Two new ene-reductases from photosynthetic extremophiles enlarge the panel of old yellow enzymes: CtOYE and GsOYE.

Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,ß-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl-cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis.

The higher plant plastid NAD(P)H dehydrogenase-like complex (NDH) is a high efficiency proton pump that increases ATP production by cyclic electron flow.

Cyclic electron flow around photosystem I (CEF) is critical for balancing the photosynthetic energy budget of the chloroplast by generating ATP without net production of NADPH. We demonstrate that the chloroplast NADPH dehydrogenase complex, a homolog to respiratory Complex I, pumps approximately two protons from the chloroplast stroma to the lumen per electron transferred from ferredoxin to plastoquinone, effectively increasing the efficiency of ATP production via CEF by 2-fold compared with CEF pathways involving non-proton-pumping plastoquinone reductases. By virtue of this proton-pumping stoichiometry, we hypothesize that NADPH dehydrogenase not only efficiently contributes to ATP production but operates near thermodynamic reversibility, with potentially important consequences for remediating mismatches in the thylakoid energy budget.

α,ß-Dicarbonyl reduction is mediated by the Saccharomyces Old Yellow Enzyme.

The undesirable flavor compounds diacetyl and 2,3-pentanedione are vicinal diketones (VDKs) formed by extracellular oxidative decarboxylation of intermediate metabolites of the isoleucine, leucine and valine (ILV) biosynthetic pathway. These VDKs are taken up by Saccharomyces and enzymatically converted to acetoin and 3-hydroxy-2-pentanone, respectively. Purification of a highly enriched diacetyl reductase fraction from Saccharomyces cerevisiae in conjunction with mass spectrometry identified Old Yellow Enzyme (Oye) as an enzyme capable of catalyzing VDK reduction. Kinetic analysis of recombinant Oye1p, Oye2p and Oye3p isoforms confirmed that all three isoforms reduced diacetyl and 2,3-pentanedione in an NADPH-dependent reaction. Transcriptomic analysis of S. cerevisiae (ale) and S. pastorianus (lager) yeast during industrial fermentations showed that the transcripts for OYE1, OYE2, arabinose dehydrogenase (ARA1), α-acetolactate synthase (ILV2) and α-acetohydroxyacid reductoisomerase (ILV5) were differentially regulated in a manner that correlated with changes in extracellular levels of VDKs. These studies provide insights into the mechanism for reducing VDKs and decreasing maturation times of beer which are of commercial importance.

Purification and characteristics of an inducible by polycyclic aromatic hydrocarbons NADP(+)-dependent naphthalenediol dehydrogenase (NDD) in Mucor circinelloides YR-1.

We detected NADP(+)-dependent dihydrodiol dehydrogenase (DD) activity in a cell-free extract from Mucor circinelloides YR-1, after high-speed centrifugation. We analyzed the enzymatic activity in the cytosolic fraction by zymograms, as described previously, and eight different DD activity bands were revealed. Five constitutive DD activities (DD1-5) were present when glucose was used as carbon source and three inducible activities (NDD, PDD1 and PDD2) when aromatic hydrocarbon compounds were used. NDD activity was induced all of the aromatic hydrocarbon compounds. The highest DD activity inducer was naphthalene and the lowest was pyrene. One of the enzymes showed higher activity with cis-naphthalene-diol rather than with trans-nahthalenediol as a substrate. We purified this particular enzyme to homogeneity and found that it had an isoelectric point of 4.6. The molecular weight for the native protein was 197.4kDa and 49.03±0.5kDa for the monomer that conforms it, suggesting a homotetrameric structure for the complete enzyme. Polyclonal antibodies were raised against it and obtained. NDD activity was almost totally inhibited when antibodies were used at low concentrations, and in native immunoblots only one band, which corresponds to the activity band detected in the zymograms, could be detected. In denaturing PAGE immunoblots only one band was detected. This band corresponds to the purified protein band of 49kDa detected in SDS-PAGE gels. The other two inducible enzymes PDD1 and PDD2 were present only when phenanthrene was used as sole carbon source in the culture media.

Structural characterization of a plant photosystem I and NAD(P)H dehydrogenase supercomplex.

Cyclic electron transport (CET) around photosystem I (PSI) plays an important role in balancing the ATP/NADPH ratio and the photoprotection of plants. The NAD(P)H dehydrogenase complex (NDH) has a key function in one of the CET pathways. Current knowledge indicates that, in order to fulfill its role in CET, the NDH complex needs to be associated with PSI; however, until now there has been no direct structural information about such a supercomplex. Here we present structural data obtained for a plant PSI-NDH supercomplex. Electron microscopy analysis revealed that in this supercomplex two copies of PSI are attached to one NDH complex. A constructed pseudo-atomic model indicates asymmetric binding of two PSI complexes to NDH and suggests that the low-abundant Lhca5 and Lhca6 subunits mediate the binding of one of the PSI complexes to NDH. On the basis of our structural data, we propose a model of electron transport in the PSI-NDH supercomplex in which the association of PSI to NDH seems to be important for efficient trapping of reduced ferredoxin by NDH.

Enzymatic characterization of an active NDH complex from Thermosynechococcus elongatus.

Although type-1 NAD(P)H dehydrogenase (NDH) complex subunit constituents and physiological functions have been reported in plants and cyanobacteria, the biochemical properties of this enzyme are not clear. We used chromatographic isolation to purify and characterize a NADPH-active NDH from the cyanobacterium Thermosynechococcus elongatus. Ferredoxin (Fd) and ferredoxin-NADP(+) oxidoreductase (FNR) were co-eluted with NDH, implying the electron donation from NADPH to NDH via the interaction with FNR. We investigated the enzymatic properties of the complex. Furthermore, the activity is competitively inhibited by rotenone, suggesting that it possesses a quinone binding site, similar to mitochondria complex I.

[The application of a "pixel method" for the quantitative assessment of the results of the histochemical studies].

The results of the application of a "pixel method" for the automated measurement of the intensity of histochemical reaction in the neurons are presented. The method is based on measuring principle, which includes the automatic counting the sum of brightnesses of all the pixels forming the image with the help of standard computer programs. The potential of this method is demonstrated on the example of NADPH-diaphorase activity study in the nitroxidergic neurons of sensory and motor nuclei of the medulla of healthy rats.

Membrane proteins in four acts: function precedes structure determination.

Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b(6)f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) ß-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B(12) binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a "fishing pole" model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to OmpF established the role of OmpF as an import channel for colicin nuclease cytotoxins. (IV) α-Synuclein, associated with the etiology of Parkinson's Disease, is an example of a protein, which is soluble and disordered in solution, but which can assume an ordered predominantly α-helical conformation upon binding to membranes. When subjected in its membrane-bound form to a trans-membrane electrical potential, α-synuclein can form voltage-gated ion channels. Summary of methods to assay functions/activities: (i) sensitive spectrophotometric assay to measure electron transfer activities; (ii) hydrophobic chromatography to deplete lipids, allowing reconstitution with specific lipids for studies on lipid-protein interactions; (iii) microbiological screen to assay high affinity binding of colicin receptor domains to Escherichia coli outer membrane receptors; (iv) electrophysiology/channel analysis (a) to select channel-occluding ligands for co-crystallization with ion channels of OmpF, and (b) to provide a unique description of voltage-gated ion channels of α-synuclein.

4-dihydrotrisporin-dehydrogenase, an enzyme of the sex hormone pathway of Mucor mucedo: purification, cloning of the corresponding gene, and developmental expression.

The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (-) mating-type-specific enzyme in the pathway from beta-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (-) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (-) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation.

Subfunctionality of hydride transferases of the old yellow enzyme family of flavoproteins of Pseudomonas putida.

To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo.