Element Coding Based Accurate Evaluation of CRISPR/Cas9 Initial Cleavage

As a powerful gene editing tool, the kinetic mechanism of CRISPR/Cas9 has been the focus to its further application. Initial cleavage events as the first domino followed by nuclease end...

Molecular mechanism of the chitinolytic peroxygenase reaction

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H2O2 as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H2O2-mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper–oxyl intermediate. The initial cleavage of H2O2 and subsequent hydrogen atom abstraction from chitin by the copper–oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO–Cu(II) to LPMO–Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO–Cu(I) is 2,000-fold faster with H2O2 than with O2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. The presence of substrate accelerated reoxidation by H2O2, whereas reoxidation by O2 became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.

Biodegradation of the Allelopathic Chemical Pterostilbene by a Sphingobium sp. Strain from the Peanut Rhizosphere

ABSTRACT Many plants produce allelopathic chemicals, such as stilbenes, to inhibit pathogenic fungi. The degradation of allelopathic compounds by bacteria associated with the plants would limit their effectiveness, but little is known about the extent of biodegradation or the bacteria involved. Screening of tissues and rhizosphere of peanut (Arachis hypogaea) plants revealed substantial enrichment of bacteria able to grow on resveratrol and pterostilbene, the most common stilbenes produced by the plants. Investigation of the catabolic pathway in Sphingobium sp. strain JS1018, isolated from the rhizosphere, indicated that the initial cleavage of pterostilbene was catalyzed by a carotenoid cleavage oxygenase (CCO), which led to the transient accumulation of 4-hydroxybenzaldehyde and 3,5-dimethoxybenzaldehyde. 4-Hydroxybenzaldehyde was subsequently used for the growth of the isolate, while 3,5-dimethoxybenzaldehyde was further converted to a dead-end metabolite with a molecular weight of 414 (C24H31O6). The gene that encodes the initial oxygenase was identified in the genome of strain JS1018, and its function was confirmed by heterologous expression in Escherichia coli. This study reveals the biodegradation pathway of pterostilbene by plant-associated bacteria. The prevalence of such bacteria in the rhizosphere and plant tissues suggests a potential role of bacterial interference in plant allelopathy. IMPORTANCE Pterostilbene, an analog of resveratrol, is a stilbene allelochemical produced by plants to inhibit microbial infection. As a potent antioxidant, pterostilbene acts more effectively than resveratrol as an antifungal agent. Bacterial degradation of this plant natural product would affect the allelopathic efficacy and fate of pterostilbene and thus its ecological role. This study explores the isolation and abundance of bacteria that degrade resveratrol and pterostilbene in peanut tissues and rhizosphere, the catabolic pathway for pterostilbene, and the molecular basis for the initial cleavage of pterostilbene. If plant allelopathy is an important process in agriculture and management of invasive plants, the ecological role of bacteria that degrade the allelopathic chemicals must be equally important.

Biodegradation of the Organic Disulfide 4,4′-Dithiodibutyric Acid by Rhodococcus spp.

ABSTRACTFourRhodococcusspp. exhibited the ability to use 4,4′-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. BecauseRhodococcus erythropolisstrain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis ofR. erythropolisMI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity ofnoxRandnox. The interruption mutant generated,R. erythropolisMI2noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently,noxwas overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB).

The Regulatory Function Of Amino Acid Region 473-487 Of Prothrombin During Coagulation

Background In the United States, every thirty nine seconds an individual dies from complications from Cardiovascular Diseases. Persistent thrombus formation at the genesis of these diseases, such as stroke and other coagulation disorders, has no full model to date. Intrinsically blood clots are produced due to excessive/unnecessary thrombin formation, which leads to the conversion of fibrinogen to fibrin. As a result the regulation of thrombin formation is critical in controlling clot generation. Upon vasculature damage, the proteolytic conversion of prothrombin (Pro) to thrombin compatible to rates of survival is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a phospholipid membrane in the presence of calcium ions. Although fXa is capable of activating Pro through the initial cleavage at Arg271 followed by the cleavage at Arg320 (pre2 pathway), it would take approximately six months to form a clot. However, the incorporation of fVa into prothrombinase results in a five-fold increase in the catalytic efficiency of fXa for thrombin generation and the order of cleavages reversed (meizo pathway). Thus the timely arrest of unwarranted bleeding is due to the assembly of prothrombinase at the site of injury. Inevitably the presence and absence of fVa dictates the pathway of Pro activation and previous studies have suggested that fXa interacts with Pro within amino acid region 473-487 in a fVa-dependent manner. Aim To evaluate the role amino acid region 473-487 of Pro has in coagulation. Methods A recombinant Pro molecule with the region 473-487 was deleted (rProΔ473-487) using site-directed mutagenesis. Methotrexate was used for selection to stably transfect BHK-21 cells with rProΔ473-487 and wild-type Pro (rProWT). The two recombinant molecules were purified according to a well-established protocol and, at the last step, Fast Performance Liquid Chromatography was used equipped with a strong anionic Mono-Q 5/50 column. Properly carboxylated rProΔ473-487 and rProWT was isolated and removed from the column by utilizing a calcium gradient. Subsequently Pro deficient plasma was used to assess the molecules clotting activities on a Diagnostica Stago STart® 4 Hemostasis Analyzer. Gel electrophoresis was used to evaluate both recombinant molecules and their ability to generate active thrombin by either the multifaceted prothrombinase or fXa alone. Further studies were then performed using generated recombinant thrombin from the recombinant Pro molecules to investigate in their ability to activate procofactors V (fV) & VIII (fVIII). Results The investigation into the Activated Partial Thromboplastin Time [APTT] revealed clotting activity for human Pro and rProWT to be comparable, whereas rProΔ473-487 was substantially limited in the process of forming a fibrin clot. Next gel electrophoresis and scanning densitometry indicated the consumption of rProΔ473-487 by prothrombinase and subsequent thrombin formation was decreased 24-fold when compared to rProWT. In contrast membrane-bound fXa alone, in the absence of fVa, exhibited a 6-fold increase in the rate of initial cleavage Arg271 and subsequent activation of rProΔ473-487. Both recombinant Pro molecules demonstrated a similar cleavage pattern of activation equivalent with human Pro suggesting no structural alterations took place in rProΔ473-487following the mutation. Furthermore, generated human thrombin and recombinant wild-type thrombin were found to activate fV and fVIII within five minutes while the recombinant mutant thrombin was impeded in the activation process out to three hours. Conclusion Overall the data demonstrate that amino acid sequence 473-487 of Pro plays a preeminent role in 1) timely activation of Pro at initial cleavage Arg320 by prothrombinase, and 2) suitable macromolecular procofactor activation. Thus there is incisive rationale why no major mutations have been identified in this dynamic region which would be problematic for inherent physiological hemostasis. Disclosures: No relevant conflicts of interest to declare.

Conformational Dynamics of Prothrombin Dictate Its Ordered Cleavage by Prothrombinase

Abstract 3359 The prothrombinase complex (membranes/Xa/Va) binds prothrombin through exosite interactions which orient and present the substrate to the protease active site for cleavage. Although both cleavage sites within the substrate are accessible, prothrombin is preferentially activated by prothrombinase through sequential cleavage at R320 followed by cleavage at R271. We investigated the exosite-dependent interaction between prothrombin and prothrombinase using fluorescence resonance energy transfer (FRET). An acceptor fluorophore was placed onto the prothrombin cleavage site by introducing the mutation R271C and fluorescently modifying the introduced thiol (IIC271*). The active site of Xa was covalently modified with a chloromethyl ketone conjugated to a donor fluorescent compound. Titrations of the fluorescently labeled prothrombinase complex with IIC271* revealed a maximum energy transfer efficiency of 70–80 % observed with two different donor probes. Resonance energy transfer arising from the substrate docking interaction was confirmed by equivalent changes in the excited state lifetime of the donors. The calculated distance between the probe at C271 and the probe tethered at the active site of Xa ranged between 34 and 40 Å. Variation in the measured distance reflects the different lengths of the tethers used to connect the donor probes. The FRET measurements, validated with two donor-acceptor pairs, indicate that the 271 site within prothrombin docked to prothrombinase is positioned distant from the active site of Xa within the enzyme complex. This distant constraint prevents R271 from effectively engaging the active site of Xa within prothrombinase, thereby providing a physical explanation for the ordered action of prothrombinase on prothrombin. It follows that initial cleavage at R320 must reorient the substrate to allow R271 access to the Xa active site. We pursued an intramolecular FRET approach in which both donor and acceptor probes were placed on the prothrombin molecule to investigate the putative conformational change of prothrombin as a result of initial cleavage. Orthogonal labeling of prothrombin was accomplished using a prothrombin variant containing C271 and a LPETG extension at its C terminus. The optimized C271 label was used for the first modification. For the second, the sequence-specific transpeptidation activity of bacterial Sortase A was used to incorporate a fluorophore at the C-terminus (IICterm*). To assess the new fluorescent modification, FRET studies of prothrombin binding to prothrombinase were repeated with the new IICterm* construct. Titrations revealed a maximal energy transfer of 16 %, confirmed by corresponding changes in excited state lifetime and yielded a Kd = 39 nM indistinguishable from the Kd= 41 nM seen for IIC271* binding. Thus, two different approaches illustrate that the affinity of prothrombin for prothrombinase is independent of active site docking by the substrate. To examine the intramolecular dynamics of prothrombin during activation, the C271 site was labeled with BADAN (donor) and the Sortase labeling method placed the Alexa532 (acceptor) at the C terminus. The orthogonal placement of BADAN and Alexa532 on the prothrombin molecule resulted in extensive energy transfer between the two probes. The cleavage of doubly-labeled prothrombin to produce meizothrombin was associated with a change in intramolecular energy transfer. The variation of the energy transfers observed between the zymogen and protease forms can be interpreted as global rearrangement of the prothrombin molecule renders the second cleavage site accessible of active site docking. The FRET studies provide a comprehensive physical explanation for the ordered cleavage of prothrombin by prothrombinase. Initial cleavage occurs at the spatially accessible cleavage site R320. The prothrombin molecule then significantly re-orients itself making the second R271 cleavage site accessible to prothrombinase in order for activation to proceed and produce thrombin. Disclosures: No relevant conflicts of interest to declare.

In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways

Using model DNA substrates and purified recombinant proteins from Pyrococcus abyssi, I have reconstituted the enzymatic reactions involved in RNA primer elimination in vitro. In my dual-labelled system, polymerase D performed efficient strand displacement DNA synthesis, generating 5′-RNA flaps which were subsequently released by Fen1, before ligation by Lig1. In this pathway, the initial cleavage event by RNase HII facilitated RNA primer removal of Okazaki fragments. In addition, I have shown that polymerase B was able to displace downstream DNA strands with a single ribonucleotide at the 5′-end, a product resulting from a single cut in the RNA initiator by RNase HII. After RNA elimination, the combined activities of strand displacement DNA synthesis by polymerase B and flap cleavage by Fen1 provided a nicked substrate for ligation by Lig1. The unique specificities of Okazaki fragment maturation enzymes and replicative DNA polymerases strongly support the existence of two pathways in the resolution of RNA fragments.

Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase

Toll-like receptor (TLR) 9 requires proteolytic processing in the endolysosome to initiate signaling in response to DNA. However, recent studies conflict as to which proteases are required for receptor cleavage. We show that TLR9 proteolysis is a multistep process. The first step removes the majority of the ectodomain and can be performed by asparagine endopeptidase (AEP) or cathepsin family members. This initial cleavage event is followed by a trimming event that is solely cathepsin mediated and required for optimal receptor signaling. This dual requirement for AEP and cathepsins is observed in all cell types that we have analyzed, including mouse macrophages and dendritic cells. In addition, we show that TLR7 and TLR3 are processed in an analogous manner. These results define the core proteolytic steps required for TLR9 function and suggest that receptor proteolysis may represent a general regulatory strategy for all TLRs involved in nucleic acid recognition.

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca2+-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC50 = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.

Meizothrombin Is Unexpectedly Zymogen-Like: Its Slow Conversion to Proteinase Dominates Thrombin Production by Prothrombinase

Abstract 2215 The proteolytic activation of prothrombin catalyzed by prothrombinase is a paradigm for zymogen activation resulting from ordered cleavage at multiple sites. Initial cleavage at Arg320 forms meizothrombin (mIIa). The associated conversion of zymogen to proteinase is instrumental in facilitating further processing at Arg271 to yield thrombin. A full kinetic explanation for this process remains obscure and controversial because of the limitations of standard kinetic approaches. We now uncover novel facets of this reaction by rapid kinetic studies to approximate single catalytic turnover. Product formation was measured continuously by stopped flow using Dansyl arginine (3-ethyl-1,5-pentanediyl) amide (DAPA) or discontinuously using rapid quenching and analysis by SDS-PAGE or peptidyl substrate cleavage following rapid mixing of preformed prothrombinase (0.3 μM) with prothrombin (0.3 μM). Prothrombin cleavage, assessed discontinuously, was essentially complete within 0.2 seconds. The results indicated initial cleavage at Arg320 to form mIIa followed by subsequent cleavage at Arg271 to produce thrombin. However, product formation measured continuously with DAPA, yielded a pronounced lag and proceeded ∼20-30-fold more slowly. The intermediate, mIIa, which is expected to bind DAPA was invisible to the continuous measurements. Analysis was further simplified using a recombinant prothrombin variant, which is exclusively cleaved at Arg320 to produce mIIa and not processed further. Continuous detection of proteinase formation by stopped flow proceeded ∼30-fold more slowly than cleavage at Arg320 measured discontinuously. These findings indicate that rapid cleavage at the Arg320 site is followed by an unexpectedly slow reaction (t½ ∼ 0.5–1 s) in which the cleaved product matures to form a competent active site. This conclusion was further tested employing stable mIIa prepared by the action of Ecarin on recombinant prothrombin variants that were not degraded further even without occluding the active site. Stopped flow kinetic studies for the binding of DAPA to these variants revealed markedly biphasic traces. The data could be globally analyzed according to a two step mechanism with an initial slow equilibrium between zymogen-like and proteinase forms in which only the proteinase form can bind the active site ligand. The zymogen- like and proteinase forms were approximately equally populated and interconverted slowly (t½ ∼ 0.5 s). These findings independently corroborate the conclusions from the kinetic studies of prothrombin cleavage. Accordingly, inclusion of this slow step could explain profiles of prothrombin depletion, transient formation of mIIa and the final appearance of thrombin seen in the action of prothrombinase on prothrombin. Zymogen-like mIIa accumulates at much higher concentrations than would be predicted from knowledge of the kinetics of the individual cleavage steps because its slow maturation to proteinase is required for further cleavage at Arg271. Thus, the rate-limiting maturation of mIIa to proteinase plays a dominant role in regulating thrombin formation. Furthermore, while mIIa is a poor catalyst for many of substrates of thrombin, it retains the ability to bind thrombomodulin and function in the anticoagulant pathway. As a result of its unexpectedly zymogen-like character and slow conversion to proteinase, mIIa produced as an intermediate by prothrombinase would be resistant to inhibition in plasma and thereby potentially be dispersed by flowing blood to exert its selectively anticoagulant functions distant from its site of production. Disclosures: No relevant conflicts of interest to declare.