Explore the vast range of titles available.

Flavonoids are potent antioxidants that play a role in defense against pathogens, UV-radiation, and the detoxification of reactive oxygen species. Dihydroflavonol 4-reductase (DFR) and flavanone 4-reductase (FNR) reduce dihydroflavonols and flavanones, respectively, using NAD(P)H to produce flavan-(3)-4-(di)ols in flavonoid biosynthesis. Anthocyanidin reductase (ANR) reduces anthocyanidins to flavan-3-ols. In addition to their sequences, the 3D structures of recombinant DFR, FNR and ANR from sorghum and switchgrass showed a high level of similarity. The catalytic mechanism, substrate-specificity and key residues of three reductases were deduced from crystal structures, site-directed mutagenesis, molecular docking, kinetics, and thermodynamic ana-lyses. Although DFR displayed its highest activity against dihydroflavonols, it also showed activity against flavanones and anthocyanidins. It was inhibited by the flavonol quercetin and high concentrations of dihydroflavonols/flavonones. SbFNR1 and SbFNR2 did not show any activity against dihydroflavonols. However, SbFNR1 displayed activity against flavanones and ANR activity against two anthocyanidins, cyanidin and pelargonidin. Therefore, SbFNR1 and SbFNR2 could be specific ANR isozymes without delphinidin activity. Sorghum has high concentrations of 3-deoxyanthocyanidins in vivo, supporting the observed high activity of SbDFR against flavonols. Mining of expression data indicated substantial induction of these three reductase genes in both switchgrass and sorghum in response to biotic stress. Key signature sequences for proper DFR/ANR classification are proposed and could form the basis for future metabolic engineering of flavonoid metabolism.

2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.

There is significant contemporary interest in the application of enzymes to replace or augment chemical reagents toward the development of more environmentally sound and sustainable processes. In particular, copper radical oxidases (CRO) from Auxiliary Activity Family 5 Subfamily 2 (AA5_2) are attractive, organic cofactor-free catalysts for the chemoselective oxidation of alcohols to the corresponding aldehydes. These enzymes were first defined by the archetypal galactose-6-oxidase (GalOx, EC 1.1.3.13) from the fungus Fusarium graminearum. The recent discovery of specific alcohol oxidases (EC 1.1.3.7) and aryl alcohol oxidases (EC 1.1.3.47) within AA5_2 has indicated a potentially broad substrate scope among fungal CROs. However, only relatively few AA5_2 members have been characterized to date. Guided by sequence similarity network and phylogenetic analysis, twelve AA5_2 homologs have been recombinantly produced and biochemically characterized in the present study. As defined by their predominant activities, these comprise four galactose 6-oxidases, two raffinose oxidases, four broad-specificity primary alcohol oxidases, and two non-carbohydrate alcohol oxidases. Of particular relevance to applications in biomass valorization, detailed product analysis revealed that two CROs produce the bioplastics monomer furan-2,5-dicarboxylic acid (FDCA) directly from 5-hydroxymethylfurfural (HMF). Furthermore, several CROs could desymmetrize glycerol (a by-product of the biodiesel industry) to d- or l-glyceraldehyde. This study furthers our understanding of CROs by doubling the number of characterized AA5_2 members, which may find future applications as biocatalysts in diverse processes.

The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4-to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates.

Dioxygen (O₂) and other gas molecules have a fundamental role in a variety of enzymatic reactions. However, it is only poorly understood which O₂ uptake mechanism enzymes employ to promote efficient catalysis and how general this is. We investigated O₂ diffusion pathways into monooxygenase and oxidase flavoenzymes, using an integrated computational and experimental approach. Enhanced-statistics molecular dynamics simulations reveal spontaneous protein-guided O₂ diffusion from the bulk solvent to preorganized protein cavities. The predicted protein-guided diffusion paths and the importance of key cavity residues for oxygen diffusion were verified by combining site-directed mutagenesis, rapid kinetics experiments, and high-resolution X-ray structures. This study indicates that monooxygenase and oxidase flavoenzymes employ multiple funnel-shaped diffusion pathways to absorb O₂ from the solvent and direct it to the reacting C4a atom of the flavin cofactor. The difference in O₂ reactivity among dehydrogenases, monooxygenases, and oxidases ultimately resides in the fine modulation of the local environment embedding the reactive locus of the flavin.

▪ Abstract  The type II fatty acid synthetic pathway is the principal route for the production of membrane phospholipid acyl chains in bacteria and plants. The reaction sequence is carried out by a series of individual soluble proteins that are each encoded by a discrete gene, and the pathway intermediates are shuttled between the enzymes as thioesters of an acyl carrier protein. The Escherichia coli system is the paradigm for the study of this system, and high-resolution X-ray and/or NMR structures of representative members of every enzyme in the type II pathway are now available. The structural biology of these proteins reveals the specific three-dimensional features of the enzymes that explain substrate recognition, chain length specificity, and the catalytic mechanisms that define their roles in producing the multitude of products generated by the type II system. These structures are also a valuable resource to guide antibacterial drug discovery.

Aliphatic ketones, such as 2-butanone and 3-hexanone, with only one carbon difference among side chains adjacent to the carbonyl carbon are difficult to be reduced enantioselectively. In this study, we utilized an acetophenone reductase from Geotrichum candidum NBRC 4597 ( Gc APRD) to reduce challenging aliphatic ketones such as 2-butanone (methyl ethyl ketone) and 3-hexanone (ethyl propyl ketone) to their corresponding ( S )-alcohols with 94% ee and > 99% ee , respectively. Through crystallographic structure determination, it was suggested that residue Trp288 limit the size of the small binding pocket. Docking simulations imply that Trp288 plays an important role to form a C-H⋯π interaction for proper orientation of ketones in the pro- S binding pose in order to produce ( S )-alcohols. The excellent ( S )-enantioselectivity is due to a non-productive pro- R binding pose, consistent with the observation that the ( R )-alcohol acts as an inhibitor of ( S )-alcohol oxidation.

3-Hydroxypropionic acid (3-HP) can be produced via the biological route involving two enzymatic reactions: dehydration of glycerol to 3-hydroxypropanal (3-HPA) and then oxidation to 3-HP. However, commercial production of 3-HP using recombinant microorganisms has been hampered with several problems, some of which are associated with the toxicity of 3-HPA and the efficiency of NAD + regeneration. We engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for the second reaction to address these issues. The residues in the binding sites for the substrates, 3-HPA and NAD + , were randomized, and the resulting libraries were screened for higher activity. Isolated KGSADH variants had significantly lower K m values for both the substrates. The enzymes also showed higher substrate specificities for aldehyde and NAD + , less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain with one of the engineered KGSADH variants exhibited less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that with the wild-type KGSADH. The flask culture of the P. denitrificans strain with the mutant KGSADH resulted in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM).

Lipid droplets (LDs) have emerged as dynamic and interactive organelles with important roles in lipid metabolism and membrane biogenesis. Here, we report that Saccharomyces cerevisiae Env9 is a novel conserved oxidoreductase involved in LD morphology. Microscopic and biochemical studies confirm localization of tagged Env9 to LDs and implicate its C-terminal hydrophobic domain (aa241–265) in its membrane association and stability. Confocal studies reveal a role for Env9 in LD morphology. Env9 positively affects both formation of large LDs upon overexpression and LD proliferation under poor carbon source. In silico bioinformatic and modeling approaches establish that ENV9 is a widely conserved member of the short-chain dehydrogenase (SDR) superfamily. Bayesian phylogenetic studies strongly support ENV9 as an ortholog of human SDR retinol dehydrogenase 12 ( RDH12 ). Dehydrogenase activity of Env9 was confirmed by in vitro oxidoreductase assays. RDH12 mutations have been linked to Leber Congenital Amaurosis. Similar site-directed point mutations in the predicted Env9 oxidoreductase active site (N146L) or cofactor-binding site (G23–24A) abolished its reductase activity in vitro, consistent with those reported in other retinol dehydrogenases. The same residues were essential for affecting LD size and number in vivo. Taken together, our results implicate oxidoreductase activity of Env9 in its cellular role in LD morphology.

Marine algae have unique defense strategies against microbial infection. However, their mechanisms of immunity remain to be elucidated and little is known about the similarity of the immune systems of marine algae and terrestrial higher plants. Here, we suggest a possible mechanism underlying algal immunity, which involves hexose oxidase (HOX)-dependent production of hydrogen peroxide (H2O2). We examined crude extracts from five different red algal species for their ability to prevent bacterial growth. The extract from one of these algae, Ptilophora subcostata, was particularly active and prevented the growth of gram-positive and -negative bacteria, which was completely inhibited by treatment with catalase. The extract did not affect the growth of either a yeast or a filamentous fungus. We partially purified from P. subcostata an enzyme involved in its antibacterial activity, which shared 50% homology with the HOX of red seaweed Chondrus crispus. In-gel carbohydrate oxidase assays revealed that P. subcostata extract had the ability to produce H2O2 in a hexose-dependent manner and this activity was highest in the presence of galactose. In addition, Bacillus subtilis growth was strongly suppressed near P. subcostata algal fronds on GYP agar plates. These results suggest that HOX plays a role in P. subcostata resistance to bacterial attack by mediating H2O2 production in the marine environment.