Increasing cleavage specificity and activity of restriction endonuclease KpnI

Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent metal ions for DNA cleavage but not for binding. The active site of KpnI can accommodate metal ions of different atomic radii for DNA cleavage. Although Mg²⁺ ion higher than 500 μM mediates promiscuous activity, Ca²⁺ suppresses the promiscuity and induces high cleavage fidelity. Here, we report that a conservative mutation of the metal-coordinating residue D148 to Glu results in the elimination of the Ca²⁺-mediated cleavage but imparting high cleavage fidelity with Mg²⁺. High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca²⁺-mediated cleavage activity by altering the geometry of the Ca²⁺-bound HNH active site. Although the D148E mutant reduces the specific activity of the enzyme, we identified a suppressor mutation that increases the turnover rate to restore the specific activity of the high fidelity mutant to the wild-type level. Our results show that active site plasticity in coordinating different metal ions is related to KpnI promiscuous activity, and tinkering the metal ion coordination is a plausible way to reduce promiscuous activity of metalloenzymes.     

Functional group requirements in the probable active site of the VS ribozyme

The VS ribozyme catalyses the site-specific cleavage of a phosphodiester linkage by a transesterification reaction that entails the attack of the neighbouring 2'-oxygen with departure of the 5'-oxygen. We have previously suggested that the A730 loop is an important component of the active site of the ribozyme, and that A756 is especially important in the cleavage reaction. Functional group modification experiments reported here indicate that the base of A756 is more important than its ribose for catalysis. A number of changes to the base, including complete ablation, lead to cleavage rates that are reduced 1000-fold, while removal of the 2'-hydroxyl group from the ribose results in tenfold slower cleavage. 2-Aminopurine fluorescence experiments indicate that this 2'-hydroxyl group is important for the structure of the A730 loop. Catalytic activity is especially sensitive to changes involving the exocyclic amine of A756; by contrast, the cleavage activity is only weakly sensitive to modification at the 7-position of the purine nucleus. These results suggest that the Watson-Crick edge of the adenine base is important in ribozyme function. We sought to test the possibility of a direct role of the nucleobase in the chemistry of the cleavage reaction. Addition of imidazole base in the medium failed to restore the activity of a ribozyme from which the nucleobase of A756 was removed. However, no restoration was obtained with exogenous adenine base either, indicating that the cavity that might result from ablation of the base was closed.     

Increased Ribozyme Activity in Crowded Solutions

Noncoding RNAs must function in the crowded environment of the cell. Previous small-angle x-ray scattering experiments showed that molecular crowders stabilize the structure of the Azoarcus group I ribozyme, allowing the ribozyme to fold at low physiological Mg²⁺ concentrations. Here, we used an RNA cleavage assay to show that the PEG and Ficoll crowder molecules increased the biochemical activity of the ribozyme, whereas sucrose did not. Crowding lowered the Mg²⁺ threshold at which activity was detected and increased total RNA cleavage at high Mg²⁺ concentrations sufficient to fold the RNA in crowded or dilute solution. After correcting for solution viscosity, the observed reaction rate was proportional to the fraction of active ribozyme. We conclude that molecular crowders stabilize the native ribozyme and favor the active structure relative to compact inactive folding intermediates.     

Modeling active RNA structures using the intersection of conformational space: application to the lead-activated ribozyme.

The Pb2+ cleavage of a specific phosphodiester bond in yeast tRNA(Phe) is the classical model of metal-assisted RNA catalysis. In vitro selection experiments have identified a tRNA(Phe) variant, the leadzyme, that is very active in cleavage by Pb2+. We present here a three-dimensional modeling protocol that was used to propose a structure for this ribozyme, and is based on the computation of the intersection of conformational space of sequence variants and the use of chemical modification data. Sequence and secondary structure data were used in a first round of computer modeling that allowed identification of conformations compatible with all known leadzyme variants. Common conformations were then tested experimentally by evaluating the activity of analogues containing modified nucleotides in the catalytic core. These experiments led to a new structural hypothesis that was tested in a second round of computer modeling. The resulting proposal for the active conformation of the leadzyme is consistent with all known structural data. The final model suggests an in-line SN2 attack mechanism and predicts two Pb2+ binding sites. The protocol presented here is generally applicable in modeling RNAs whenever the catalytic or binding activity of structural analogues is known.     

Reconstitution of human azurocidin catalytic triad and proteolytic activity by site-directed mutagenesis

Azurocidin belongs to the serprocidin family, but it is devoid of proteolytic activity due to a substitution of His and Ser residues in the catalytic triad. The aim of this study was to reconstitute the active site of azurocidin by site-directed mutagenesis, analyze its processing and restored proteolytic activity. Azurocidin expressed in Sf9 insect cells possessing the reconstituted His41-Asp89-Ser175 triad exhibited significant proteolytic activity toward casein with a pH optimum of approximately 8-9, but a reconstitution of only one active site amino acid did not result in proteolytically active protein. Enzymatically active recombinant azurocidin caused cleavage of the C-terminal fusion tag with the primary cleavage site after lysine at Lys-Leu and after alanine at Ala-Ala, and the secondary cleavage site after arginine at Arg-Gln, as well as with low efficiency caused cleavage of insulin chain B after leucine at Leu-Tyr and Leu-Cys, and after alanine at Ala-Leu. We demonstrate that cleavage of the azurocidin C-terminal tripeptide is not necessary for its enzymatic activity. The first isoleucine present in mature azurocidin can be replaced by similar amino acids, such as leucine or valine, but its substitution by histidine or arginine decreases proteolytic activity.     

Reconstitution of enzymatic activity by the association of the cap and catalytic domains of human topoisomerase I

When human topoisomerase I binds DNA, two opposing lobes in the enzyme, the cap region (amino acid, residues 175-433) and the catalytic domain (Deltacap, residues 433 to the COOH terminus) clamp tightly around the DNA helix to form the precleavage complex. Although Deltacap contains all of the residues known to be important for catalysis and binds DNA with an affinity similar to that of the intact enzyme, this fragment lacks catalytic activity. However, a mixture of Deltacap and topo31 (residues 175-433) reconstitutes enzymatic activity as measured by plasmid DNA relaxation and suicide cleavage assays. Although the formation of an active complex between topo31 and Deltacap is too unstable to be detected by pull-down experiments even in the presence of DNA, the association of topo31 with Deltacap persists and is detectable after the complex catalyzes the covalent attachment of the DNA to Deltacap by suicide cleavage. Removal of topo31 from Deltacap-DNA after suicide cleavage reveals that, unlike the cleavage reaction, religation does not require the cap region of the protein. These results suggest that activation of the catalytic domain of the enzyme for cleavage requires both DNA binding and the presence of the cap region of the protein.     

Presence of a coordinated metal ion in a trans-acting antigenomic delta ribozyme.

We have investigated the cleavage induced by metal ions in an antigenomic form of a trans-acting delta ribozyme. A specific Mg(2+)-induced cleavage at position G(52)at the bottom of the P2 stem was observed to occur solely within catalytically active ribozyme-substrate complexes (i.e. those that performed the essential conformational transition step). Only the divalent cations which support catalytic activity permitted the detection of specific induced cleavages in this region. Using various mutant ribozymes and substrates, we demonstrated a correlation between enzymatic activity and the Mg(2+)-induced cleavage pattern. We show that the efficiency of the coordination of the magnesium to its binding site is related to the nature of the base pair in the middle of the P1 stem (i.e. Rz(23)-S(8)). Together with additional evidence from nuclease probing experiments that indicates the occurrence of a structural rearrangement involving the bottom of the P2 stem upon formation of the P1 helix, these results show that an intimate relationship exists between the folding and the catalytic activity of the delta ribozyme.     

Activation of prothrombin Barcelona evidence for active high molecular weight intermediates

Prothrombin Barcelona is a varient of human prothrombin. Its activation by factor Xa is characterized by two abnormal features. The first one is the absence of one of the two Xa-catalyzed cleavages, and the second one the generation of a thrombin-like activity responsible for the thrombin-catalyzed cleavages on the prothrombin molecule and for the activity toward small substrates but without clotting activity.The molecular species exhibiting the thrombin-like activity have been characterized Diisopropyl phosphofluoridate incorporation experiments show that upon activation, two high molecular weight intermediates bearing an active site are formed: one has the molecular weight of prothrombin, the other of prethrombin 1. In the presence of hirudin, only the first intermediate is formed due to a single cleavage by factor Xa which is therefore responsible for the unmasking of the active site.     

Intracellular ribozyme-catalyzed trans-cleavage of RNA monitored by fluorescence resonance energy transfer

Small catalytic RNAs like the hairpin ribozyme are proving to be useful intracellular tools; however, most attempts to demonstrate trans-cleavage of RNA by ribozymes in cells have been frustrated by rapid cellular degradation of the cleavage products. Here, we describe a fluorescence resonance energy transfer (FRET) assay that directly monitors cleavage of target RNA in tissue-culture cells. An oligoribonucleotide substrate was modified to inhibit cellular ribonuclease degradation without interfering with ribozyme cleavage, and donor (fluorescein) and acceptor (tetramethylrhodamine) fluorophores were introduced at positions flanking the cleavage site. In simple buffers, the intact substrate produces a strong FRET signal that is lost upon cleavage, resulting in a red-to-green shift in dominant fluorescence emission. Hairpin ribozyme and fluorescent substrate were microinjected into murine fibroblasts under conditions in which substrate cleavage can occur only inside the cell. A strong FRET signal was observed by fluorescence microscopy when substrate was injected, but rapid decay of the FRET signal occurred when an active, cognate ribozyme was introduced with the substrate. No acceleration in cleavage rates was observed in control experiments utilizing a noncleavable substrate, inactive ribozyme, or an active ribozyme with altered substrate specificity. Subsequently, the fluorescent substrates were injected into clonal cell lines that expressed cognate or noncognate ribozymes. A decrease in FRET signal was observed only when substrate was microinjected into cells expressing its cognate ribozyme. These results demonstrate trans-cleavage of RNA within mammalian cells, and provide an experimental basis for quantitative analysis of ribozyme activity and specificity within the cell.     

Histidyl-tRNA synthetase urzymes: Class I and II aminoacyl tRNA synthetase urzymes have comparable catalytic activities for cognate amino acid activation

Four minimal (119-145 residue) active site fragments of Escherichia coli Class II histidyl-tRNA synthetase were constructed, expressed as maltose-binding protein fusions, and assayed for histidine activation as fusion proteins and after TEV cleavage, using the (32)PP(i) exchange assay. All contain conserved Motifs 1 and 2. Two contain an N-terminal extension of Motif 1 and two contain Motif 3. Five experimental results argue strongly for the authenticity of the observed catalytic activities: (i) active site titration experiments showing high (～0.1-0.55) fractions of active molecules, (ii) release of cryptic activity by TEV cleavage of the fusion proteins, (iii) reduced activity associated with an active site mutation, (iv) quantitative attribution of increased catalytic activity to the intrinsic effects of Motif 3, the N-terminal extension and their synergistic effect, and (v) significantly altered K(m) values for both ATP and histidine substrates. It is therefore plausible that neither the insertion domain nor Motif 3 were essential for catalytic activity in the earliest Class II aminoacyl-tRNA synthetases. The mean rate enhancement of all four cleaved constructs is ～10(9) times that of the estimated uncatalyzed rate. As observed for the tryptophanyl-tRNA synthetase (TrpRS) Urzyme, these fragments bind ATP tightly but have reduced affinity for cognate amino acids. These fragments thus likely represent Urzymes (Ur = primitive, original, earliest + enzyme) comparable in size and catalytic activity and coded by sequences proposed to be antisense to that coding the previously described Class I TrpRS Urzyme. Their catalytic activities provide metrics for experimental recapitulation of very early evolutionary events.     

Activity of crystalline turkey egg white lysozyme.

Hexagonal crystals of turkey egg white lysozyme have been examined for activity in order to evaluate their potential for use in time-resolved X-ray crystallographic experiments. Substrates used in this study were hexa-N-acetylglucosamine (hexa-GlcNAc) and a modified analogue of hexa-GlcNAc where the terminal sugar ring was opened by reduction with tritiated sodium borohydride. This gave a labeled beta-N-acetylglucosaminitol unit at the sixth position of the sugar chain and allowed easy quantitation of enzymatic cleavage on TLC plates. Using these substrates, it has been shown that turkey egg white lysozyme is enzymatically active in the crystal. Enzyme dispersed in the buffer surrounding the crystal does not show detectable activity under conditions relevant to an X-ray experiment. Unmodified hexa-GlcNAc is hydrolyzed into di-, tri-, and tetrasaccharides in the crystal. This cleavage pattern is different from that obtained with hen egg white lysozyme in solution and likely causes of the differences are discussed. The reduced radiolabeled oligosaccharide has a unique cleavage pattern with trisaccharides as the products. The specific activity of the enzyme with the radiolabelled analogue was 9.8 (+/- 1.0) x 10(-7) mmol/min/mg protein at 22 degrees C in the crystal.     

A small region in the angiotensin-converting enzyme distal ectodomain is required for cleavage-secretion of the protein at the plasma membrane

Both germinal and somatic isoforms of ACE are type I ectoproteins expressed on the cell surface from where the enzymatically active ectodomains are released to circulation by a regulated cleavage-secretion process. Our previous studies have shown that ACE-secretase activity is regulated by the ACE distal ectodomain and not by sequences at or around the cleavage site. In the current study we have identified that the ACE residues encompassing 343 to 655 of the germinal form are needed for its cleavage-secretion. To narrow down this region further, we have examined the cleavage-secretion of ACE-CD4 chimeric proteins in mammalian cells and Pichia pastoris. These experiments identified five residues (HGEKL) in the ACE region of the chimeric proteins that were essential for their cleavage-secretion. When the corresponding residues were substituted by alanine in native germinal and somatic ACE, the mutant proteins were not cleaved, although they were displayed on the cell surface and enzymatically active. These results demonstrated that a small region in the ectodomain of ACE is required for its cleavage at the juxtamembrane domain. This conclusion was further supported by our observation that secreted ACE inhibited cell-bound ACE cleavage-secretion, although the secreted form did not contain the cleavage site.     

The active site of the junction-resolving enzyme T7 endonuclease I

Endonuclease I is a junction-resolving enzyme encoded by bacteriophage T7, that selectively binds and cleaves four-way DNA junctions. We have recently solved the structure of this dimeric enzyme at atomic resolution, and identified the probable catalytic residues. The putative active site comprises the side-chains of three acidic amino acids (Glu20, Asp55 and Glu65) together with a lysine residue (Lys67), and shares strong similarities with a number of type II restriction enzymes. However, it differs from a typical restriction enzyme as the proposed catalytic residues in both active sites are contributed by both polypeptides of the dimer. Mutagenesis experiments confirm the importance of all the proposed active site residues. We have carried out in vitro complementation experiments using heterodimers formed from mutants in different active site residues, showing that Glu20 is located on a different monomer from the remaining amino acid residues comprising the active site. These experiments confirm that the helix-exchanged architecture of the enzyme creates a mixed active site in solution. Such a composite active site structure should result in unilateral cleavage by the complemented heterodimer; this has been confirmed by the use of a cruciform substrate. Based upon analogy with closely similar restriction enzyme active sites and our mutagenesis experiments, we propose a two-metal ion mechanism for the hydrolytic cleavage of DNA junctions.     

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived.     

Independently cloned halves of cytomegalovirus assemblin, An and Ac, can restore proteolytic activity to assemblin mutants by intermolecular complementation.

Herpesviruses encode an essential serine proteinase called assemblin that is responsible for cleaving the precursor assembly protein during the process of capsid formation. In cytomegalovirus (CMV), assemblin undergoes autoproteolysis at an internal (I) site located near the middle of the molecule. I-site cleavage converts the enzyme to an active two-chain form consisting of the subunits An and Ac. We have recently shown that the recombinant An and Ac subunits can spontaneously associate within eukaryotic cells to yield active two-chain proteinase. This finding indicates that the subunits are able to independently assume their correct functional conformations and led us to test whether they are capable of intermolecular complementation. This was done by coexpressing inactive mutant (point, deletion, and insertion) forms of assemblin together with the wild-type subunit (either An or Ac) corresponding to the domain of assemblin that was mutated. Results of these experiments showed that both An and Ac are able to rescue the enzymatic activity of assemblin mutants. I-site cleavage of the mutated assemblin occurred during complementation but was not absolutely required, as shown by effective complementation of inactive assemblins with noncleavable I sites. We have also shown that intermolecular complementation can rescue the activity of an inactive mutant full-length proteinase precursor and can occur between different species of CMV (e.g., human CMV subunit can rescue activity of mutant simian CMV assemblin). These results indicate that assemblin is able to form active multimeric structures that may be of functional importance.     

Proteolysis of ProPTHrP(1-141) by "prohormone thiol protease" at multibasic residues generates PTHrP-related peptides: implications for PTHrP peptide production in lung cancer cells

The parathyroid hormone-related protein (PTHrP) precursor requires proteolytic processing to generate PTHrP-related peptide products that possess regulatory functions in the control of PTH-like (parathyroid-like) actions and cell growth, calcium transport, and osteoclast activity. Biologically active peptide domains within the PTHrP precursor are typically flanked at their NH2- and COOH-termini by basic residue cleavage sites consisting of multibasic, dibasic, and monobasic residues. These basic residues are predicted to serve as proteolytic cleavage sites for converting the PTHrP precursor into active peptide products. The coexpression of the prohormone processing enzyme PTP ("prohormone thiol protease") in PTHrP-containing lung cancer cells, and the lack of PTP in cell lines that contain little PTHrP, implicate PTP as a candidate processing enzyme for proPTHrP. Therefore, in this study, PTP cleavage of recombinant proPTHrP(1-141) precursor was evaluated by MALDI mass spectrometry to identify peptide products and cleavage sites. PTP cleaved the PTHrP precursor at the predicted basic residue cleavage sites to generate biologically active PTHrP-related peptides that correspond to the NH2-terminal domain (residues 1-37) that possesses PTH-like and growth regulatory activities, the mid-region domain (residues 38-93) that regulates calcium transport, and the COOH-terminal domain (residues 102-141) that modulates osteoclast activity. Lack of cleavage at other types of amino acids demonstrated the specificity of PTP processing at basic residue cleavage sites. Overall, these results demonstrate the ability of PTP to cleave the PTHrP precursor at multibasic, dibasic, and monobasic residue cleavage sites to generate active PTHrP-related peptides. The presence of PTP immunoreactivity in PTHrP-containing lung cancer cells suggests PTP as a candidate processing enzyme for the PTHrP precursor.