A novel multifunctional GH9 enzyme from Paenibacillus curdlanolyticus B-6 exhibiting endo/exo functions of cellulase, mannanase and xylanase activities.

PcMulGH9, a novel glycoside hydrolase family 9 (GH9) from Paenibacillus curdlanolyticus B-6, was successfully expressed in Escherichia coli. It is composed of a catalytic domain of GH9, two domains of carbohydrate-binding module family 3 (CBM3) and two domains of fibronectin type 3 (Fn3). The PcMulGH9 enzyme showed broad activity towards the ß-1,4 glycosidic linkages of cellulose, mannan and xylan, including cellulose and xylan contained in lignocellulosic biomass, which is rarely found in GH9. The enzyme hydrolysed substrates with bifunctional endo-/exotypes cellulase, mannanase and xylanase activities, but predominantly exhibited exo-activities. This enzyme released cellobiose as a major product from cellohexaose, while mannotriose and xylotriose were major hydrolysis products from mannohexaose and xylohexaose, respectively. Moreover, PcMulGH9 could hydrolyse untreated corn hull and rice straw into xylo- and cello-oligosaccharides. Enzyme kinetics, site-directed mutagenesis and molecular docking revealed that Met394, located at the binding subsite + 2, was involved in broad substrate specificity of PcMulGH9 enzyme. This study offers new knowledge of the multifunctional cellulase/mannanase/xylanase in GH9. The PcMulGH9 enzyme showed a novel function of GH9, which increases its potential for saccharification of lignocellulosic biomass into value-added products, especially oligosaccharides.

Convergent Cascade Catalyzed by Monooxygenase-Alcohol Dehydrogenase Fusion Applied in Organic Media.

With the aim of applying redox-neutral cascade reactions in organic media, fusions of a type II flavin-containing monooxygenase (FMO-E) and horse liver alcohol dehydrogenase (HLADH) were designed. The enzyme orientation and expression vector were found to influence the overall fusion enzyme activity. The resulting bifunctional enzyme retained the catalytic properties of both individual enzymes. The lyophilized cell-free extract containing the bifunctional enzyme was applied for the convergent cascade reaction consisting of cyclobutanone and butane-1,4-diol in different microaqueous media with only 5 % (v/v) aqueous buffer without any addition of external cofactor. Methyl tert-butyl ether and cyclopentyl methyl ether were found to be the best organic media for the synthesis of γ-butyrolactone, resulting in about 27 % analytical yield.

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.

Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol.

Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6-6.5, low salt concentrations and 3 mM Mg2+ or 0.3-3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37ºC. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.

Biochemical characterization of a bifunctional acetaldehyde-alcohol dehydrogenase purified from a facultative anaerobic bacterium Citrobacter sp. S-77.

Acetaldehyde-alcohol dehydrogenase (ADHE) is a bifunctional enzyme consisting of two domains of an N-terminal acetaldehyde dehydrogenase (ALDH) and a C-terminal alcohol dehydrogenase (ADH). The enzyme is known to be important in the cellular alcohol metabolism. However, the role of coenzyme A-acylating ADHE responsible for ethanol production from acetyl-CoA remains uncertain. Here, we present the purification and biochemical characterization of an ADHE from Citrobacter sp. S-77 (ADHE(S77)). Interestingly, the ADHE(S77) was unable to be solubilized from membrane with detergents either 1% Triton X-100 or 1% Sulfobetaine 3-12. However, the enzyme was easily dissociated from membrane by high-salt buffers containing either 1.0 M NaCl or (NH(4))(2)SO(4) without detergents. The molecular weight of a native protein was estimated as approximately 400 kDa, consisting of four identical subunits of 96.3 kDa. Based on the specific activity and kinetic analysis, the ADHES77 tended to have catalytic reaction towards acetaldehyde elimination rather than acetaldehyde formation. Our experimental observation suggests that the ADHES77 may play a pivotal role in modulating intracellular acetaldehyde concentration.

Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases from Thermotoga maritima and Thermotoga hypogea.

Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

Expression, crystallization and preliminary X-ray analysis of McbB, a multifunctional enzyme involved in ß-carboline skeleton biosynthesis.

ß-Carboline alkaloids (ßCs), with tricyclic pyrido[3,4-b]indole rings, have important pharmacological and therapeutic value. In the biosynthesis of ßCs, the Pictet-Spengler (PS) cyclization reaction is responsible for the formation of ring structures. McbB is one of a few enzymes that are known to catalyse PS cyclization. It can also catalyse decarboxylation and oxidation. Here, the expression, crystallization and preliminary data analysis of McbB are reported. The crystals diffracted to 2.10 Šresolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 66.06, b = 85.48, c = 106.19 Å, α = 90.00, ß = 106.77, γ = 90.00°. These results provide a basis for solving the crystal structure and elucidating the catalytic mechanism for McbB.

Kinetic analysis of human PrimPol DNA polymerase activity reveals a generally error-prone enzyme capable of accurately bypassing 7,8-dihydro-8-oxo-2'-deoxyguanosine.

Recent studies have identified human PrimPol as a new RNA/DNA primase and translesion DNA synthesis polymerase (TLS pol) that contributes to nuclear and mitochondrial DNA replication. We investigated the mechanism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg²âº as a cofactor, PrimPol binds primer-template DNA with low affinity K(d,DNA) values (∼200-1200 nM). DNA binding is enhanced 34-fold by Mn²âº (K(d,DNA) = 27 nM). The pol activity of PrimPol is increased 400-1000-fold by Mn²âº compared to Mg²âº based on steady-state kinetic parameters. PrimPol makes a mistake copying undamaged DNA once every ∼100-2500 insertions events, which is comparable to other TLS pols, and the fidelity of PrimPol is ∼1.7-fold more accurate when Mg²âº is the cofactor compared to Mn²âº. PrimPol inserts dCMP opposite 8-oxo-dG with 2- (Mn²âº) to 6-fold (Mg²âº) greater efficiency than dAMP misinsertion. PrimPol-catalyzed dCMP insertion opposite 8-oxo-dG proceeds at ∼25% efficiency relative to unmodified template dG, and PrimPol readily extends from dC:8-oxo-dG base pairs (bps) with ∼2-fold greater efficiency than dA:8-oxo-dG bps. A tetrahydrofuran (THF) abasic-site mimic decreases PrimPol activity to ∼0.04%. In summary, PrimPol exhibits the fidelity typical of other TLS pols, is rather unusual in the degree of activation afforded by Mn²âº, and accurately bypasses 8-oxo-dG, a DNA lesion of special relevance to mitochondrial DNA replication and transcription.