Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A.

Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective strategies for bioethanol production. Cel7A from the fungus Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. Here, we used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A molecules binding to and moving processively along immobilized cellulose. Individual enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzyme molecules bound to cellulose in a static state and dissociated without detectable movement, whereas a minority of molecules moved processively for an average distance of 39 nm at an average speed of 3.2 nm/s. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either static or processive states and can reversibly switch between states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state, either by dissociation from the cellulose surface or by initiation of a processive run. Thus, accelerating the transition of Cel7A out of its static state is a potential avenue for improving cellulase efficiency.

Gene expression and protein synthesis of esterase from Streptococcus mutans are affected by biodegradation by-product from methacrylate resin composites and adhesives.

An esterase from S. mutans UA159, SMU_118c, was shown to hydrolyze methacrylate resin-based dental monomers. OBJECTIVE: To investigate the association of SMU_118c to the whole cellular hydrolytic activity of S. mutans toward polymerized resin composites, and to examine how the bacterium adapts its hydrolytic activity in response to environmental stresses triggered by the presence of a resin composites and adhesives biodegradation by-product (BBP). MATERIALS AND METHODS: Biofilms of S. mutans UA159 parent wild strain, SMU_118c knockout strain (ΔSMU_118c), and SMU_118c complemented strain (ΔSMU_118cC) were incubated with photo-polymerized resin composite. High performance liquid chromatography was used to quantify the amount of a universal 2,2-Bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane (bisGMA)-derived BBP, bishydroxy-propoxy-phenyl-propane (bisHPPP) in the media. Fluorescence in situ hybridization (FISH) and quantitative proteomic analysis were used to measure SMU_118c gene expression and production of SMU_118c protein, respectively, from biofilms of S. mutans UA159 wild strain that were cultured with bisHPPP. RESULTS: The levels of bisHPPP released from composite were similar for ΔSMU_118c and media control, and these were significantly lower compared to the parent wild-strain UA159 and complemented strain (ΔSMU_118cC) (p < 0.05). Gene expression of SMU_118c and productions of SMU_118c protein were higher for bisHPPP incubated biofilms (p < 0.05). SIGNIFICANCE: This study suggests that SMU_118c is a dominant esterase in S. mutans and capable of catalyzing the hydrolysis of the resinous matrix of polymerized composites and adhesives. In turn, the bacterial response to BBP was to increase the expression of the esterase gene and enhance esterase production, potentially accelerating the biodegradation of the restoration, adhesive and restoration-tooth interface, ultimately contributing to premature restoration failure. STATEMENT OF SIGNIFICANCE: We recently reported (Huang et al., 2018) on the isolation and initial characterization of a specific esterase (SMU_118c) from S. mutans that show degradative activity toward the hydrolysis of dental monomers. The current study further characterize this enzyme and shows that SMU_118c is a dominant degradative esterase activity in the cariogenic bacterium S. mutans and is capable of catalyzing the hydrolysis of the resinous matrix of polymerized composites and adhesives. In turn, the bacterial response to biodegradation by-products from composites and adhesives was to increase the expression of the esterase gene and enhance esterase production, accelerating the biodegradation of the restoration, adhesive and the restoration-tooth interface, potentially contributing to the pathogenesis of recurrent caries around resin composite restorations.

Cleavage-resistant fusion proteins of the M(2) muscarinic receptor and Gα(i1). Homotropic and heterotropic effects in the binding of ligands.

BACKGROUND: G protein-coupled receptors fused to a Gα-subunit are functionally similar to their unfused counterparts. They offer an intriguing view into the nature of the receptor-G protein complex, but their usefulness depends upon the stability of the fusion. METHODS: Fusion proteins of the M(2) muscarinic receptor and the α-subunit of G(i1) were expressed in CHO and Sf9 cells, extracted in digitonin-cholate, and examined for their binding properties and their electrophoretic mobility on western blots. RESULTS: Receptor fused to native α(i1) underwent proteolysis near the point of fusion to release a fragment with the mobility of α(i1). The cleavage was prevented by truncation of the α-subunit at position 18. Binding of the agonist oxotremorine-M to the stable fusion protein from Sf9 cells was biphasic, and guanylylimidodiphosphate promoted an apparent interconversion of sites from higher to lower affinity. With receptor from CHO cells, the apparent capacity for N-[(3)H]methylscopolamine was 60% of that for [(3)H]quinuclidinylbenzilate; binding at saturating concentrations of the latter was inhibited in a noncompetitive manner at low concentrations of unlabeled N-methylscopolamine. CONCLUSIONS: A stable fusion protein of the M(2) receptor and truncated α(i1) resembles the native receptor-G protein complex with respect to the guanyl nucleotide-sensitive binding of agonists and the noncompetitive binding of antagonists. GENERAL SIGNIFICANCE: Release of the α-subunit is likely to occur with other such fusion proteins, rendering the data ambiguous or misleading. The properties of a chemically stable fusion protein support the notion that signaling proceeds via a stable multimeric complex of receptor and G protein.

X-ray crystal structures of rabbit N-acetylglucosaminyltransferase I (GnT I) in complex with donor substrate analogues.

The Golgi-resident glycosyltransferase, UDP-N-acetyl-d-glucosamine:alpha-3-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I), initiates the conversion of high-mannose oligosaccharides to complex and hybrid structures in the biosynthesis of N-linked glycans. Reported here are the X-ray crystal structures of GnT I in complex with UDP-CH2-GlcNAc (a non-hydrolyzable C-glycosidic phosphonate), UDP-2-deoxy-2-fluoro-glucose, UDP-glucose and UDP. Collectively, these structures provide evidence for the importance of the GlcNAc moiety and its N-acetyl group in donor substrate binding, as well as insight into the role played by the flexible 318-330 loop in substrate binding and product release. In addition, the UDP-CH2-GlcNAc complex reveals a well-defined glycerol molecule poised for nucleophilic attack on the C1 atom of the donor substrate analogue. The position and orientation of this glycerol molecule have allowed us to model the binding of the Manalpha1,3Manbeta1 moiety of the acceptor substrate and, based on the model, to suggest a rationalization for the main determinants of GnT I acceptor specificity.

Mathematical basis for the optimization of single cell-line screening on multi-well plates.

Screening of stably transfected cells on multi-well plates is most efficient when a maximum number of wells contain a single colony. The multinomial distribution has been used to derive a novel equation that relates the number of wells containing a specified number of cell-lines and the total number of viable cells loaded on the plate. To test its validity, Chinese hamster ovary cells were transfected with a gene coding for the M2 muscarinic cholinergic receptor and screened on 96-well plates. The observed and predicted numbers of wells containing a single cell-line were indistinguishable. This approach therefore can be used to optimize the conditions for screening transfected cells.

Induction of laccase activity in the edible straw mushroom, Volvariella volvacea.

Volvariella volvacea, strain V14, produces multiple forms of extracellular laccase when grown in submerged culture in a defined medium with glucose as sole carbon source, and on cotton waste 'compost' representative of the conditions used for industrial-scale mushroom cultivation. In liquid culture, enzyme synthesis is associated with the onset of secondary growth, and is positively regulated by copper (up to 200 microM CuSO(4)) and by various aromatic compounds. In solid-state systems, only low levels of laccase are detectable during the vegetative growth phase but enzyme activity increases sharply at the onset of fruiting and during sporophore development.