A chromosomal locus required for copper resistance, competitive fitness, and cytochrome c biogenesis in Pseudomonas fluorescens.

A chromosomal locus required for copper resistance and competitive fitness was cloned from a strain of Pseudomonas fluorescens isolated from copper-contaminated agricultural soil. Sequence analysis of this locus revealed six open reading frames with homology to genes involved in cytochrome c biogenesis in other bacteria, helC, cycJ, cycK, tipB, cycL, and cycH, with the closest similarity being to the aeg-46.5(yej) region of the Escherichia coli chromosome. The proposed functions of these genes in other bacteria include the binding, transport, and coupling of heme to apocytochrome c in the periplasm of these Gram-negative bacteria. Putative heme-binding motifs were present in the predicted products of cycK and cycL, and TipB contained a putative disulfide oxidoreductase active site proposed to maintain the heme-binding site of the apocytochrome in a reduced state for ligation of heme. Tn3-gus mutagenesis showed that expression of the genes was constitutive but enhanced by copper, and confirmed that the genes function both in copper resistance and production of active cytochrome c. However, two mutants in cycH were copper-sensitive and oxidase-positive, suggesting that the functions of these genes, rather than cytochrome c oxidase itself, were required for resistance to copper.

An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective.

Overactivation of calcium-activated neutral protease (calpain) has been implicated in the pathophysiology of several degenerative conditions, including stroke, myocardial ischemia, neuromuscular degeneration, and cataract formation. Alpha-mercaptoacrylate derivatives (exemplified by PD150606), with potent and selective inhibitory actions against calpain, have been identified. PD150606 exhibits the following characteristics: (i) Ki values for mu- and m-calpains of 0.21 microM and 0.37 microM, respectively, (ii) high specificity for calpains relative to other proteases, (iii) uncompetitive inhibition with respect to substrate, and (iv) it does not shield calpain against inactivation by the active-site inhibitor trans-(epoxysuccinyl)-L-leucyl-amido-3-methylbutane, suggesting a nonactive site action for PD150606. The recombinant calcium-binding domain from each of the large or small subunits of mu-calpain was found to interact with PD150606. In low micromolar range, PD15O6O6 inhibited calpain activity in two intact cell systems. The neuroprotective effects of this class of compound were also demonstrated by the ability of PD150606 to attenuate hypoxic/hypoglycemic injury to cerebrocortical neurons in culture and excitotoxic injury to Purkinje cells in cerebellar slices.

Cloning and characterization of cDNAs for matrix metalloproteinases of regenerating newt limbs.

Matrix metalloproteinases (MMPs) of regenerating urodele limbs have been suggested to play crucial roles in the process of the dedifferentiation of cells in the damaged tissues and the ensuing blastema formation because the activation of MMPs is an early and conspicuous event occurring in the amputated limb. MMP cDNAs were cloned as products of the reverse transcription-PCR from cDNA libraries of newt limbs, and their structures were characterized. Three cDNAs encoding newt MMPs (2D-1, 2D-19, and 2D-24) have been cloned from second day postamputation regenerating limbs, and a cDNA (EB-1) was cloned from early bud-stage regenerating limbs. These cDNAs included the full-length coding regions. The deduced amino acid sequences of 2D-1, 2D-19, 2D-24, and EB-1 had a homology with mammalian MMP9, MMP3/10, MMP3/10, and MMP13, respectively. The basic motif of these newt MMP genes was similar to mammalian counterparts and contained regions encoding a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. However, some unique molecular evolutionary features were also found in the newt MMPs. cDNAs of 2D-19 and 2D-24 contained a specific insertion and deletion, respectively. The insertion of 2D-19 is threonine-rich, similar to the threonine cluster found in the collagenase-like sea urchin hatching enzyme. Northern blot analysis showed that the expression levels of the newt MMPs were dramatically increased after amputation, suggesting that they play an important role(s) in tissue remodeling of the regenerating limb.

Insights into antibody catalysis: structure of an oxygenation catalyst at 1.9-angstrom resolution.

The x-ray crystal structures of the sulfide oxidase antibody 28B4 and of antibody 28B4 complexed with hapten have been solved at 2.2-angstrom and 1.9-angstrom resolution, respectively. To our knowledge, these structures are the highest resolution catalytic antibody structures to date and provide insight into the molecular mechanism of this antibody-catalyzed monooxygenation reaction. Specifically, the data suggest that entropic restriction plays a fundamental role in catalysis through the precise alignment of the thioether substrate and oxidant. The antibody active site also stabilizes developing charge on both sulfur and periodate in the transition state via cation-pi and electrostatic interactions, respectively. In addition to demonstrating that the active site of antibody 28B4 does indeed reflect the mechanistic information programmed in the aminophosphonic acid hapten, these high-resolution structures provide a basis for enhancing turnover rates through mutagenesis and improved hapten design.

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.

Role of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.

Bovine pyruvate dehydrogenase phosphatase (PDP) is a Mg2+-dependent and Ca2+-stimulated heterodimer that is a member of the protein phosphatase 2C family and is localized to mitochondria. Insight into the function of the regulatory subunit of PDP (PDPr) has been gained. It decreases the sensitivity of the catalytic subunit of PDP (PDPc) to Mg2+. The apparent Km of PDPc for Mg2+ is increased about 5-fold, from about 0.35 mM to 1.6 mM. The polyamine spermine increases the sensitivity of PDP but not PDPc to Mg2+, apparently by interacting with PDPr. PDPc but not PDP can use the phosphopeptide RRAT(P)VA as a substrate. These observations are interpreted to indicate that PDPr blocks or distorts the active site of PDPc and that spermine produces a conformational change in PDPr that reverses its inhibitory effect. These findings suggest that PDPr may be involved in the insulin-induced activation of the mitochondrial PDP in adipose tissue, which is characterized by a decrease in its apparent Km for Mg2+.

Differences in the RNA binding sites of iron regulatory proteins and potential target diversity.

Posttranscriptional regulation of genes of mammalian iron metabolism is mediated by the interaction of iron regulatory proteins (IRPs) with RNA stem-loop sequence elements known as iron-responsive elements (IREs). There are two identified IRPs, IRP1 and IRP2, each of which binds consensus IREs present in eukaryotic transcripts with equal affinity. Site-directed mutagenesis of IRP1 and IRP2 reveals that, although the binding affinities for consensus IREs are indistinguishable, the contributions of arginine residues in the active-site cleft to the binding affinity are different in the two RNA binding sites. Furthermore, although each IRP binds the consensus IRE with high affinity, each IRP also binds a unique alternative ligand, which was identified in an in vitro systematic evolution of ligands by exponential enrichment procedure. Differences in the two binding sites may be important in the function of the IRE-IRP regulatory system.

The C-terminal region of human angiogenin has a dual role in enzymatic activity.

The ribonucleolytic activity of angiogenin (Ang) is essential to Ang's capacity to induce blood vessel formation. Previous x-ray diffraction and mutagenesis results have shown that the active site of the human protein is obstructed by Gln-117 and imply that the C-terminal region of Ang must undergo a conformational rearrangement to allow substrate binding and catalysis. As a first step toward structural characterization of this conformational change, additional site-directed mutagenesis and kinetic analysis have been used to examine the intramolecular interactions that stabilize the inactive conformation of the protein. Two residues of this region, Ile-119 and Phe-120, are found to make hydrophobic interactions with the remainder of the protein and thereby help to keep Gln-117 in its obstructive position. Furthermore, the suppression of activity by the intramolecular interactions of Ile-119 and Phe-120 is counterbalanced by an effect of the adjacent residues, Arg-121, Arg-122, and Pro-123 which do not appear to form contacts with the rest of the protein structure. They contribute to enzymatic activity, probably by constituting a peripheral subsite for binding polymeric substrates. The results reveal the nature of the conformational change in human Ang and assign a key role to the C-terminal region both in this process and, presumably, in the regulation of human Ang function.

Homing events in the gyrA gene of some mycobacteria.

The A subunit of DNA gyrase in Mycobacterium leprae, unlike its counterpart in Mycobacterium tuberculosis, is produced by protein splicing as its gene, gyrA, harbors a 1260-bp in-frame insertion encoding an intein, a putative homing endonuclease. Analysis of the gyrA locus from different mycobacterial species revealed the presence of inteins in Mycobacterium flavescens, Mycobacterium gordonae and Mycobacterium kansasii but not in 10 other pathogenic or saprophytic mycobacteria. In all four cases where intein coding sequences were found, they were localized in the same position in gyrA, immediately downstream of the codon for the key active-site residue Tyr-130. The intein products were similar, but not identical, in sequence and the splice junctions displayed all the features found in other polypeptides known to be produced by protein splicing from a precursor protein. Paired motifs, found in homing endonucleases encoded by some group I RNA introns, and inteins showing endonuclease activity, were present in the gyrA inteins as were other intein-specific signatures. Some strains of M. flavescens, M. gordonae, and M. kansasii were shown by PCR analysis to have inteinless gyrA genes, in contrast to the situation in M. leprae where all the isolates possessed insertions in gyrA. Sequencing of the corresponding regions revealed that, although the GyrA protein sequence was conserved, the nucleotide sequences differed in gyrA genes with and without inteins, suggesting that the homing endonuclease displays sequence specificity.

Batimastat, a potent matrix mealloproteinase inhibitor, exhibits an unexpected mode of binding.

Matrix metalloproteinase enzymes have been implicated in degenerative processes like tumor cell invasion, metastasis, and arthritis. Specific metalloproteinase inhibitors have been used to block tumor cell proliferation. We have examined the interaction of batimastat (BB-94) with a metalloproteinase [atrolysin C (Ht-d), EC 3.4.24.42] active site at 2.0-angstroms resolution (R = 16.8%). The title structure exhibits an unexpected binding geometry, with the thiophene ring deeply inserted into the primary specificity site. This unprecedented binding geometry dramatizes the significance of the cavernous primary specificity site, pointing the way for the design of a new generation of potential antitumor drugs.

Visualization of intermediate and transition-state structures in protein-tyrosine phosphatase catalysis.

Engineering site-specific amino acid substitutions into the protein-tyrosine phosphatase (PTPase) PTP1 and the dual-specific vaccinia H1-related phosphatase (VHR), has kinetically isolated the two chemical steps of the reaction and provided a rare opportunity for examining transition states and directly observing the phosphoenzyme intermediate. Changing serine to alanine in the active-site sequence motif HCXXGXXRS shifted the rate-limiting step from intermediate formation to intermediate hydrolysis. Using phosphorus 31P NMR, the covalent thiol-phosphate intermediate was directly observed during catalytic turnover. The importance of the conserved aspartic acid (D92 in VHR and D181 in PTP1) in both chemical steps was established. Kinetic analysis of D92N and D181N mutants indicated that aspartic acid acts as a general acid by protonating the leaving-group phenolic oxygen. Structure-reactivity experiments with native and aspartate mutant enzymes established that proton transfer is concomitant with P-O cleavage, such that no charge develops on the phenolic oxygen. Steady- and presteady-state kinetics, as well as NMR analysis of the double mutant D92N/S131A (VHR), suggested that the conserved aspartic acid functions as a general base during intermediate hydrolysis. As a general base, aspartate would activate a water molecule to facilitate nucleophilic attack. The amino acids involved in transition-state stabilization for cysteinylphosphate hydrolysis were confirmed by the x-ray structure of the Yersinia PTPase complexed with vanadate, a transition-state mimic that binds covalently to the active-site cysteine. Consistent with the NMR, x-ray, biochemical, and kinetic data, a unifying mechanism for catalysis is proposed.

Multiplex selection technique (MuST): an approach to clone transcription factor binding sites.

We have used a multiplex selection approach to construct a library of DNA-protein interaction sites recognized by many of the DNA-binding proteins present in a cell type. An estimated minimum of two-thirds of the binding sites present in a library prepared from activated Jurkat T cells represent authentic transcription factor binding sites. We used the library for isolation of "optimal" binding site probes that facilitated cloning of a factor and to identify binding activities induced within 2 hr of activation of Jurkat cells. Since a large fraction of the oligonucleotides obtained appear to represent "optimal" binding sites for sequence-specific DNA-binding proteins, it is feasible to construct a catalog of consensus binding sites for DNA-binding proteins in a given cell type. Qualitative and quantitative comparisons of the catalogs of binding site sequences from various cell types could provide valuable insights into the process of differentiation acting at the level of transcriptional control.

A two-subunit type I DNA topoisomerase (reverse gyrase) from an extreme hyperthermophile.

A recently described reverse gyrase from the hyperthermophilic methanogen Methanopyrus kandleri is the only known example of a heterodimeric type I topoisomerase. The enzyme is made up of a 42-kDa subunit which covalently interacts with DNA (RgyA) and a 138-kDa subunit which binds ATP (RgyB). We have now cloned and sequenced the genes for both subunits of this enzyme. Surprisingly, the universally conserved type I topoisomerase domain [Lima, C. D., Wang, J. C. & Mondragon, A. (1994) Nature (London) 367, 138-146] which has been found as a contiguous polypeptide in the prokaryotes and eukaryotes is shared between the protomers. The subdomain with the active-site tyrosine is entirely within RgyA, whereas the subdomain implicated in noncovalent binding of the cleaved DNA strand is contained entirely in RgyB. The appearance of this unique structure in a highly conserved enzyme family supports the hypothesis that the methanogens branched from other prokaryotes and eukaryotes very early in evolution.

Phosphinate analogs of D-, D-dipeptides: slow-binding inhibition and proteolysis protection of VanX, a D-, D-dipeptidase required for vancomycin resistance in Enterococcus faecium.

VanX is a D-Ala-D-Ala dipeptidase that is essential for vancomycin resistance in Enterococcus faecium. Contrary to most proteases and peptidases, it prefers to hydrolyze the amino substrate but not the related kinetically and thermodynamically more favorable ester substrate D-Ala-D-lactate. The enzymatic activity of VanX was previously found to be inhibited by the phosphinate analogs of the proposed tetrahedral intermediate for hydrolysis of D-Ala-D-Ala. Here we report that such phosphinates are slow-binding inhibitors. D-3-[(1-Aminoethyl)phosphinyl]-D-2-methylpropionic acid I showed a time-dependent onset of inhibition of VanX and a time-dependent return to uninhibited steady-state rates upon dilution of the enzyme/inhibitor mixture. The initial inhibition constant Ki after immediate addition of VanX to phosphinate I to form the E-I complex is 1.5 microM but is then lowered by a relatively slow isomerization step to a second complex, E-I*, with a final K*i of 0.47 microM. This slow-binding inhibition reflects a Km/K*i ratio of 2900:1. The rate constant for the slow dissociation of complex E-I* is 0.24 min-1. A phosphinate analog with an ethyl group replacing what would be the side chain of the second D-alanyl residue in the normal tetrahedral adduct gives a K*i value of 90 nM. Partial proteolysis of VanX reveals two protease-sensitive loop regions that are protected by the intermediate analog phosphinate, indicating that they may be part of the VanX active site.

The vertebrate alcohol dehydrogenase system: variable class II type form elucidates separate stages of enzymogenesis.

A mixed-class alcohol dehydrogenase has been characterized from avian liver. Its functional properties resemble the classical class I type enzyme in livers of humans and animals by exhibiting low Km and kcat values with alcohols (Km = 0.7 mM with ethanol) and low Ki values with 4-methylpyrazole (4 microM). These values are markedly different from corresponding parameters of class II and III enzymes. In contrast, the primary structure of this avian liver alcohol dehydrogenase reveals an overall relationship closer to class II and to some extent class III (69 and 65% residue identities, respectively) than to class I or the other classes of the human alcohol dehydrogenases (52-61%), the presence of an insertion (four positions in a segment close to position 120) as in class II but in no other class of the human enzymes, and the presence of several active site residues considered typical of the class II enzyme. Hence, the avian enzyme has mixed-class properties, being functionally similar to class I, yet structurally similar to class II, with which it also clusters in phylogenetic trees of characterized vertebrate alcohol dehydrogenases. Comparisons reveal that the class II enzyme is approximately 25% more variable than the "variable" class I enzyme, which itself is more variable than the "constant" class III enzyme. The overall extreme, and the unusual chromatographic behavior may explain why the class II enzyme has previously not been found outside mammals. The properties define a consistent pattern with apparently repeated generation of novel enzyme activities after separate gene duplications.