Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion

The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection

Proteomic Analysis of Fusarium solani Isolated from the Asian Longhorned Beetle, Anoplophora glabripennis

Wood is a highly intractable food source, yet many insects successfully colonize and thrive in this challenging niche. Overcoming the lignin barrier of wood is a key challenge in nutrient acquisition, but full depolymerization of intact lignin polymers has only been conclusively demonstrated in fungi and is not known to occur by enzymes produced by insects or bacteria. Previous research validated that lignocellulose and hemicellulose degradation occur within the gut of the wood boring insect, Anoplophora glabripennis (Asian longhorned beetle), and that a fungal species, Fusarium solani (ATCC MYA 4552), is consistently associated with the larval stage. While the nature of this relationship is unresolved, we sought to assess this fungal isolate's ability to degrade lignocellulose and cell wall polysaccharides and to extract nutrients from woody tissue. This gut-derived fungal isolate was inoculated onto a wood-based substrate and shotgun proteomics using Multidimensional Protein Identification Technology (MudPIT) was employed to identify 400 expressed proteins. Through this approach, we detected proteins responsible for plant cell wall polysaccharide degradation, including proteins belonging to 28 glycosyl hydrolase families and several cutinases, esterases, lipases, pectate lyases, and polysaccharide deacetylases. Proteinases with broad substrate specificities and ureases were observed, indicating that this isolate has the capability to digest plant cell wall proteins and recycle nitrogenous waste under periods of nutrient limitation. Additionally, several laccases, peroxidases, and enzymes involved in extracellular hydrogen peroxide production previously implicated in lignin depolymerization were detected. In vitro biochemical assays were conducted to corroborate MudPIT results and confirmed that cellulases, glycosyl hydrolases, xylanases, laccases, and Mn- independent peroxidases were active in culture; however, lignin- and Mn- dependent peroxidase activities were not detected While little is known about the role of filamentous fungi and their associations with insects, these findings suggest that this isolate has the endogenous potential to degrade lignocellulose and extract nutrients from woody tissue

Harnessing the potential of ligninolytic enzymes for lignocellulosic biomass pretreatment

Abundant lignocellulosic biomass from various industries provides a great potential feedstock for the production of value-added products such as biofuel, animal feed, and paper pulping. However, low yield of sugar obtained from lignocellulosic hydrolysate is usually due to the presence of lignin that acts as a protective barrier for cellulose and thus restricts the accessibility of the enzyme to work on the cellulosic component. This review focuses on the significance of biological pretreatment specifically using ligninolytic enzymes as an alternative method apart from the conventional physical and chemical pretreatment. Different modes of biological pretreatment are discussed in this paper which is based on (i) fungal pretreatment where fungi mycelia colonise and directly attack the substrate by releasing ligninolytic enzymes and (ii) enzymatic pretreatment using ligninolytic enzymes to counter the drawbacks of fungal pretreatment. This review also discusses the important factors of biological pretreatment using ligninolytic enzymes such as nature of the lignocellulosic biomass, pH, temperature, presence of mediator, oxygen, and surfactant during the biodelignification process

Partial characterization of the human serum transferrin epitope reactive with the monoclonal antibody TRC-2

A murine monoclonal antibody (MAb) (TRC-2) specific for human serum transferrin (Tf-h) was developed. This antibody was depressive on cell growth in serum-free medium in the presence of limiting amounts of Tfh, but it did not inhibit the binding of Tf-h-alkaline phosphatase (AP) conjugate to the Tf-receptor (TfR) in a cellular enzyme-linked immunosorbent assay (CELISA) system. On the other hand, the immune complex Tf-h-TRC-2 was implicated to bind to the receptor in indirect CELISA. Moreover, the detectability of Tf-h-TfR on the cell surface via Tf-bound TRC-2 suggested that the antibody may inhibit the rapid internalization of this complex. To map the TRC-2-specific epitope, Tf-h was subjected to proteolytic degradation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The treatment with trypsin gave rise to, among others, a fragment of about 42 kDa, which was reactive with TRC-2. Through sequence analysis by automated Edman degradation, the N-terminal sequence of the 42 kDa-tryptic fragment was aligned to the N-terminus of mature transferrin (VPDKTVR). The N-terminal sequence of an immunoreactive CNBr-fragment of about 13 kDa was, in turn, identical with the sequence (NQLRGKK) corresponding to the residues 110-116 on Tf-h

On the mode of inhibition of eukaryotic protein synthesis by ADP-ribosylation of elongation factor 2

The exchange of flee guanine nucleotides with guanine nucleotides bound to elongation factor 2 (EF-2) and to the EF-2-ribosome complex, and the effect of ADP-ribosylation of the EF-2 thereon, were investigated by nitrocellulose filter assay. Under the experimental conditions, stoichiometric amounts of guanine nucleotides were bound, in particular, to ternary complexes of EF-2 with biphasic kinetics. The exchange kinetics were similarly biphasic in all cases. Ribosomes appeared to have variable effects on the exchange kinetics, depending on the type of nucleotide bound. Thus, in their presence, the rate and magnitude of the fast exchange of nucleotides revealed increasing values in the order GTP (GXP) > GTP gamma S > GDP, ADP-ribosylation had no inhibitory effect on the binding of guanine nucleotides to EF-2 or to the EF-2-ribosome complex but reduced significantly the fast exchange of GTP (GXP) and GTP gamma S bound to the EF-2-ribosome complex. The effect of ADP-ribosylation on the fast exchange of GDP in binary and ternary complexes mas less pronounced. The mechanism of inhibition of protein synthesis by ADP-ribosylation of EF-2 is discussed in view of these data

ADP-ribosylation of serum proteins: Evaluation as a potential tumor marker

Serum samples from cancer patients revealed elevated levels of in vitro ADP-ribosylation through non-enzymic binding of ADP-ribose to free acceptor sites on serum proteins. Low concentrations of serum ADP-ribose caused by high NAD glycohydrolase activity together with elevated rates of ADP-ribose transport into erythrocytes appeared to account for undersaturation of the acceptor sites on serum proteins. ADP-ribosylation of serum proteins was assessed as an indicator of cancer disease, and an attempt was made to determine the correlation of ADP-ribosylation levels with carcinoembryonic antigen (CEA) values. Based on positive test results for all tumor patients and negative test results for all healthy controls, sensitivity and specificity of ADP-ribosylation as a tumor indicator were estimated as 67% and 95%, respectively. A close correlation appeared to exist with CEA (r = 0.67; P < 0.001). Similarly, the changes in the levels of ADP-ribosylation correlated with the changes in the levels of CEA during the clinical course (r = 058; P < 0.05)