Short-chain Dehydrogenase Research Articles

In vitro screening and in silico prediction of antifungal metabolites from rhizobacterium Achromobacter kerstersii JKP9.

The main objective of this study was to identify the antifungal metabolites from Achromobacter kerstersii JKP9, a rhizosphere bacterium isolated from tomato cultivations, inhibiting the melanin biosynthetic pathways in vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici (Fol). To achieve this objective, all the rhizobacterial morphotypes were screened for plant-growth-promoting and antagonistic activities. Ethyl acetate extract of Achromobacter kerstersii JKP9 was purified in HPLC and predicted for antifungals in GC-MS equipped with Wiley library. After identification, molecular docking of useful ligands with modeled Short-chain Dehydrogenase/ Reductase (SDR) of Fol (Locus: FOXG_00472). Results were indicated that the potential strain Achromobacter kerstersii JKP9 exclusively secreted five pyrrole analogs notable for their antifungal role with no extracellular antifungal enzyme production as seen in other rhizobacterial isolates. In silico docking studies identified, Pyrrolo[1, 2-a]pyrazine-1,4-dione, hexahydro- as effective for SDR in Fol. From these results, we conclude that bacterial pyrroles can be used as an effective fungicide to control Fusarium wilt in tomatoes. In the future, these pyrrole derivatives can directly be employed as eco-friendly fungicides or may be used as antifungal supplements in agrochemical products for the sustainable production of tomatoes.

Adaptation of pine wood nematode Bursaphelenchus xylophilus to \u03b2-pinene stress

BackgroundThe pine wood nematode (PWN; Bursaphelenchus xylophilus) is the most damaging biological pest in pine forest ecosystems in China. However, the pathogenic mechanism remains unclear. Tracheid cavitation induced by excess metabolism of volatile terpenes is a typical characteristic of pine trees infected by B. xylophilus. β-pinene, one of the main volatile terpenes, influences PWN colonization and reproduction, stimulating pathogenicity during the early stages of infection. To elucidate the response mechanism of PWN to β-pinene, pathogenesis, mortality, and reproduction rate were investigated under different concentrations of β-pinene using a transcriptomics approach.ResultsA low concentration of β-pinene (BL, C < 25.74 mg/ml) inhibited PWN reproduction, whereas a high concentration (BH, C > 128.7 mg/ml) promoted reproduction. Comparison of PWN expression profiles under low (BL, 21.66 mg/ml) and high (BH, 214.5 mg/ml) β-pinene concentrations at 48 h identified 659 and 418 differentially expressed genes (DEGs), respectively, compared with controls. Some key DEGs are potential regulators of β-pinene via detoxification metabolism (cytochrome P450, UDP-glucuronosyltransferases and short-chain dehydrogenases), ion channel/transporter activity (unc and ATP-binding cassette families), and nuclear receptor -related genes. Gene Ontology enrichment analysis of DEGs revealed metabolic processes as the most significant biological processes, and catalytic activity as the most significant molecular function for both BL and BH samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) analysis showed that xenobiotics biodegradation and metabolism, carbohydrate metabolism, lipid metabolism, amino acid metabolism, metabolism of cofactors and vitamins, and transport and catabolism were the dominant terms in metabolism categories.ConclusionIn addition to detoxification via reduction/oxidation (redox) activity, PWN responds to β-pinene through amino acid metabolism, carbohydrate metabolism, and other pathways including growth regulation and epidermal protein changes to overcome β-pinene stress. This study lays a foundation for further exploring the pathogenic mechanism of PWN.

Crossing the Border: From Keto- to Imine Reduction in Short-Chain Dehydrogenases/Reductases.

The family of NAD(P)H‐dependent short‐chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto‐reducing activity, yet negligible imine‐reducing capability, and mining the Short‐Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active‐site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine‐reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto‐reducing activity was engineered into a promiscuous enzyme with imine‐reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the “classical” SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine‐reducing enzymes.

Tobacco RNA-dependent RNA polymerase 1 affects the expression of defence-related genes in Nicotiana benthamiana upon Tomato leaf curl Gujarat virus infection.

RNA-dependent RNA polymerase 1 of Nicotiana tabacum modulates ToLCGV pathogenesis by influencing a number of defence-related genes in N. benthamiana plants. Key means of plants protecting themselves from the invading viruses is through RNA silencing. RNA-dependent RNA polymerase-1 (RDR1) is one of the crucial proteins of the RNA silencing pathway, which is induced after infection by viruses. RDR1 functions in the generation of small interfering RNAs (siRNAs) against the viral genome, thus it is antiviral in nature. Here, we used the transgenic Nicotiana benthamiana plant expressing N. tabacum NtRDR1 and observed reduced susceptibility towards Tomato leaf curl Gujarat virus (ToLCGV) infection compared to the wild-type N. benthamiana plants. To understand the reason for such reduced susceptibility, we prepared high-definition small RNA (sRNA) cDNA libraries from ToLCGV-infected wild-type N. benthamiana and NtRDR1 expressing N. benthamiana lines and carried out next-generation sequencing (NGS). We found that upon ToLCGV infection the majority of siRNAs generated from the host genome were of the 24 nucleotide (nt) class, while viral siRNAs (vsiRNAs) were of the 21-22-nt class, indicating that transcriptional gene silencing (TGS) is the major pathway for silencing of host genes while viral genes are silenced, predominantly, by post transcriptional gene silencing (PTGS) pathways. We estimated the changes in the expression of various defence-related genes, such as Constitutively Photomorphogenic-9 (COP9) signalosome (CSN) complex subunit-7, Pentatricopeptide repeat containing protein (PPRP), Laccase-3, Glutathione peroxidase-1 (GPX-1), Universal stress protein (USP) A-like protein, Heat shock transcription factor B4 (HSTF-B4), Auxin response factor-18 (ARF18), WRKY-6 and Short chain dehydrogenase reductase-3a. The differential expression of these genes might be linked with the enhanced tolerance of NtRDR1 N. benthamiana transgenic plants to ToLCGV. Our study suggests that reduced expression of subunit-7 of CSN complex and WRKY6, and increased expression of USPA-like protein might be linked with the reduced susceptibility of NtRDR1-transgenic N. benthamiana plants to ToLCGV.

Biological pincer complexes.

At least two types of pincer complexes are known to exist in biology. A metal-pyrroloquinolone quinone (PQQ) cofactor was first identified in bacterial methanol dehydrogenase, and later also found in selected short-chain alcohol dehydrogenases of other microorganisms. The PQQ-associated metal can be calcium, magnesium, or a rare earth element depending on the enzyme sequence. Synthesis of this organic ligand requires a series of accessory proteins acting on a small peptide, PqqA. Binding of metal to PQQ yields an ONO-type pincer complex. More recently, a nickel-pincer nucleotide (NPN) cofactor was discovered in lactate racemase, LarA. This cofactor derives from nicotinic acid adenine dinucleotide via action of a carboxylase/hydrolase, sulfur transferase, and nickel insertase, resulting in an SCS-type pincer complex. The NPN cofactor likely occurs in selected other racemases and epimerases of bacteria, archaea, and a few eukaryotes.

The Progesterone 5β-Reductase/Iridoid Synthase Family: A Catalytic Reservoir for Specialized Metabolism across Land Plants.

Iridoids are plant-derived terpenoids with a rich array of bioactivities. The key step in iridoid skeleton formation is the reduction of 8-oxogeranial by certain members of the progesterone 5β-reductase/iridoid synthase (PRISE) family of short-chain alcohol dehydrogenases. Other members of the PRISE family have previously been implicated in the biosynthesis of the triterpenoid class of cardenolides, which requires the reduction of progesterone. Here, we explore the occurrence and activity of PRISE across major lineages of plants. We observed trace activities toward either 8-oxogeranial or progesterone in all PRISEs, including those from nonseed plants and green algae. Phylogenetic analysis, coupled with enzymatic assays, show that these activities appear to have become specialized in specific angiosperm lineages. This broad analysis of the PRISE family provides insight into how these enzymes evolved in plants and also suggests that iridoid synthase activity is an ancestral trait in all land plants, which might have contributed to the rise of iridoid metabolites.

Systematic Evaluation of Imine-Reducing Enzymes: Common Principles in Imine Reductases, β-Hydroxy Acid Dehydrogenases, and Short-Chain Dehydrogenases/ Reductases.

The enzymatic, asymmetric reduction of imines is catalyzed by imine reductases (IREDs), members of the short‐chain dehydrogenase/reductase (SDR) family, and β‐hydroxy acid dehydrogenase (βHAD) variants. Systematic evaluation of the structures and substrate‐binding sites of the three enzyme families has revealed four common principles for imine reduction: structurally conserved cofactor‐binding domains; tyrosine, aspartate, or glutamate as proton donor; at least four characteristic flanking residues that adapt the donor's pK a and polarize the substrate; and a negative electrostatic potential in the substrate‐binding site to stabilize the transition state. As additional catalytically relevant positions, we propose alternative proton donors in IREDs and βHADs as well as proton relays in IREDs, βHADs, and SDRs. The functional role of flanking residues was experimentally confirmed by alanine scanning of the imine‐reducing SDR from Zephyranthes treatiae. Mutating the “gatekeeping” phenylalanine at standard position 200 resulted in a tenfold increase in imine‐reducing activity.

Differential expression and functional analysis of two short-chain alcohol dehydrogenases/reductases in Hedychium coronarium

Abstract In this study, the full cDNA sequences of HcADH2 and HcADH3 were cloned from Hedychium coronarium. The amino acid sequences encoded by them contained three most conserved motifs of short-chain alcohol dehydrogenase (ADH), namely NAD+ binding domain, TGxxx[AG]xG and active site YxxxK. The highest similarity between two genes and ADH from other plants was 70%. Phylogenetic analysis showed that they belonged to a member of the short-chain dehydrogenases/reductases 110C subfamily, but they were distinctly clustered in different clades. Real-time polymerase chain reaction analyses showed that HcADH2 was specifically expressed in bract, and it was expressed higher in no-scented Hedychium forrestii than other Hedychium species, but was undetectable in Hedychium coccineum. HcADH3 was expressed higher in the lateral petal of the flower than in other vegetative organs, and it was expressed the most in H. coronarium that is the most scented among Hedychium species, and its expression levels peaked at the half opening stage. HcADH2 and HcADH3 had almost no significant expression in leaves, but HcADH2 was expressed in response to external stimuli. The mechanical injury and methyl jasmonate (MeJA) treatment could induce expression of HcADH2 in leaves, whereas HcADH3 could have an induced expression only by MeJA. The recombinant HcADH3 protein, but not HcADH2, expressed in Escherichia coli-catalysed conversion of geraniol into citral. It was speculated that HcADH3 had an induced expression in vegetative organ of H. coronarium and took part in monoterpenoid biosynthesis in H. coronarium flowers, but the role of HcADH2 is relevant only for defensive reactions.

Protein\u2013protein interaction of Rv0148 with Htdy and its predicted role towards drug resistance in Mycobacterium tuberculosis

BackgroundMycobacterium tuberculosis resides inside host macrophages during infection and adapts to resilient stresses generated by the host immune system. As a response, M. tuberculosis codes for short-chain dehydrogenases/reductases (SDRs). These SDRs are nicotinamide adenine dinucleotide-reliant oxidoreductases involved in cell homeostasis. The precise function of oxidoreductases in bacteria especially M. tuberculosis were not fully explored. This study aimed to know the detail functional role of one of the oxidoreductase Rv0148 in M. tuberculosis.ResultsIn silico analysis revealed that Rv0148 interacts with Htdy (Rv3389) and the protein interactions were confirmed using far western blot. Gene knockout mutant of Rv0148 in M. tuberculosis was constructed by specialized transduction. Macrophage cell line infection with this knockout mutant showed increased expression of pro-inflammatory cytokines. This knockout mutant is sensitive to oxidative, nitrogen, redox and electron transport inhibitor stress agents. Drug susceptibility testing of the deletion mutant showed resistance to first-line drugs such as streptomycin and ethambutol and second-line aminoglycosides such as amikacin and kanamycin. Based on interactorme analysis for Rv0148 using STRING database, we identified 220 most probable interacting partners for Htdy protein. In the Rv0148 knockout mutants, high expression of htdy was observed and we hypothesize that this would have perturbed the interactome thus resulting in drug resistance. Finally, we propose that Rv0148 and Htdy are functionally interconnected and involved in drug resistance and cell homeostasis of M. tuberculosis.ConclusionsOur study suggests that Rv0148 plays a significant role in various functional aspects such as intermediatory metabolism, stress, homeostasis and also in drug resistance.

Biocatalytic Asymmetric Reduction of γ‐Keto Esters to Access Optically Active γ‐Aryl‐γ‐butyrolactones

AbstractAn efficient stereoselective syntheses of a series of functionalized optically active γ‐aryl‐γ‐butyrolactones is achieved by enzymatic asymmetric reduction of the corresponding sterically demanding γ‐keto esters employing wild‐type and recombinant alcohol dehydrogenases. The best stereoselectivities for the reduction via hydrogen transfer was obtained with two short chain dehydrogenases (SDRs) of complementary stereospecificity from Aromatoleum aromaticum, namely the Prelog‐specific NADH‐dependent (S)‐1‐phenylethanol dehydrogenase [(S)‐PED] and the anti‐Prelog‐specific (R)‐1‐(4‐hydroxyphenyl)‐ethanol dehydrogenase [(R)‐HPED], respectively. Biotransformations catalyzed by both enzymes, followed by TFA‐catalyzed cyclization of the resulting γ‐hydroxy esters, furnished the respective (S)‐ and (R)‐configured products with exquisite optical purity (up to &gt;99% ee). The synthetic value was demonstrated on preparative scale for the asymmetric bioreduction of the model compound, methyl 4‐oxo‐4‐phenylbutanoate, affording optically pure (S)‐γ‐phenyl‐γ‐butyrolactone (&gt;99% ee) in 67–74% isolated yield at 89–95% conversion depending on the applied scale.magnified image

Crystal structures of cyanobacterial light-dependent protochlorophyllide oxidoreductase

The reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is the penultimate step of chlorophyll biosynthesis. In oxygenic photosynthetic bacteria, algae, and plants, this reaction can be catalyzed by the light-dependent Pchlide oxidoreductase (LPOR), a member of the short-chain dehydrogenase superfamily sharing a conserved Rossmann fold for NAD(P)H binding and the catalytic activity. Whereas modeling and simulation approaches have been used to study the catalytic mechanism of this light-driven reaction, key details of the LPOR structure remain unclear. We determined the crystal structures of LPOR from two cyanobacteria, Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus Structural analysis defines the LPOR core fold, outlines the LPOR-NADPH interaction network, identifies the residues forming the substrate cavity and the proton-relay path, and reveals the role of the LPOR-specific loop. These findings provide a basis for understanding the structure-function relationships of the light-driven Pchlide reduction.

Altered proteome of a Burkholderia pseudomallei mutant defective in short-chain dehydrogenase affects cell adhesion, biofilm formation and heat stress tolerance.

Burkholderia pseudomallei is a Gram-negative bacillus that causes melioidosis and is recognized as an important public health problem in southeast Asia and northeast Australia. The treatment of B. pseudomallei infection is hampered by resistance to a wide range of antimicrobial agents and no vaccine is currently available. At present, the underlying mechanisms of B. pseudomallei pathogenesis are poorly understood. In our previous study, we reported that a B. pseudomallei short-chain dehydrogenase (SDO; BPSS2242) mutant constructed by deletion mutagenesis showed reduced B. pseudomallei invasion and initial intracellular survival. This indicated that SDO is associated with the pathogenesis of melioidosis. In the present study, the role of B. pseudomallei SDO was further investigated using the SDO deletion mutant by a proteomic approach. The protein profiles of the SDO mutant and wild-type K96243 were investigated through gel-based proteomic analysis. Quantitative intensity analysis of three individual cultures of the B. pseudomallei SDO mutant revealed significant down-regulation of five protein spots compared with the wild-type. Q-TOF MS/MS identified the protein spots as a glutamate/aspartate ABC transporter, prolyl-tRNA synthetase, Hsp70 family protein, quinone oxidoreductase and a putative carboxypeptidase. Functional assays were performed to investigate the role of these differentially expressed proteins in adhesion to host cells, biofilm induction and survival under heat stress conditions. The SDO deletion mutant showed a decreased ability to adhere to host cells. Moreover, biofilm formation and the survival rate of bacteria under heat stress conditions were also reduced in the mutant strain. Our findings provide insight into the role of SDO in the survival and pathogenesis of B. pseudomallei at the molecular level, which may be applied to the prevention and control of B. pseudomallei infection.

Biochemical analysis of a sugar 4,6‐dehydratase from Acanthamoeba polyphaga Mimivirus

The exciting discovery of the giant DNA Mimivirus in 2003 challenged the conventional description of viruses in a radical way, and since then, dozens of additional giant viruses have been identified. It has now been demonstrated that the Mimivirus genome encodes for the two enzymes required for the production of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, namely a 4,6-dehydratase and an aminotransferase. In light of our long-standing interest in the bacterial 4,6-dehydratases and in unusual sugars in general, we conducted a combined structural and functional analysis of the Mimivirus 4,6-dehydratase referred to as R141. For this investigation, the three-dimensional X-ray structure of R141 was determined to 2.05 Å resolution and refined to an R-factor of 18.3%. The overall fold of R141 places it into the short-chain dehydrogenase/reductase (SDR) superfamily of proteins. Whereas its molecular architecture is similar to that observed for the bacterial 4,6-dehydratases, there are two key regions where the polypeptide chain adopts different conformations. In particular, the conserved tyrosine that has been implicated as a catalytic acid or base in SDR superfamily members is splayed away from the active site by nearly 12 Å, thereby suggesting that a major conformational change must occur upon substrate binding. In addition to the structural analysis, the kinetic parameters for R141 using either dTDP-d-glucose or UDP-d-glucose as substrates were determined. Contrary to a previous report, R141 demonstrates nearly identical catalytic efficiency with either nucleotide-linked sugar. The data presented herein represent the first three-dimensional model for a viral 4,6-dehydratase and thus expands our understanding of these fascinating enzymes.

Gene family expansion of pinewood nematode to detoxify its host defence chemicals.

Gene gain/loss in the context of gene family dynamics plays an important role in evolutionary processes as organisms, particularly invasive species, adapt to new environments or niches. One notable example of this is the duplication of digestive proteases in some parasitic insects and helminths to meet nutritional requirements during animal parasitism. However, whether gene family expansion participates in the adaptation of a plant parasite nematode to its host remains unknown. Here, we compared the newly sequenced genomes of the pinewood nematode, Bursaphelenchus xylophilus, with the genomes of free-living, animal-parasitic and plant-parasitic nematodes. The results showed gene expansions occurring in 51 gene families in B. xylophilus, especially in xenobiotic detoxification pathways, including flavin monooxygenase (FMO), cytochrome P450 (CYP450), short chain dehydrogenase (SDR), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST). Although a majority of these expansions probably resulted from gene duplications, nine ADH genes were potentially acquired by horizontal gene transfer (HGT) from fungi. From the transcriptomes of B. xylophilus treated with pine saplings and terpenes, candidate xenobiotic detoxification genes were identified. We propose that host defence chemicals led to gene family expansions of xenobiotic detoxification pathways in B. xylophilus facilitating its survival in pine resin ducts. This study contributes to a better understanding of how a parasitic nematode adapts to its host.

Enzymatic synthesis of an orlistat intermediate using a mutant short-chain dehydrogenase from Novosphingobium aromaticivorans

Methyl (R)-3-hydroxytetradeconoate ((R)-MHOT) is a crucial chiral intermediate for the chemical synthesis of the anti-obesity drug, orlistat. Here, (R)-MHOT was prepared from methyl 3-oxotetradecanoate (MOT) using a mutant of the short-chain dehydrogenase/reductase (SDR) from Novosphingobium aromaticivorans (NaSDR). Mutant NaSDR-G145A/I199L had a 3.23 times greater kcat value than that of wild type toward MOT. The conditions for the expression of recombinant NaSDR-G145A/I199L were further investigated and obtained cells were used for gram-scale preparation of (R)-MHOT with 50 g/L of MOT. The target product was extracted and confirmed by gas chromatography; the enantiomeric excess value of (R)-MHOT was 99.0 %. Molecular docking analysis was used to reveal the molecular basis of the enhanced catalytic activity of NaSDR-G145A/I199L; NaSDR-G145A/I199L presented a more effective docking posture than NaSDR. This is the first reported use of SDR for preparing (R)-MHOT via the reduction of MOT. Our study provides a foundation for greener preparation of (R)-MHOT.

Metabolic engineering Saccharomyces cerevisiae for de novo production of the sesquiterpenoid (+)-nootkatone

Background(+)-Nootkatone is a highly valued sesquiterpenoid compound, exhibiting a typical grapefruit aroma and various desired biological activities for use as aromatics and pharmaceuticals. The high commercial demand of (+)-nootkatone is predominately met by chemical synthesis, which entails the use of environmentally harmful reagents. Efficient synthesis of (+)-nootkatone via biotechnological approaches is thus urgently needed to satisfy its industrial demand. However, there are only a limited number of studies that report the de novo synthesis of (+)-nootkatone from simple carbon sources in microbial cell factories, and with relatively low yield.ResultsAs the direct precursor of (+)-nootkatone biosynthesis, (+)-valencene was first produced in large quantities in Saccharomyces cerevisiae by overexpressing (+)-valencene synthase CnVS of Callitropsis nootkatensis in combination with various mevalonate pathway (MVA) engineering strategies, including the expression of CnVS and farnesyl diphosphate synthase (ERG20) as a fused protein, overexpression of a truncated form of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (tHMG1), and downregulating the squalene synthase enzyme (ERG9). These approaches altogether brought the production of (+)-valencene to 217.95 mg/L. Secondly, we addressed the (+)-valencene oxidation by overexpressing the Hyoscyamus muticus premnaspirodiene oxygenase (HPO) variant (V482I/A484I) and cytochrome P450 reductase (ATR1) from Arabidopsis thaliana. However, (+)-valencene was predominantly oxidized to β-nootkatol and only minor amounts of (+)-nootkatone (9.66 mg/L) were produced. We further tackled the oxidation of β-nootkatol to (+)-nootkatone by screening various dehydrogenases. Our results showed that the short-chain dehydrogenase/reductase (SDR) superfamily dehydrogenases ZSD1 of Zingiber zerumbet and ABA2 of Citrus sinensis were capable of effectively catalyzing β-nootkatol oxidation to (+)-nootkatone. The yield of (+)-nootkatone increased to 59.78 mg/L and 53.48 mg/L by additional overexpression of ZSD1 and ABA2, respectively.ConclusionWe successfully constructed the (+)-nootaktone biosynthesis pathway in S. cerevisiae by overexpressing the (+)-valencene synthase CnVS, cytochrome P450 monooxygenase HPO, and SDR family dehydrogenases combined with the MVA pathway engineering, providing a solid basis for the whole-cell production of (+)-nootkatone. The two effective SDR family dehydrogenases tested in this study will serve as valuable enzymatic tools in further optimizing (+)-nootkatone production.

An atypical short-chain dehydrogenase-reductase functions in the relaxation of photoprotective qH in Arabidopsis.

Photosynthetic organisms experience wide fluctuations in light intensity and regulate light harvesting accordingly to prevent damage from excess energy. The antenna quenching component qH is a sustained form of energy dissipation that protects the photosynthetic apparatus under stress conditions. This photoprotective mechanism requires the plastid lipocalin LCNP and is prevented by SUPPRESSOR OF QUENCHING1 (SOQ1) under non-stress conditions. However, the molecular mechanism of qH relaxation has yet to be resolved. Here, we isolated and characterized RELAXATION OF QH1 (ROQH1), an atypical short-chain dehydrogenase-reductase that functions as a qH-relaxation factor in Arabidopsis. The ROQH1 gene belongs to the GreenCut2 inventory specific to photosynthetic organisms, and the ROQH1 protein localizes to the chloroplast stroma lamellae membrane. After a cold and high-light treatment, qH does not relax in roqh1 mutants and qH does not occur in leaves overexpressing ROQH1. When the soq1 and roqh1 mutations are combined, qH can neither be prevented nor relaxed and soq1 roqh1 displays constitutive qH and light-limited growth. We propose that LCNP and ROQH1 perform dosage-dependent, antagonistic functions to protect the photosynthetic apparatus and maintain light-harvesting efficiency in plants.

Structural characterization of the Pseudomonas aeruginosa dehydrogenase AtuB involved in citronellol and geraniol catabolism

Pseudomonas aeruginosa can metabolize acyclic monoterpenoids (such as citronellol and geraniol) as the only carbon and energy sources. A total of seven proteins (AtuA, AtuB, AtuCF, AtuD, AtuE, AtuG, AtuH) have been identified in Pseudomonas aeruginosa as participating in the acyclic terpene utilization pathway. AtuB is a dehydrogenase enzyme responsible for citronellol and geraniol catabolism in the acyclic terpene utilization (Atu) pathway, although its structure and function have not been characterized to date. Here we report the crystal structure of AtuB from Pseudomonas aeruginosa PAO1 (PaAtuB) to 1.8 Å resolution. PaAtuB crystallizes in the space group F222 with a single monomer in the asymmetric unit. Analytical ultracentrifugation data shows that PaAtuB forms a stable tetramer in solution, which is consistent with the structure. Structural analysis confirms that AtuB belongs to the short-chain dehydrogenase/reductase (SDR) family. AtuB is predicted to bind NADP(H) from the crystal structure, which is confirmed by MicroScale Thermophoresis analysis that shows PaAtuB binds NADP(H) with a Kd value of 258 μM. This work provides a starting point to explore potential biotechnology and pharmaceutical applications of AtuB.

SdrA, an NADP(H)-regenerating enzyme, is crucial for Coxiella burnetii to resist oxidative stress and replicate intracellularly.

Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is a Gram-negative bacterium that replicates inside macrophages within a highly oxidative vacuole. Screening of a transposon mutant library suggested that sdrA, which encodes a putative short-chain dehydrogenase, is required for intracellular replication. Short-chain dehydrogenases are NADP(H)-dependent oxidoreductases, and SdrA contains a predicted NADP+ binding site, suggesting it may facilitate NADP(H) regeneration by C. burnetii, a key process for surviving oxidative stress. Purified recombinant 6×His-SdrA was able to convert NADP+ to NADP(H) in vitro. Mutation to alanine of a conserved glycine residue at position 12 within the predicted NADP binding site abolished significant enzymatic activity. Complementation of the sdrA mutant (sdrA::Tn) with plasmid-expressed SdrA restored intracellular replication to wild-type levels, but expressing enzymatically inactive G12A_SdrA did not. The sdrA::Tn mutant was more susceptible in vitro to oxidative stress, and treating infected host cells with L-ascorbate, an anti-oxidant, partially rescued the intracellular growth defect of sdrA::Tn. Finally, stable isotope labelling studies demonstrated a shift in flux through metabolic pathways in sdrA::Tn consistent with the presence of increased oxidative stress, and host cells infected with sdrA::Tn had elevated levels of reactive oxygen species compared with C. burnetii NMII.

Application of human organotypic skin raft cultures for analysis of retinoid metabolism, retinoic acid signaling, and screening of bioactive rexinoids.

Several human enzymes of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins exhibit catalytic oxidoreductive activity toward retinoid substrates in vitro. For some retinoid-active enzymes, their physiological significance for retinoid metabolism is supported by phenotypes linked to naturally occurring mutations in human genes or by targeted gene knockout studies of their murine homologs. However, for those enzymes that are not well conserved or display properties different from their murine counterparts, evaluation of their physiological roles can be challenging. Here, we describe the adaptation of stratified organotypic culture of human epidermis for evaluating the contribution of human putative SDR retinol dehydrogenases to biosynthesis of all-trans-retinoic acid in a three-dimensional cellular model highly sensitive to the levels of all-trans-retinol and all-trans-retinoic acid. In addition to providing a valuable readout of metabolic changes occurring in the presence or absence of the enzyme of interest, this model allows for evaluation of the effects of various retinoid and rexinoid therapeutic compounds on retinoic acid signaling, cell growth and differentiation.