Structural and Computational Bases for Dramatic Skeletal Rearrangement in Anditomin Biosynthesis.

AndA, an Fe(II)/α-ketoglutarate (αKG)-dependent enzyme, is the key enzyme that constructs the unique and congested bridged-ring system of anditomin (1), by catalyzing consecutive dehydrogenation and isomerization reactions. Although we previously characterized AndA to some extent, the means by which the enzyme facilitates this drastic structural reconstruction have remained elusive. In this study, we have solved three X-ray crystal structures of AndA, in its apo form and in the complexes with Fe(II), αKG, and two substrates. The crystal structures and mutational experiments identified several key amino acid residues important for the catalysis and provided insight into how AndA controls the reaction. Furthermore, computational calculations validated the proposed reaction mechanism for the bridged-ring formation and also revealed the requirement of a series of conformational changes during the transformation.

Structural basis of enzymatic benzene ring reduction.

In chemical synthesis, the widely used Birch reduction of aromatic compounds to cyclic dienes requires alkali metals in ammonia as extremely low-potential electron donors. An analogous reaction is catalyzed by benzoyl-coenzyme A reductases (BCRs) that have a key role in the globally important bacterial degradation of aromatic compounds at anoxic sites. Because of the lack of structural information, the catalytic mechanism of enzymatic benzene ring reduction remained obscure. Here, we present the structural characterization of a dearomatizing BCR containing an unprecedented tungsten cofactor that transfers electrons to the benzene ring in an aprotic cavity. Substrate binding induces proton transfer from the bulk solvent to the active site by expelling a Zn(2+) that is crucial for active site encapsulation. Our results shed light on the structural basis of an electron transfer process at the negative redox potential limit in biology. They open the door for biological or biomimetic alternatives to a basic chemical synthetic tool.

A combined approach for enhancing the stability of recombinant cis-dihydrodiol naphthalene dehydrogenase from Pseudomonas putida G7 allowed for the structural and kinetic characterization of the enzyme.

The second enzyme of the naphthalene degradation pathway in Pseudomonas putida G7 is NahB, a dehydrogenase that converts cis-1,2-dihydroxy-1,2-dihydronaphthalene to 1,2-dihydroxynaphthalene. We report the cloning, optimization of expression, purification, kinetic studies and preliminary structural characterization of the recombinant NahB. The nahB gene was cloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli Rosetta (DE3) as an N-terminal hexa-histidine-tagged protein (6xHis-NahB). Using methods of enhancing protein stability in solution, we tested different expression, cell lysis, and purification protocols with and without ligand supplementation. The protein stability was evaluated by dynamic light scattering and circular dichroism spectroscopy assays. Best-derived protocols (expression at 18 °C, cell lysis with homogenizer, and three purification steps) were used to produce 20 mg of homogeneous 6xHis-NahB per liter of culture. The secondary and quaternary structures of 6xHis-NahB were assessed by circular dichroism and size-exclusion chromatography experiments, respectively. The enzyme was NAD+-dependent and active at pH 7.0 and 9.4 for the oxidation of the substrate. The Michaelis-Menten parameters determined at pH 7.0 and 25 °C for the substrate and cofactor, presented respective Km values of 6 and 350 µM, and a kcat value of 8.3 s-1. Furthermore, we identified conditions for the crystallization of 6xHis-NahB. X-ray diffraction data were collected from a single 6xHis-NahB crystal which diffracted to 2.21 Å. The crystal belongs to space group I222, with unit-cell parameters a = 63.62, b = 69.50, and c = 117.47 Å. The tertiary structure of 6xHis-NahB was determined using the molecular replacement method. Further structural refinement is currently underway.

Mining Novel Allergens from Coconut Pollen Employing Manual De Novo Sequencing and Homology-Driven Proteomics.

Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish "b" ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its in vitro allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins.

Identification, purification and characterization of furfural transforming enzymes from Clostridium beijerinckii NCIMB 8052.

Generation of microbial inhibitory compounds such as furfural and 5-hydroxymethylfurfural (HMF) is a formidable roadblock to fermentation of lignocellulose-derived sugars to butanol. Bioabatement offers a cost effective strategy to circumvent this challenge. Although Clostridium beijerinckii NCIMB 8052 can transform 2-3 g/L of furfural and HMF to their less toxic alcohols, higher concentrations present in biomass hydrolysates are intractable to microbial transformation. To delineate the mechanism by which C. beijerinckii detoxifies furfural and HMF, an aldo/keto reductase (AKR) and a short-chain dehydrogenase/reductase (SDR) found to be over-expressed in furfural-challenged cultures of C. beijerinckii were cloned and over-expressed in Escherichia coli Rosetta-gami™ B(DE3)pLysS, and purified by histidine tag-assisted immobilized metal affinity chromatography. Protein gel analysis showed that the molecular weights of purified AKR and SDR are close to the predicted values of 37 kDa and 27 kDa, respectively. While AKR has apparent Km and Vmax values of 32.4 mM and 254.2 mM s(-1) respectively, using furfural as substrate, SDR showed lower Km (26.4 mM) and Vmax (22.6 mM s(-1)) values on the same substrate. However, AKR showed 7.1-fold higher specific activity on furfural than SDR. Further, both AKR and SDR were found to be active on HMF, benzaldehyde, and butyraldehyde. Both enzymes require NADPH as a cofactor for aldehydes reduction. Based on these results, it is proposed that AKR and SDR are involved in the biotransformation of furfural and HMF by C. beijerinckii.

An enoate reductase Achr-OYE4 from Achromobacter sp. JA81: characterization and application in asymmetric bioreduction of C=C bonds.

A putative enoate reductase, Achr-OYE4, was mined from the genome of Achromobacter sp. JA81, expressed in Escherichia coli, and was characterized. Sequence analysis and spectral properties indicated that Achr-OYE4 is a typical flavin mononucleotide-dependent protein; it preferred NADH over NADPH as a cofactor. The heterologously expressed protein displayed good activity and excellent stereoselectivity toward some activated alkenes in the presence of NADH, NADPH, or their recycling systems. The glucose dehydrogenase-based recycling system yielded the best results in most cases, with a product yield of up to 99 % and enantiopurity of >99 % ee. Achr-OYE4 is an important addition to the asymmetric reduction reservoir as an "old yellow enzyme" from Achromobacter.

Characterization of BciB: a ferredoxin-dependent 8-vinyl-protochlorophyllide reductase from the green sulfur bacterium Chloroherpeton thalassium.

Two enzymes, BciA and BciB, are known to reduce the C-8 vinyl group of 8-vinyl protochlorophyllide, producing protochlorophyllide a, during the synthesis of chlorophylls and bacteriochlorophylls in chlorophototrophic bacteria. BciA from the green sulfur bacterium Chlorobaculum tepidum reduces the C-8 vinyl group using NADPH as the reductant. Cyanobacteria and some other chlorophototrophs have a second, nonhomologous type of 8-vinyl reductase, BciB, but the biochemical properties of this enzyme have not yet been described. In this study, the bciB gene of the green sulfur bacterium Chloroherpeton thalassium was expressed in Escherichia coli , and the recombinant protein was purified and characterized. Recombinant BciB binds a flavin adenine dinucleotide cofactor, and EPR spectroscopy as well as quantitative analyses of bound iron and sulfide suggest that BciB binds two [4Fe-4S] clusters, one of which may not be essential for the activity of the enzyme. Using electrons provided by reduced ferredoxin or dithionite, recombinant BciB was active and reduced the 8-vinyl moiety of the substrate, 8-vinyl protochlorophyllide, producing protochlorophyllide a. A structural model for BciB based on a recent structure for the FrhB subunit of F420-reducing [NiFe]-hydrogenase of Methanothermobacter marburgensis is proposed. Possible reasons for the occurrence and distribution of BciA and BciB among various chlorophototrophs are discussed.

Purification, crystallization and preliminary X-ray diffraction analysis of enoyl-acyl carrier protein reductase (FabK) from Streptococcus mutans strain UA159.

A triclosan-resistant flavoprotein termed FabK is the sole enoyl-acyl carrier protein reductase in Streptococcus pneumoniae and Streptococcus mutans. In this study, FabK from S. mutans strain UA159 was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.40 Å resolution using a synchrotron-radiation source. The crystal belonged to space group P6(2), with unit-cell parameters a = b = 105.79, c = 44.15 Å. The asymmetric unit contained one molecule, with a corresponding V(M) of 2.05 Å(3) Da(-1) and a solvent content of 39.9%.

Bilirubin oxidase from Magnaporthe oryzae: an attractive new enzyme for biotechnological applications.

A novel bilirubin oxidase (BOD), from the rice blast fungus Magnaporthe oryzae, has been identified and isolated. The 64-kDa protein containing four coppers was successfully overexpressed in Pichia pastoris and purified to homogeneity in one step. Protein yield is more than 100 mg for 2 L culture, twice that of Myrothecium verrucaria. The k(cat)/K(m) ratio for conjugated bilirubin (1,513 mM⁻¹ s⁻¹) is higher than that obtained for the BOD from M. verrucaria expressed in native fungus (980 mM⁻¹ s⁻¹), with the lowest K(m) measured for any BOD highly desirable for detection of bilirubin in medical samples. In addition, this protein exhibits a half-life for deactivation >300 min at 37 °C, high stability at pH 7, and high tolerance towards urea, making it an ideal candidate for the elaboration of biofuel cells, powering implantable medical devices. Finally, this new BOD is efficient in decolorizing textile dyes such as Remazol brilliant Blue R, making it useful for environmentally friendly industrial applications.

Expression, purification and preliminary X-ray crystallographic analysis of cyanobacterial biliverdin reductase.

Biliverdin reductase (BVR) catalyzes the conversion of biliverdin IX α to bilirubin IX α with concomitant oxidation of an NADH or NADPH cofactor. This enzyme also binds DNA and enhances the transcription of specific genes. Recombinant cyanobacterial BVR was overexpressed in Escherichia coli, purified and crystallized. A native data set was collected to 2.34 Šresolution on beamline BL38B1 at SPring-8. An SeMet data set was collected from a microcrystal (300×10×10 µm) on the RIKEN targeted protein beamline BL32XU and diffraction spots were obtained to 3.0 Šresolution. The native BVR crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=58.8, b=88.4, c=132.6 Å. Assuming that two molecules are present in the asymmetric unit, VM (the Matthews coefficient) was calculated to be 2.37 Å3 Da(-1) and the solvent content was estimated to be 48.1%. The structure of cyanobacterial BVR may provide insights into the mechanisms of its enzymatic and physiological functions.

Enzymes of naphthalene metabolism by Pseudomonas fluorescens 26K strain.

The ability of Pseudomonas fluorescens 26K strain to utilize naphthalene at concentrations up to 600 mg/liter as the sole source of carbon and energy in mineral liquid media was shown. Using HPLC, TLC, and mass-spectrometry, the intermediates of naphthalene transformation by this strain were identified as naphthalene cis-1,2-dihydrodiol, salicylaldehyde, salicylate, catechol, 2-hydroxymuconic semialdehyde, and 1-naphthol. Catechol 2,3-dioxygenase (a homotetramer with native molecular mass 125 kDa) and NAD+-dependent homohexameric naphthalene cis-1,2-dihydrodiol dehydrogenase with native molecular mass 160 kDa were purified from crude extract of the strain and characterized. NAD+-dependent homodimeric salicylaldehyde dehydrogenase with molecular mass 110 kDa was purified and characterized for the first time. Based on the data, a pathway of naphthalene degradation by P. fluorescens 26K is suggested.

Structure, dynamics and function of the evolutionarily changing biliverdin reductase B family.

Biliverdin reductase B (BLVRB) family members are general flavin reductases critical in maintaining cellular redox with recent findings revealing that BLVRB alone can dictate cellular fate. However, as opposed to most enzymes, the BLVRB family remains enigmatic with an evolutionarily changing active site and unknown structural and functional consequences. Here, we applied a multi-faceted approach that combines X-ray crystallography, NMR and kinetics methods to elucidate the structural and functional basis of the evolutionarily changing BLVRB active site. Using a panel of three BLVRB isoforms (human, lemur and hyrax) and multiple human BLVRB mutants, our studies reveal a novel evolutionary mechanism where coenzyme 'clamps' formed by arginine side chains at two co-evolving positions within the active site serve to slow coenzyme release (Positions 14 and 78). We find that coenzyme release is further slowed by the weaker binding substrate, resulting in relatively slow turnover numbers. However, different BLVRB active sites imposed by either evolution or mutagenesis exhibit a surprising inverse relationship between coenzyme release and substrate turnover that is independent of the faster chemical step of hydride transfer also measured here. Collectively, our studies have elucidated the role of the evolutionarily changing BLVRB active site that serves to modulate coenzyme release and has revealed that coenzyme release is coupled to substrate turnover.

b-type dihydroorotate dehydrogenase is purified as a H2O2-forming NADH oxidase from Bifidobacterium bifidum.

Our previous report showed the existence of microaerophilic Bifidobacterium species that can grow well under aerobic conditions rather than anoxic conditions in a liquid shaking culture. The difference in the aerobic growth properties between the O(2)-sensitive and microaerophilic species is due to the existence of a system to produce H(2)O(2) in the growth medium. In this study, we purified and characterized the NADH oxidase that is considered to be a key enzyme in the production of H(2)O(2). Bifidobacterium bifidum, an O(2)-sensitive bacterium and the type species of the genus Bifidobacterium, possessed one dominant active fraction of NADH oxidase and a minor active fraction of NAD(P)H oxidase activity detected in the first step of column chromatography for purification of the enzyme. The dominant active fraction was further purified and determined from its N-terminal sequence to be a homologue of b-type dihydroorotate dehydrogenase (DHOD), composed of PyrK (31 kDa) and PyrDb (34 kDa) subunits. The genes that encode PyrK and PryDb are tandemly located within an operon structure. The purified enzyme was found to be a heterotetramer showing the typical spectrum of a flavoprotein, and flavin mononucleotide and flavin adenine dinucleotide were identified as cofactors. The purified enzyme was characterized as the enzyme that catalyzes the DHOD reaction and also catalyzes a H(2)O(2)-forming NADH oxidase reaction in the presence of O(2). The kinetic parameters suggested that the enzyme could be involved in H(2)O(2) production in highly aerated environments.

Factors accelerating pyrimidine production in Deinococcus radiophilus.

In studying the pyrimidine synthesising pathway in Deinococcus radiophilus two instances of anomalous behaviour were observed. One was the strikingly different results obtained for two types of assay for carbamoyl phosphate synthetase. Both depend on the fixation of 14C from the substrate bicarbonate to give radioactive products. In the coupled assay the carbamoyl phosphate product of the enzyme is converted to carbamoyl aspartate in the presence of aspartate and aspartate transcarbamoylase. In the direct assay aspartate is omitted from the reaction mixture and the carbamoyl phosphate is converted to urea. It was found that the radioactive counts in the direct assay were about 5% of those measured in the coupled assay. The second anomaly was that omission of glutamine from both assay mixtures had no significant effect on the fixation of radioactive carbon. These results suggested that aspartate amino-N could be the source of nitrogen for glutamine synthesis by a substrate-channelled pathway which delivered glutamine to carbamoyl phosphate synthetase, and that externally added glutamine could not access its binding site on the enzyme.

Expression, purification, crystallization and preliminary X-ray analysis of maleylacetate reductase from Burkholderia sp. strain SJ98.

Maleylacetate reductase (EC 1.3.1.32) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 A. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 A resolution, respectively.

Purification and characteristics of an enzyme with both bilirubin oxidase and laccase activities from mycelium of the basidiomycete Pleurotus ostreatus.

A homogenous enzyme with both bilirubin oxidase and laccase activities was isolated from a submerged culture of the basidiomycete Pleurotus ostreatus mycelium and characterized. The yield of the enzyme was 127 microg/g dry biomass of the mycelium. The specific activity of the enzyme was 21 and 261 U/mg to bilirubin and to a laccase substrate ABTS, respectively. The intracellular phenol oxidase from the P. ostreatus mycelium was identified as bilirubin oxidase with the amino acid sequence highly homologous to that of the pox2 gene-encoded product. The enzyme displayed the maximal laccase activity at 50-55 degrees C to all substrates examined, whereas the pH optimum was substrate-dependent and changed from 3.0 for ABTS to 7.0 for syringaldazine and guaiacol. The enzyme maintained catalytic activity within a broad pH range but was inactivated at pH 4.0. The enzyme was thermostable but very sensitive to metal chelating inhibitors. Trypan Blue (5 mg/liter) was completely decolorizated upon 3 h of incubation with the bilirubin oxidase (20 mU/ml) at room temperature.

Cloning, overexpression, purification, and characterization of the maleylacetate reductase from Sphingobium chlorophenolicum strain ATCC 53874.

The final enzyme in the pentachlorophenol (PCP) biodegradation pathway in Sphingobium chlorophenolicum is maleylacetate reductase (PcpE), which catalyzes the reductive dehalogenation of 2-chloromaleylacetate to maleylacetate and the subsequent reduction of malyelacetate to 3-oxoadipate. In this study, the pcpE gene was cloned from S. chlorophenolicum strain ATCC 53874 and overexpressed in Escherichia coli BL21-AI cells. The recombinant PcpE, purified to higher than 95% purity using affinity chromatography, exhibited optimal activity at pH 7.0. The kinetic parameters k(cat) and K(m) were 1.2 +/- 0.3 s(-1) and 0.09 +/- 0.04 mM, respectively, against maleylacetate under the optimal pH. In addition, the purified PcpE was able to restore PCP-degrading capability to S. chlorophenolicum strain ATCC 39723, implicating that there was no functional PcpE in the ATCC 39723 strain.

Crystallization and preliminary X-ray diffraction studies of maleylacetate reductase from Rhizobium sp. strain MTP-10005.

Maleylacetate reductase (EC 1.3.1.32), which catalyzes the reduction of maleylacetate to 3-oxoadipate, plays an important role in the aerobic microbial catabolism of resorcinol. The enzyme has been crystallized at 293 K by the sitting-drop vapour-diffusion method supplemented with a microseeding technique, using ammonium sulfate as the precipitating agent. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 56.85, b = 121.13, c = 94.09 A, beta = 101.48 degrees , and contained one dimeric molecule in the asymmetric unit. It diffracted to 1.79 A resolution.

Isolation and characterization of a Delta5-desaturase from Oblongichytrium sp.

We isolated a cDNA clone with homology to known desaturase genes from Oblongichytrium sp., recently classified as a new genus of thraustochytrids (Labyrinthulomycetes), and found that it encoded Delta5-desaturase by its heterologous expression in yeast. The enzyme had higher activity toward 20:4n-3 than 20:3n-6, indicating that this Delta5-desaturase can be used in the production of n-3 polyunsaturated fatty acids in transgenic organisms.

Crystallization and preliminary X-ray diffraction analysis of Leishmania major dihydroorotate dehydrogenase.

Dihydroorotate dehydrogenases (DHODHs) are flavin-containing enzymes that catalyze the oxidation of L-dihydroorotate to orotate, the fourth step in the de novo pyrimidine nucleotide synthesis pathway. In this study, DHODH from Leishmania major has been crystallized by the vapour-diffusion technique using lithium sulfate as the precipitating agent. The crystals belong to space group P6(1), with unit-cell parameters a = 143.7, c = 69.8 A. X-ray diffraction data were collected to 2.0 A resolution using an in-house rotating-anode generator. Analysis of the solvent content and the self-rotation function indicate the presence of two molecules in the asymmetric unit. The structure has been solved by the molecular-replacement technique.