Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases

The 2.15-Å resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3′ to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-SP phosphoryl oxygen on the adjacent 3′ phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.

In vitro selection for altered divalent metal specificity in the RNase P RNA

The ribozyme RNase P absolutely requires divalent metal ions for catalytic function. Multiple Mg2+ ions contribute to the optimal catalytic efficiency of RNase P, and it is likely that the tertiary structure of the ribozyme forms a specific metal-binding pocket for these ions within the active-site. To identify base moieties that contribute to catalytic metal-binding sites, we have used in vitro selection to isolate variants of the Escherichia coli RNase P RNA with altered specificities for divalent metal. RNase P RNA variants with increased activity in Ca2+ were enriched over 18 generations of selection for catalysis in the presence of Ca2+, which is normally disfavored relative to Mg2+. Although a wide spectrum of mutations was found in the generation-18 clones, only a single point mutation was common to all clones: a cytosine-to-uracil transition at position 70 (E. coli numbering) of RNase P. Analysis of the C70U point mutant in a wild-type background confirmed that the identity of the base at position 70 is the sole determinant of Ca2+ selectivity. It is noteworthy that C70 lies within the phylogenetically well conserved J3/4-P4-J2/4 region, previously implicated in Mg2+ binding. Our finding that a single base change is sufficient to alter the metal preference of RNase P is further evidence that the J3/4-P4-J2/4 domain forms a portion of the ribozyme’s active site.

Apg7p/Cvt2p: A Novel Protein-activating Enzyme Essential for Autophagy

In the yeast Saccharomyces cerevisiae, the Apg12p–Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc–tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p–Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p–Apg5p conjugation.

Protein Kinase Activity and Identification of a Toxic Effector Domain of the Target of Rapamycin TOR Proteins in Yeast

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.

Regulation of product chain length by isoprenyl diphosphate synthases

An analysis of the x-ray structure of homodimeric avian farnesyl diphosphate synthase (geranyltransferase, EC 2.5.1.10) coupled with information about conserved amino acids obtained from a sequence alignment of 35 isoprenyl diphosphate synthases that synthesize farnesyl (C15), geranylgeranyl (C20), and higher chain length isoprenoid diphosphates suggested that the side chains of residues corresponding to F112 and F113 in the avian enzyme were important for determining the ultimate length of the hydrocarbon chains. This hypothesis was supported by site-directed mutagenesis to transform wild-type avian farnesyl diphosphate synthase (FPS) into synthases capable of producing geranylgeranyl diphosphate (F112A), geranylfarnesyl (C25) diphosphate (F113S), and longer chain prenyl diphosphates (F112A/F113S). An x-ray analysis of the structure of the F112A/F113S mutant in the apo state and with allylic substrates bound produced the strongest evidence that these mutations caused the observed change in product specificity by directly altering the size of the binding pocket for the growing isoprenoid chain in the active site of the enzyme. The proposed binding pocket in the apo mutant structure was increased in depth by 5.8 Å as compared with that for the wild-type enzyme. Allylic diphosphates were observed in the holo structures, bound through magnesium ions to the aspartates of the first of two conserved aspartate-rich sequences (D117–D121), with the hydrocarbon tails of all the ligands growing down the hydrophobic pocket toward the mutation site. A model was constructed to show how the growth of a long chain prenyl product may proceed by creation of a hydrophobic passageway from the FPS active site to the outside surface of the enzyme.

Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A

An important question in the cell cycle field is how cyclin-dependent kinases (cdks) target their substrates. We have studied the role of a conserved hydrophobic patch on the surface of cyclin A in substrate recognition by cyclin A-cdk2. This hydrophobic patch is ≈35Å away from the active site of cdk2 and contains the MRAIL sequence conserved among a number of mammalian cyclins. In the x-ray structure of cyclin A-cdk2-p27, this hydrophobic patch contacts the RNLFG sequence in p27 that is common to a number of substrates and inhibitors of mammalian cdks. We find that mutation of this hydrophobic patch on cyclin A eliminates binding to proteins containing RXL motifs without affecting binding to cdk2. This docking site is critical for cyclin A-cdk2 phosphorylation of substrates containing RXL motifs, but not for phosphorylation of histone H1. Impaired substrate binding by the cyclin is the cause of the defect in RXL substrate phosphorylation, because phosphorylation can be rescued by restoring a cyclin A–substrate interaction in a heterologous manner. In addition, the conserved hydrophobic patch is important for cyclin A function in cells, contributing to cyclin A’s ability to drive cells out of the G1 phase of the cell cycle. Thus, we define a mechanism by which cyclins can recruit substrates to cdks, and our results support the notion that a high local concentration of substrate provided by a protein–protein interaction distant from the active site is critical for phosphorylation by cdks.

The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm

Thioredoxin 1 is a major thiol-disulfide oxidoreductase in the cytoplasm of Escherichia coli. One of its functions is presumed to be the reduction of the disulfide bond in the active site of the essential enzyme ribonucleotide reductase. Thioredoxin 1 is kept in a reduced state by thioredoxin reductase. In a thioredoxin reductase null mutant however, most of thioredoxin 1 is in the oxidized form; recent reports have suggested that this oxidized form might promote disulfide bond formation in vivo. In the Escherichia coli periplasm, the protein disulfide isomerase DsbC is maintained in the reduced and active state by the membrane protein DsbD. In a dsbD null mutant, DsbC accumulates in the oxidized form. This oxidized form is then able to promote disulfide bond formation. In both these cases, the inversion of the function of these thiol oxidoreductases appears to be due to an altered redox balance of the environment in which they find themselves. Here, we show that thioredoxin 1 attached to the alkaline phosphatase signal sequence can be exported into the E. coli periplasm. In this new environment for thioredoxin 1, we show that thioredoxin 1 can promote disulfide bond formation and, therefore, partially complement a dsbA strain defective for disulfide bond formation. Thus, we provide evidence that by changing the location of thioredoxin 1 from cytoplasm to periplasm, we change its function from a reductant to an oxidant. We conclude that the in vivo redox function of thioredoxin 1 depends on the redox environment in which it is localized.

Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage

Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76Δ) ribozymes in D2O and H2O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76Δ mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under kcat/KM conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (β) of 0.51 was determined for the base-rescued reaction of C76Δ. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage.

A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control

The p53 tumor suppressor protein and the MDM2 oncoprotein form a feedback-control loop that up-regulates cellular MDM2 production, blocks p53 activity, and promotes p53 decay. tsg101 was discovered as a gene whose deficiency results in neoplastic transformation of NIH 3T3 cells and the ability to generate metastatic tumors in nude mice. Its protein product contains a domain, Ubc, characteristic of the catalytic domain of ubiquitin conjugase (E2) enzymes but lacking an active-site cysteine crucial for ubiquitin conjugase activity. Here we report that TSG101 participates with MDM2 in an autoregulatory loop that modulates the cellular levels of both proteins, and also of p53, by affecting protein decay. We show that the Ubc domain of TSG101 interferes with ubiquitination of MDM2, that TSG101 inhibits MDM2 decay and elevates its steady-state level, and that these events are associated with down-regulation of p53 protein. Conversely, pulse–chase and Western blot experiments in wild-type and mutant fibroblasts indicate that elevation of MDM2 by overexpression of wild-type p53, by amplification of the endogenous MDM2 gene, or by transfection of MDM2-expressing constructs promotes TSG101 loss, which we show occurs by 26S proteasome-dependent decay. Our results identify TSG101 as both a regulator of, and target of, MDM2/p53 circuitry.

Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA

DNMT2 is a human protein that displays strong sequence similarities to DNA (cytosine-5)-methyltransferases (m5C MTases) of both prokaryotes and eukaryotes. DNMT2 contains all 10 sequence motifs that are conserved among m5C MTases, including the consensus S-adenosyl-l-methionine-binding motifs and the active site ProCys dipeptide. DNMT2 has close homologs in plants, insects and Schizosaccharomyces pombe, but no related sequence can be found in the genomes of Saccharomyces cerevisiae or Caenorhabditis elegans. The crystal structure of a deletion mutant of DNMT2 complexed with S-adenosyl-l-homocysteine (AdoHcy) has been determined at 1.8 Å resolution. The structure of the large domain that contains the sequence motifs involved in catalysis is remarkably similar to that of M.HhaI, a confirmed bacterial m5C MTase, and the smaller target recognition domains of DNMT2 and M.HhaI are also closely related in overall structure. The small domain of DNMT2 contains three short helices that are not present in M.HhaI. DNMT2 binds AdoHcy in the same conformation as confirmed m5C MTases and, while DNMT2 shares all sequence and structural features with m5C MTases, it has failed to demonstrate detectable transmethylase activity. We show here that homologs of DNMT2, which are present in some organisms that are not known to methylate their genomes, contain a specific target-recognizing sequence motif including an invariant CysPheThr tripeptide. DNMT2 binds DNA to form a denaturant-resistant complex in vitro. While the biological function of DNMT2 is not yet known, the strong binding to DNA suggests that DNMT2 may mark specific sequences in the genome by binding to DNA through the specific target-recognizing motif.

Methylation of the Protein Phosphatase 2A Catalytic Subunit Is Essential for Association of Bα Regulatory Subunit But Not SG2NA, Striatin, or Polyomavirus Middle Tumor Antigen

Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Bα subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Bα subunit binding without greatly affecting methylation, indicating that Bα subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Bα subunit but not the amount that could be coimmunoprecipitated with Aα subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Bα subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Bα subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.

A ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Chlorobium tepidum that is involved with sulfur metabolism and the response to oxidative stress

A gene encoding a product with substantial similarity to ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) was identified in the preliminary genome sequence of the green sulfur bacterium Chlorobium tepidum. A highly similar gene was subsequently isolated and sequenced from Chlorobium limicola f.sp. thiosulfatophilum strain Tassajara. Analysis of these amino acid sequences indicated that they lacked several conserved RubisCO active site residues. The Chlorobium RubisCO-like proteins are most closely related to deduced sequences in Bacillus subtilis and Archaeoglobus fulgidus, which also lack some typical RubisCO active site residues. When the C. tepidum gene encoding the RubisCO-like protein was disrupted, the resulting mutant strain displayed a pleiotropic phenotype with defects in photopigment content, photoautotrophic growth and carbon fixation rates, and sulfur metabolism. Most important, the mutant strain showed substantially enhanced accumulation of two oxidative stress proteins. These results indicated that the C. tepidum RubisCO-like protein might be involved in oxidative stress responses and/or sulfur metabolism. This protein might be an evolutional link to bona fide RubisCO and could serve as an important tool to analyze how the RubisCO active site developed.

An isoleucine/leucine residue in the carboxyltransferase domain of acetyl-CoA carboxylase is critical for interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors

cDNA fragments encoding the carboxyltransferase domain of the multidomain plastid acetyl-CoA carboxylase (ACCase) from herbicide-resistant maize and from herbicide-sensitive and herbicide-resistant Lolium rigidum were cloned and sequenced. A Leu residue was found in ACCases from herbicide-resistant plants at a position occupied by Ile in all ACCases from sensitive grasses studied so far. Leu is present at the equivalent position in herbicide-resistant ACCases from other eukaryotes. Chimeric ACCases containing a 1000-aa fragment of two ACCase isozymes found in a herbicide-resistant maize were expressed in a yeast ACC1 null mutant to test herbicide sensitivity of the enzyme in vivo and in vitro. One of the enzymes was resistant/tolerant, and one was sensitive to haloxyfop and sethoxydim, rendering the gene-replacement yeast strains resistant and sensitive to these compounds, respectively. The sensitive enzyme has an Ile residue, and the resistant one has a Leu residue at the putative herbicide-binding site. Additionally, a single Ile to Leu replacement at an equivalent position changes the wheat plastid ACCase from sensitive to resistant. The effect of the opposite substitution, Leu to Ile, makes Toxoplasma gondii apicoplast ACCase resistant to haloxyfop and clodinafop. In this case, inhibition of the carboxyltransferase activity of ACCase (second half-reaction) of a large fragment of the Toxoplasma enzyme expressed in Escherichia coli was tested. The critical amino acid residue is located close to a highly conserved motif of the carboxyltransferase domain, which is probably a part of the enzyme active site, providing the basis for the activity of fop and dim herbicides.

Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases.

Residue 225 in serine proteases is typically Pro or Tyr and specifies an important and unanticipated functional aspect of this class of enzymes. Proteases with Y225, like thrombin, are involved in highly specialized functions like blood coagulation and complement that are exclusively found in vertebrates. In these proteases, the catalytic activity is enhanced allosterically by Na+ binding. Proteases with P225, like trypsin, are typically involved in digestive functions and are also found in organisms as primitive as eubacteria. These proteases have no requirement for Na+ or other monovalent cations. The molecular origin of this physiologically important difference is remarkably simple and is revealed by a comparison of the Na+ binding loop of thrombin with the homologous region of trypsin. The carbonyl O atom of residue 224 makes a key contribution to the coordination shell of the bound Na+ in thrombin, but is oriented in a manner incompatible with Na+ binding in trypsin because of constraints imposed by P225 on the protein backbone. Pro at position 225 is therefore incompatible with Na+ binding and is a direct predictor of the lack of allosteric regulation in serine proteases. To directly test this hypothesis, we have engineered the thrombin mutant Y225P. This mutant has lost the ability to bind Na+ and behaves like the allosteric slow (Na(+)-free) form. The Na(+)-induced allosteric regulation also bears on the molecular evolution of serine proteases. A strong correlation exists between residue 225 and the codon used for the active site S195. Proteases with P225 typically use a TCN codon for S195, whereas proteases with Y225 use an AGY codon. It is proposed that serine proteases evolved from two main lineages: (i) TCN/P225 with a trypsin-like ancestor and (ii) AGY/Y225 with a thrombin-like ancestor. We predict that the Na(+)-induced allosteric regulation of catalytic activity can be introduced in the TCN/P225 lineage using the P225Y replacement.

Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach to this problem: the uncatalyzed hydrolysis of GTP is analyzed, and the understanding derived is applied to the Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from this chemical perspective suggests that proton abstraction from the attacking water by a general base and stabilization of charge development on the gamma-phosphoryl oxygen atoms would not be catalytic. Rather, this analysis focuses attention on the GDP leaving group, including the beta-gamma bridge oxygen of GTP, the atom that undergoes the largest change in charge in going from the ground state to the transition state. This leads to a new catalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-nonbridging phosphoryl oxygens and a network of interactions that positions the nucleophilic water molecule and gamma-phosphoryl group with respect to one another may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the beta-gamma bridge oxygen with an Arg side chain that is provided in trans by GAP. The conclusions for Ras and related G proteins are expected to apply more widely to other enzymes that catalyze phosphoryl (-PO(3)2-) transfer, including kinases and phosphatases.