased contents of various isoprenoids (chlorophyll, carotenoids, and gibberellins), but with reduced level of ABA, indicating that GmDXRs participate in the control of differential isoprenoid biosynthesis of the MEP pathway. This work provides a new insight into the wide presence of multicopies of DXR enzyme in plants.

Cloning and Functional Analysis of Enoyl-CoA Reductase Gene BnECR from Oilseed Rape ( Brassica napus L.)

Very-long-chain fatty acids (VLCFAs) are critical components in cuticular waxes, sphingolipids, and triacylglycerols in higher plants. Biosynthesis of VLCFAs is catalyzed by the fatty acyl-CoA elongase, a membrane-bound enzymatic complex containing 3-ketoacyl-CoA synthase (KCS), 3-ketoacyl-CoA reductase (KCR), 3-hydroxacyl-CoA dehydratase (HCD), and trans-2,3-enoyl-CoA reductase (ECR). In this study, the primers were designed based on multiple alignments of trans-2,3-enoyl-CoA reductase (ECR) gene sequences from Arabidopsis thaliana and other plants, and the full-length cDNA, here designated BnECR, and the corresponding genomic sequences were isolated from Brassica napus using rapid amplification of cDNA ends method (GenBank accession numbers FJ899705 and FJ899705). The sequence of BnECR cDNA was 1328 bp in length excluding the poly dA tail, and the corresponding genomic sequence was 2093 bp. BnECR was composed of 4 exons and contained a 163 bp 5′ untranslated region (5′ UTR) and a 233 bp 3′ UTR. The deduced BnECR protein was 310 amino acid in length with a molecular weight of 735.78 kD and an isoelectric point of 9.52. The critical functional sites K 144 and R 145 in AtECR were unchanged in BnECR. The G 225SGGYQIPR/HG 234, which presented a non-classical NADPH-binding motif, was found in the C-terminal of BnECR. NCBI Blastn, multiple alignments and conserved domain search showed that BnECR had the highest homology to A. thaliana AtECR. RT-PCR analysis showed that BnECR was ubiquitously expressed in B. napus and preferentially expressed in the stem. The transcript level of BnECR at mid and late stages of seed development in low erucic acid rapeseed cultivar was obviously lower than that in high erucic acid rapeseed cultivar, suggesting that BnECR was involved in biosynthesis of erucic acid. The 933 bp open reading frame of BnECR was subcloned into the yeast- Escherichia coli shuttle vector pYES2.0. The recombinant plasmid was transformed into Saccharomyces cerevisiae wild type strain By4743 and the mutant strain YDL015c. With galactose as inducer, the transformant was cultured to induce the expression of BnECR. The gas chromatographic result indicated that BnECR was overexpressed effectively in S. cerevisiae, and the content of erucic acid reached 1.34% of the total fatty acid in the recombinant strain, an increase of 52% over the control. Functional complementation of BnECR in an ECR-deficient mutant yeast demonstrated that BnECR mediated the biosynthesis of VLCFAs. The results suggest that BnECR should be functional orthologue of AtECR.

Characterization of two cotton cDNAs encoding trans-2-enoyl-CoA reductase reveals a putative novel NADPH-binding motif

Very long chain fatty acids are important components of plant lipids, suberins, and cuticular waxes. Trans-2-enoyl-CoA reductase (ECR) catalyses the fourth reaction of fatty acid elongation, which is NADPH dependent. In the present study, the expression of two cotton ECR (GhECR) genes revealed by quantitative RT-PCR analysis was up-regulated during cotton fibre elongation. GhECR1 and 2 each contain open reading frames of 933 bp in length, both encoding proteins consisting of 310 amino acid residues. GhECRs show 32% identity to Saccharomyces cerevisiae Tsc13p at the deduced amino acid level, and the GhECR genes were able to restore the viability of the S. cerevisiae haploid tsc13-deletion strain. A putative non-classical NADPH-binding site in GhECR was predicted by an empirical approach. Site-directed mutagenesis in combination with gas chromatography–mass spectrometry analysis suggests that G(5X)IPXG presents a putative novel NADPH-binding motif of the plant ECR family. The data suggest that both GhECR genes encode functional enzymes harbouring non-classical NADPH-binding sites at their C-termini, and are involved in fatty acid elongation during cotton fibre development.