[Show abstract][Hide abstract] ABSTRACT: Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.
Nucleic Acids Research 11/2013; 42(4). DOI:10.1093/nar/gkt1101 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Integrases, such as that of the Streptomyces temperate bacteriophage C31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. C31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a "subunit rotation" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of C31 integrase-mediated recombination in a topologically constrained experimental system using hybrid "phes" recombination sites, each of which comprises a C31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive "360° rotation" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory "gating" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.
Proceedings of the National Academy of Sciences 11/2012; 109(48). DOI:10.1073/pnas.1210964109 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The roles of Mg(2+) and Zn(2+) ions in promoting phosphoryl transfer catalysed by alkaline phosphatase are yet to be fully characterised. We investigated the divalent metal ion requirements for the monoesterase and diesterase activities of calf intestinal alkaline phosphatase. The synergistic effect of Mg(2+) and Zn(2+) in promoting the hydrolysis of para-nitrophenyl phosphate (monoesterase reaction) by alkaline phosphatase is not observed in the hydrolysis of the diesterase substrate, bis-para-nitrophenyl phosphate. Indeed, the diesterase reaction is inhibited by concentrations of Mg(2+) that were optimal for the monoesterase reaction. This study reveals that the substrate specificities of alkaline phosphatases and related bimetalloenzymes are subject to regulation by changes in the nature and availability of cofactors, and the different cofactor requirements of the monoesterase and diesterase reactions of mammalian alkaline phosphatases could have significance for the biological functions of the enzymes.
The Open Biochemistry Journal 12/2011; 5:67-72. DOI:10.2174/1874091X01105010067
[Show abstract][Hide abstract] ABSTRACT: Zinc-finger recombinases (ZFRs) are chimaeric proteins comprising a serine recombinase catalytic domain linked to a zinc-finger DNA binding domain. ZFRs can be tailored to promote site-specific recombination at diverse 'Z-sites', which each comprise a central core sequence flanked by zinc-finger domain-binding motifs. Here, we show that purified ZFRs catalyse efficient high-specificity reciprocal recombination between pairs of Z-sites in vitro. No off-site activity was detected. Under different reaction conditions, ZFRs can catalyse Z-site-specific double-strand DNA cleavage. ZFR recombination activity in Escherichia coli and in vitro is highly dependent on the length of the Z-site core sequence. We show that this length effect is manifested at reaction steps prior to formation of recombinants (binding, synapsis and DNA cleavage). The design of the ZFR protein itself is also a crucial variable affecting activity. A ZFR with a very short (2 amino acids) peptide linkage between the catalytic and zinc-finger domains has high activity in vitro, whereas a ZFR with a very long linker was less recombination-proficient and less sensitive to variations in Z-site length. We discuss the causes of these phenomena, and their implications for practical applications of ZFRs.
Nucleic Acids Research 08/2011; 39(21):9316-28. DOI:10.1093/nar/gkr652 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Site-specific recombinases are enzymes that promote precise rearrangements of DNA sequences. They do this by cutting and rejoining the DNA strands at specific positions within a pair of target sites recognized and bound by the recombinase. One group of these enzymes, the serine recombinases, initiates strand exchange by making double-strand breaks in the DNA of the two sites, in an intermediate built around a catalytic tetramer of recombinase subunits. However, these catalytic steps are only the culmination of a complex pathway that begins when recombinase subunits recognize and bind to their target sites as dimers. To form the tetramer-containing reaction intermediate, two dimer-bound sites are brought together by protein dimer-dimer interactions. During or after this initial synapsis step, the recombinase subunit and tetramer conformations change dramatically by repositioning of component subdomains, bringing about a transformation of the enzyme from an inactive to an active configuration. In natural serine recombinase systems, these steps are subject to elaborate regulatory mechanisms in order to ensure that cleavage and rejoining of DNA strands only happen when and where they should, but we and others have identified recombinase mutants that have lost dependence on this regulation, thus facilitating the study of the basic steps leading to catalysis. We describe how our studies on activated mutants of two serine recombinases, Tn3 resolvase and Sin, are providing us with insights into the structural changes that occur before catalysis of strand exchange, and how these steps in the reaction pathway are regulated.
Biochemical Society Transactions 04/2011; 39(2):617-22. DOI:10.1042/BST0390617 · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The active-site interactions involved in the catalysis of DNA site-specific recombination by the serine recombinases are still incompletely understood. Recent crystal structures of synaptic gammadelta resolvase-DNA intermediates and biochemical analysis of Tn3 resolvase mutants have provided new insights into the structure of the resolvase active site, and how interactions of the catalytic residues with the DNA substrate might promote the phosphoryl transfer reactions.
Biochemical Society Transactions 04/2010; 38(2):417-21. DOI:10.1042/BST0380417 · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To characterize the residues that participate in the catalysis of DNA cleavage and rejoining by the site-specific recombinase Tn3 resolvase, we mutated conserved polar or charged residues in the catalytic domain of an activated resolvase variant. We analysed the effects of mutations at 14 residues on proficiency in binding to the recombination site ('site I'), formation of a synaptic complex between two site Is, DNA cleavage and recombination. Mutations of Y6, R8, S10, D36, R68 and R71 resulted in greatly reduced cleavage and recombination activity, suggesting crucial roles of these six residues in catalysis, whereas mutations of the other residues had less dramatic effects. No mutations strongly inhibited binding of resolvase to site I, but several caused conspicuous changes in the yield or stability of the synapse of two site Is observed by non-denaturing gel electrophoresis. The involvement of some residues in both synapsis and catalysis suggests that they contribute to a regulatory mechanism, in which engagement of catalytic residues with the substrate is coupled to correct assembly of the synapse.
Nucleic Acids Research 09/2009; 37(22):7590-602. DOI:10.1093/nar/gkp797 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of two hydrazine derivatives (isoniazid and hydralazine) on the inactivation of myeloperoxidase by H 2 O 2 were investigated. Incubation of 20 nM myeloperoxidase with 0.25 mM H 2 O 2 alone caused a time-dependent irreversible loss of tetramethylbenzidine oxidation activity with a pseudo-first order inactivation rate constant of 0.057 min -1 . The hydrazine derivatives increased the inactivation rate in a concentration-dependent manner. Inactivation of the enzyme by H 2 O 2 with or without the hydrazides showed a saturation kinetics pattern. Steady state kinetics analysis suggests that the hydrazides likely inactivate myeloperoxidase using a similar inactivating species as does H 2 O 2 . A bimolecular rate constant, specific inactivation rate enhancement factor (k* enh) is proposed as a formal description of the inactivation rate stimulation by the hydrazides. This parameter potentially avoids confounding the finite inactivation due to H 2 O 2 with that caused by the presence of the hydrazides. The relevance of these findings and the constants derived to the analysis of suicide inactivation of peroxidases by reductant substrates are discussed.
The Open Enzyme Inhibition Journal 08/2009; 2(1). DOI:10.2174/1874940200902010028
[Show abstract][Hide abstract] ABSTRACT: The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with ‘activating’ mutations, which can catalyse recombination at binding site I of res when the rest of res is absent. Site I × site I recombination promoted by these variants can be as fast as res
res recombination promoted by wild-type resolvase. Activated variants have reduced topological selectivity and no longer require the 2–3′ interface between subunits that is essential for wild-type resolvase-mediated recombination. They also promote formation of a stable synapse comprising a resolvase tetramer and two copies of site I. Cleavage of the DNA strands by the activated mutants is slow relative to the rate of synapsis. Stable resolvase tetramers were not detected in the absence of DNA or bound to a single site I. Our results lead us to conclude that the synapse is assembled by sequential binding of resolvase monomers to site I followed by interaction of two site I-dimer complexes. We discuss the implications of our results for the mechanisms of synapsis and regulation in recombination by wild-type resolvase.
Nucleic Acids Research 12/2008; 36(22):7181-91. DOI:10.1093/nar/gkn885 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The interactions of Mg2+ and Zn2+ ions in the activation of non-specific tissue alkaline phosphatase were investigated using crude extracts of rat kidney. Activation of alkaline phosphatase by the metal ions was accompanied by changes in the kinetic parameters of p-nitrophenylphosphate hydrolysis. The results suggest some synergistic interactions between Mg2+ and Zn2+ ionsin promoting the hydrolysis of p-nitrophenylphosphate by alkaline phosphatase. The results show that assays of alkaline phosphatase activity in homogenised tissue samples will give better responses if both Mg2+ and Zn2+ ions are included in the reactions
[Show abstract][Hide abstract] ABSTRACT: The concentration-dependent stimulation of rat liver alkaline phosphatase (ALP) catalyzed hydrolysis of para- nitrophenylphosphate (pNPP) was studied. ALP displayed some activity even in the absence of exogenous Mg2+. Kinetic analyses show that activation by Mg2+ is exerted at the Vmax level without necessarily enhancing the affinity of the enzyme for the ion. However, the hyperbolic activation operates only within the optimal level of 0 to 5mM concentrations of the metal ion. Higher concentrations were actually inhibitory in a pure non-competitive manner. Mg2+, either as an activator (optimal concentrations) or inhibitor (supra-optimal levels) exerts its action via a Vmax effect with only negligible effect on Kmfor the substrate.
[Show abstract][Hide abstract] ABSTRACT: Effects of repeated administration of varying concentrations of the ethanolic extract of Bougainvillea spectabilis leaves on some hepatic and renal function indices were examined in albino rats (Rattus novergicus). The experimental animals were randomly divided into three groups: those administered distilled water (control), those administered 100 mg/kg body weight of the extract (E1) and those administered 200 mg/kg body weight of the extract (E2). The animals were administered the extract daily for seven days after which venous blood was collected. The extract significantly reduced (P < 0.05) serum albumin and calcium ion concentrations while it significantly increased (P < 0.05) serum phosphate ion, urea and creatinine concentrations compared with control. The extract did not have any significant effect (P > 0.05) on the serum sodium ion concentration while it reduced serum potassium ion concentration significantly (P < 0.05) at the dose of 200 mg/ kg body weight compared with controls. Generally, activities of liver ALP, AST, ALT, and Ca 2+ -Mg 2+ ATPase were not significantly affected (P > 0.05) D: Salasar/Vol.-17/chp-12/Proof-3/Date : 8 // 2006
[Show abstract][Hide abstract] ABSTRACT: The combined effect of two linear inhibitors of rat liver alkaline phosphatase (ALP). vanadate (Van) and L-phenylalanine (L-phe) were studied using a modification of the common Yonetani-Theorell procedure proposed for studying synergistic inhibition. The modes of inhibition of ALP by Van and L-phe as analysed using the double reciprocal plots of the Michaelis-Menten equation were mixed and uncompetitive inhibition respectively. Analysis of the combined effect of the two inhibitors showed that their inhibitory effects were mutually enhanced. The mechanistic aspects and practical applications of the procedure are discussed.
[Show abstract][Hide abstract] ABSTRACT: The effect of promethazine on peroxidase-catalyzed oxidation of 3,3', 5,5'-tetramethylbenzidine was investigated at pH 5.4. Promethazine dose dependently introduced a lag in the appearance of tetramethylbenzidine charge-transfer complex monitored at 652 nm. Increasing concentrations of tetramethylbenzidine however decreased the lag period proportional to the tetramethylbenzidine concentration. Addition of promethazine to preformed charge transfer complex caused rapid bleaching of the blue-colored complex. Titration of promethazine with the yellow-colored diimine gave rise to the blue charge-transfer complex and the complete reduction of the species to the colorless parent amine compound. The available evidence suggests that promethazine is oxidized via redox mediation by tetramethylbenzidine peroxidase-oxidized products.
Archives of Biochemistry and Biophysics 09/2000; 380(2):251-6. DOI:10.1006/abbi.2000.1710 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The inactivation of peroxidases by its oxidant substrate H 2 O 2 limits the usefulness of these versatile enzymes. Here, we investigated the effect of reaction conditions on inactivation of horseradish peroxidase by excess H 2 O 2 . Inactivation was more pronounced at pH extremes, indicating that reactions in which the oxidation products induce significant changes in reaction pH could accentuate the loss of peroxidase activity. In reactions carried out in sodium acetate buffer, higher inactivation rates were observed when the buffer ion concentration was increased, an indication that peroxidase might be generating reactive radicals from the buffer molecules. Promethazine exerted a modest protective effect against inactivation; however, higher concentrations of the redox mediator caused a slight increase in inactivation, likely due to the formation of reactive promethazine radicals, which in turn attack the protein via a mechanism different from that caused by excess H 2 O 2 . These findings will help in defining the optimal reaction conditions that preserve the activity of the peroxidase molecules.