The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases

Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.     

番茄‘Ls-89’和‘坂砧2号’幼苗活性氧代谢对南方根结线虫侵染的反应

为了解番茄(Lycopersicon esculentum)砧木幼苗活性氧代谢与抗南方根结线虫(Meloidogyne incognita)的关系,以高感品种‘Ls-89’与高抗品种‘坂砧2号’为材料,采用盆栽人工接种法,研究了南方根结线虫侵染对番茄砧木幼苗活性氧代谢的影响。结果表明,未接种南方根结线虫的番茄砧木幼苗,其根系与叶片活性氧水平及相关酶活性在品种间没有显著差异。接种南方根结线虫后,两品种幼苗根系与叶片的O_2·~–生成速率和H_2O_2含量均升高,且‘坂砧2号’显著高于‘Ls-89’,但‘Ls-89’的MDA含量则显著高于‘坂砧2号’。两品种幼苗根系与叶片的SOD活性均在侵染早期降低,以‘坂砧2号’降幅较大;但POD、CAT活性在侵染早期变化不大,至侵染中后期则显著升高。因此,番茄抗性品种砧木幼苗的膜脂抗氧化能力较强,活性氧水平较高,且SOD活性对南方根结线虫侵染敏感。     

Isolation and Characterization of a New Extracellular Bacteriolytic Endopeptidase of <Emphasis Type="Italic">Lysobacter</Emphasis> sp. XL1

The previously unstudied bacteriolytic enzyme L(4) was isolated from the culture liquid of the bacterium Lysobacter sp. XL1 in electrophoretically homogeneous state. The enzyme L(4) is a diaminopimelinoyl-alanine endopeptidase relative to peptidoglycan of Lysobacter sp. XL1. The enzyme is an alkaline protein of approximately 21 kD. The N-terminal amino acid sequence of the enzyme has been determined - A V V N G V N Y V Gx T T A ... The maximal activity of the enzyme was observed in 0.05 M Tris-HCl at pH 8.0 and 50-55 degrees C. The half-inactivation temperature of the enzyme is 52 degrees C. The endopeptidase L(4) is not a metalloenzyme since it is not affected by EDTA. The enzyme is inhibited by p-chloromercuribenzoic acid by 72% and by phenylmethylsulfonyl fluoride by 43%, which indicates the involvement of serine and thiol groups in its functioning.     

Prechemistry versus preorganization in DNA replication fidelity

The molecular origin of nucleotide insertion catalysis and fidelity of DNA polymerases is explored by means of computational simulations. Special attention is paid to the examination of the validity of proposals that invoke prechemistry effects, checkpoints concepts, and dynamical effects. The simulations reproduce the observed fidelity in Pol β, starting with the relevant observed X-ray structures of the complex with the right (R) and wrong (W) nucleotides. The generation of free energy surfaces for the R and W systems also allowed us to analyze different proposals about the origin of the fidelity and to reach several important conclusions. It is found that the potential of mean force (PMF) obtained by proper sampling does not support QM/MM-based proposals of a large barrier before the prechemistry state. Furthermore, examination of dynamical proposals by the renormalization approach indicates that the motions from open to close configurations do not contribute to catalysis or fidelity. Finally we discuss and analyze the induced fit concept and show that, despite its importance, it does not explain fidelity. That is, the fidelity is apparently due to the change in the preorganization of the chemical site, as a result of the relaxation of the binding site upon binding of the incorrect nucleotide. Finally and importantly, since the issue is the barrier associated with the enzyme-substrate (ES)/DNA complex at the chemical transition state and not the path to this complex formation (unless this path involves rate determining steps), it is also not useful to invoke checkpoints while discussing fidelity.