Crystal structure of Y34F mutant human mitochondrial manganese superoxide dismutase and the functional role of tyrosine 34

Tyrosine 34 is a prominent and conserved residue in the active site of the manganese superoxide dismutases in organisms from bacteria to man. We have prepared the mutant containing the replacement Tyr 34 --> Phe (Y34F) in human manganese superoxide dismutase (hMnSOD) and crystallized it in two different crystal forms, orthorhombic and hexagonal. Crystal structures of hMnSOD Y34F have been solved to 1.9 A resolution in a hexagonal crystal form, denoted as Y34Fhex, and to 2.2 A resolution in an orthorhombic crystal form, denoted as Y34Fortho. Both crystal forms give structures that are closely superimposable with that of wild-type hMnSOD, with the phenyl rings of Tyr 34 in the wild type and Phe 34 in the mutant very similar in orientation. Therefore, in Y34F, a hydrogen-bonded relay that links the metal-bound hydroxyl to ordered solvent (Mn-OH to Gln 143 to Tyr 34 to H2O to His 30) is broken. Surprisingly, the loss of the Tyr 34 hydrogen bonds resulted in large increases in stability (measured by Tm), suggesting that the Tyr 34 hydroxyl does not play a role in stabilizing active-site architecture. The functional role of the side chain hydroxyl of Tyr 34 can be evaluated by comparison of the Y34F mutant with the wild-type hMnSOD. Both wild-type and Y34F had kcat/Km near 10(9) M-1 s-1, close to diffusion-controlled; however, Y34F showed kcat for maximal catalysis smaller by 10-fold than the wild type. In addition, the mutant Y34F was more susceptible to product inhibition by peroxide than the wild-type enzyme. This activity profile and the breaking of the hydrogen-bonding chain at the active site caused by the replacement Tyr 34 --> Phe suggest that Tyr 34 is a proton donor for O2* - reduction to H2O2 or is involved indirectly by orienting solvent or other residues for proton transfer. Up to 100 mM buffers in solution failed to enhance catalysis by either Y34F or the wild-type hMnSOD, suggesting that protonation from solution cannot enhance the release of the inhibiting bound peroxide ion, likely reflecting the enclosure of the active site by conserved residues as shown by the X-ray structures. The increased thermostability of the mutant Y34F and equal diffusion-controlled activity of Y34F and wild-type enzymes with normal superoxide levels suggest that evolutionary conservation of active-site residues in metalloenzymes reflects constraints from extreme rather than average cellular conditions. This new hypothesis that extreme rather than normal substrate concentrations are a powerful constraint on residue conservation may apply most strongly to enzyme defenses where the ability to meet extreme conditions directly affects cell survival.     

Identification of a phosphate regulatory site and a low affinity binding site for glucose 6-phosphate in the N-terminal half of human brain hexokinase

Crystal structures of human hexokinase I reveal identical binding sites for phosphate and the 6-phosphoryl group of glucose 6-phosphate in proximity to Gly87, Ser88, Thr232, and Ser415, a binding site for the pyranose moiety of glucose 6-phosphate in proximity to Asp84, Asp413, and Ser449, and a single salt link involving Arg801 between the N- and C-terminal halves. Purified wild-type and mutant enzymes (Asp84 --> Ala, Gly87 --> Tyr, Ser88 --> Ala, Thr232 --> Ala, Asp413 --> Ala, Ser415 --> Ala, Ser449 --> Ala, and Arg801 --> Ala) were studied by kinetics and circular dichroism spectroscopy. All eight mutant hexokinases have kcat and Km values for substrates similar to those of wild-type hexokinase I. Inhibition of wild-type enzyme by 1,5-anhydroglucitol 6-phosphate is consistent with a high affinity binding site (Ki = 50 microM) and a second, low affinity binding site (Kii = 0.7 mM). The mutations of Asp84, Gly87, and Thr232 listed above eliminate inhibition because of the low affinity site, but none of the eight mutations influence Ki of the high affinity site. Relief of 1,5-anhydroglucitol 6-phosphate inhibition by phosphate for Asp84 --> Ala, Ser88 --> Ala, Ser415 --> Ala, Ser449 --> Ala and Arg801 --> Ala mutant enzymes is substantially less than that of wild-type hexokinase and completely absent in the Gly87 --> Tyr and Thr232 --> Ala mutants. The results support several conclusions. (i) The phosphate regulatory site is at the N-terminal domain as identified in crystal structures. (ii) The glucose 6-phosphate binding site at the N-terminal domain is a low affinity site and not the high affinity site associated with potent product inhibition. (iii) Arg801 participates in the regulatory mechanism of hexokinase I.     

DAMGO recognizes four residues in the third extracellular loop to discriminate between mu- and kappa-opioid receptors

Previously, we reported that replacement of the region from the fifth transmembrane domain to the C-terminus of kappa-opioid receptor with the corresponding region of mu-opioid receptor gives high affinity for [D-Ala2, N-MePhe4, Gly-ol5]enkephalin (DAMGO), a mu-opioid receptor-selective ligand, to the resultant chimeric receptor, suggesting that the difference in the amino acid sequence within this region is critical for the discrimination between mu- and kappa-opioid receptors by DAMGO. In the present study, we constructed further six mu/kappa-chimeric receptors and revealed that at least two separate regions around the third extracellular loop are critical for the discrimination between mu- and kappa-opioid receptors by DAMGO. Furthermore, we constructed several mutant receptors by a site-directed mutagenesis technique and found that the difference between Glu297 of kappa-opioid receptor and Lys303 of mu-opioid receptor in one region, and the difference between Ser310, Tyr312 and Tyr313 of kappa-opioid receptor and Val316, Trp318 and His319 of mu-opioid receptor in the other region, are critical for the discrimination between these receptors by DAMGO. The mutant receptor, kappa (E297K + Y313H + Y312W + S310V), in which the Glu297, Ser310, Tyr312 and Tyr313 of kappa-opioid receptor were changed to Lys, Val, Trp and His, respectively, bound to DAMGO with high affinity (Kd = 8.7 +/- 1.2 nM) and efficiently mediated the inhibitory effect of DAMGO on intracellular cAMP accumulation. The present results showed that these four amino acid residues act as determinants for the discrimination between mu- and kappa-opioid receptors by DAMGO.     

Core mutations that promote the calcium-induced allosteric transition of bovine recoverin

Recoverin is a small calcium binding protein involved in regulation of the phototransduction cascade in retinal rod cells. It functions as a calcium sensor by undergoing a cooperative, ligand-dependent conformational change, resulting in the extrusion of the N-terminal myristoyl group from a hydrophobic pocket. To test the role of certain core residues in tuning this allosteric switch, we have made and characterized two mutants: W31K, which replaces Trp31 with Lys; and a double mutant, I52A/Y53A, in which Ile52 and Tyr53 are both replaced by Ala. These mutations decrease the hydrophobicity of the myristoyl binding pocket. They are thus expected to make sequestering of the myristoyl group less favorable and destabilize the Ca2+-free state. As predicted, the myristoylated forms of the mutants exhibit increased affinity for Ca2+, whether monitored by equilibrium binding of 45Ca2+ (Kd = 17.2, 7.9, and 8.1 microM for wild type, W31K, and I52A/Y53A, respectively) or by the change in tryptophan fluorescence associated with the conformational change (Kd = 17.9, 3.6, and 4.4 microM for wild type, W31K, and I52A/Y53A, respectively). The mutants also exhibit decreased cooperativity of binding (Hill coefficient = 1.2 and 1.0 for W31K and I52A/Y53A vs 1. 4 for wild type). Binding of the mutant proteins to rod outer segment membranes occurs at lower Ca2+ concentrations compared to wild-type protein (K1/2 = 5.6, 2.2, and 1.0 microM for wild type, W31K, and I52A/Y53A, respectively). The unmyristoylated forms of the mutants exhibit biphasic Ca2+ binding curves, nearly identical to that observed for wild type. The binding data for the two mutants can be explained by a concerted allosteric model in which the mutations affect only the equilibrium constant L between the two allosteric forms, T (the Ca2+-free form) and R (the Ca2+-bound form), without affecting the intrinsic binding constants for the two Ca2+ sites. Two-dimensional NMR spectra of the Ca2+-free forms of the mutants have been compared to the wild-type spectrum, whose peaks have been assigned to specific residues (1). Many resonances assigned to residues in the C-terminal domain (residues 100-202) in the wild-type spectrum are identical in the mutant spectra, suggesting that the backbone structure of the C-terminal domain is probably unchanged in both mutants. The N-terminal domain, in which both mutations are located, reveals in each case numerous changes of undetermined spatial extent.     

Role of tyrosine 129 in the active site of spinach glycolate oxidase

The enzymatic properties and the three-dimensional structure of spinach glycolate oxidase which has the active-site Tyr129 replaced by Phe (Y129F glycolate oxidase) has been studied. The structure of the mutant is unperturbed which facilitates interpretation of the biochemical data. Y129F glycolate oxidase has an absorbance spectrum with maxima at 364 and 450 nm (epsilon max = 11400 M-1 cm-1). The spectrum indicates that the flavin is in its normal protonated form, i.e. the Y129F mutant does not lower the pKa of the N(3) of oxidized flavin as does the wild-type enzyme [Macheroux, P., Massey, V., Thiele, D. J., and Volokita, M. (1991) Biochemistry 30, 4612-4619]. This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pKa is above pH 9. In contrast to wild-type glycolate oxidase, oxalate does not perturb the absorbance spectrum of Y129F glycolate oxidase. Moreover oxalate does not inhibit the enzymatic activity of the mutant enzyme. Typical features of wild-type glycolate oxidase that are related to a positively charged lysine side chain near the flavin N(1)-C(2 = O), such as stabilization of the anionic flavin semiquinone and formation of tight N(5)-sulfite adducts, are all conserved in the Y129F mutant protein. Y129F glycolate oxidase exhibited about 3.5% of the wild-type activity. The lower turnover number for the mutant of 0.74 s-1 versus 20 s-1 for the wild-type enzyme amounts to an increase of the energy of the transition state of about 7.8 kJ/mol. Steady-state analysis gave Km values of 1.5 mM and 7 microM for glycolate and oxygen, respectively. The Km for glycolate is slightly higher than that found for wild-type glycolate oxidase (1 mM) whereas the Km for oxygen is much lower. As was the case for wild-type glycolate oxidase, reduction was found to be the rate-limiting step in catalysis, with a rate of 0.63 s-1. The kinetic properties of Y129F glycolate oxidase provide evidence that the main function of the hydroxyl group of Tyr129 is the stabilization of the transition state.     

Interaction of pyridoxal 5'-phosphate with tryptophan-139 at the subunit interface of dimeric D-amino acid transaminase

The crystal structure of dimeric bacterial D-amino acid transaminase shows that the indole rings of the two Trp-139 side chains face each other in the subunit interface about 10 angstroms from the coenzyme, pyridoxal 5'-phosphate. To determine whether it has a role in the catalytic efficiency of the enzyme or interacts with the coenzyme, Trp-139 has been substituted by several different types of amino acids, and the properties of these recombinant mutant enzymes have been compared to the wild-type enzyme. In the native wild-type holoenzyme, the fluorescence of one of the three Trp residues per monomer is almost completely quenched, probably due to its interaction with PLP since in the native wild-type apoenzyme devoid of PLP, tryptophan fluorescence is not quenched. Upon reconstitution of this apoenzyme with PLP, the tryptophan fluorescence is quenched to about the same extent as it is in the native wild-type enzyme. The site of fluorescence quenching is Trp-139 since the W139F mutant in which Trp-139 is replaced by Phe has about the same amount of fluorescence as the wild-type enzyme. The circular dichroism spectra of the holo and the apo forms of both the wild-type and the W139F enzymes in the far-ultraviolet show about the same degree of ellipticity, consistent with the absence of extensive global changes in protein structure. Furthermore, comparison of the circular dichroism spectrum of the W139F enzyme at 280 nm with the corresponding spectral region of the wild-type enzyme suggests a restricted microenvironment for Trp-139 in the latter enzyme. The functional importance of Trp-139 is also demonstrated by the finding that its replacement by Phe, His, Pro, or Ala gives mutant enzymes that are optimally active at temperatures below that of the wild-type enzyme and undergo the E-PLP --> E-PMP transition as a function of D-Ala concentration with reduced efficiency. The results suggest that a fully functional dimeric interface with the two juxtaposed indole rings of Trp-139 is important for optimal catalytic function and maximum thermostability of the enzyme and, furthermore, that there might be energy transfer between Trp-139 and coenzyme PLP.     

Quantitation of hepatitis G virus RNA in allogeneic bone marrow transplant recipients. Stem Cell Transplantation Team

In thermolysin, tryptophan 115 seems to be at the S2 subsite. Trp-115 was replaced with tyrosine, phenylalanine, leucine, and valine during site-directed mutagenesis in order to evaluate the role of Trp-115 in the proteolytic activity of thermolysin. The mutant enzymes with Tyr-115 or Phe-115 had as much proteolytic activity as the wild-type enzyme, but the other two mutant enzymes had no activity. We found earlier that the substitution of Trp-115 with alanine, glutamic acid, lysine, and glutamine causes the enzyme to lose all activity, so an aromatic amino acid at position 115 seems to be essential for thermolysin.     

Circular dichroism and electron microscopy of a core Y61F mutant of the F1 gene 5 single-stranded DNA-binding protein and theoretical analysis of CD spectra of four Tyr --> Phe substitutions

A core Y61F mutant of the gene 5 single-stranded DNA-binding protein (g5p) of f1 bacterial virus aggregated when expressed from a plasmid, but, after refolding in vitro, it behaved much like wild-type and may be a stability or folding mutant. Circular dichroism (CD) titrations showed the same cooperative polynucleotide binding modes for Y61F and wild-type g5p. There are n = 4 and n congruent with 2.5 modes for binding to poly[d(A)] at low ionic strengths, but n = 4, n = 3, and n congruent with 2-2.5 modes for binding to fd single-stranded viral DNA (fd ssDNA), where n is the number of nucleotides occluded by each bound g5p monomer in a given mode. Y61F g5p has slightly reduced affinity in the n = 4 mode. Electron microscopy showed that Y61F g5p forms left-handed nucleoprotein superhelices indistinguishable from wild-type. Progression from binding to fd ssDNA in the n = 4 to n = 3 to n congruent with 2-2.5 mode is accompanied by an increase in the number of helical turns, an increase from (7.7 +/- 0.3) to (9.5 +/- 0.3) to ( approximately 10-13) g5p dimers per turn, and a decrease in the number of DNA nucleotides per turn. From CD spectra for four of five possible Y --> F g5p mutants, we infer that the fifth tyrosine, Tyr 56, contributes strongly to the CD. Retention of a strong 229 nm CD band in all mutants indicates that all retain elements of the native structure. Spectra of Y26F, Y34F, and Y61F g5p imply limited mobility of the replacement Phe. Comparison of measured with calculated CD spectra also suggests limited mobility for Tyr 26 and Tyr 34 in g5p in solution, and provides new information that the g5p structure in solution may be dominated by Tyr 41 rotamers differing from that stabilized in the crystal.     

A hydrophobic cluster between transmembrane helices 5 and 6 constrains the thyrotropin-releasing hormone receptor in an inactive conformation

We have studied the role of a highly conserved tryptophan and other aromatic residues of the thyrotropin-releasing hormone (TRH) receptor (TRH-R) that are predicted by computer modeling to form a hydrophobic cluster between transmembrane helix (TM)5 and TM6. The affinity of a mutant TRH-R, in which Trp279 was substituted by alanine (W279A TRH-R), for most tested agonists was higher than that of wild-type (WT) TRH-R, whereas its affinity for inverse agonists was diminished, suggesting that W279A TRH-R is constitutively active. We found that W279A TRH-R exhibited 3.9-fold more signaling activity than WT TRH-R in the absence of agonist. This increased basal activity was inhibited by the inverse agonist midazolam, confirming that the mutant receptor is constitutively active. Computer-simulated models of the unoccupied WT TRH-R, the TRH-occupied WT TRH-R, and various TRH-R mutants predict that a hydrophobic cluster of residues, including Trp279 (TM6), Tyr282, and Phe199 (TM5), constrains the receptor in an inactive conformation. In support of this model, we found that substitution of Phe199 by alanine or of Tyr282 by alanine or phenylalanine, but not of Tyr200 (by alanine or phenylalanine), resulted in a constitutively active receptor. We propose that a hydrophobic cluster including residues in TM5 and TM6 constrains the TRH-R in an inactive conformation via interhelical interactions. Disruption of these constraints by TRH binding or by mutation leads to changes in the relative positions of TM5 and TM6 and to the formation of an active form of TRH-R.     

The binding of bromophenol blue to aspartate aminotransferase

The effects of solvent polarity, heavy atoms and sulphur-containing compounds on the fluorescence and phosphorescence of indole and tryptophan have been investigated. The low-temperature luminescence of a group of dipeptides containing tryptophan is also reported. The phosphorescence to fluorescence quantum yield ratio and the phosphorescence lifetime are shown to be particularly sensitive to environmental factors. The relevance of these results to the interpretation of protein luminescence spectra is briefly discussed.     

Analysis of the human factor VIII A2 inhibitor epitope by alanine scanning mutagenesis

Antibodies directed to the A2 domain of factor VIII (fVIII) are usually an important component of the polyclonal response in patients who have clinically significant inhibitory antibodies to fVIII. A major determinant of the A2 epitope has been located by homolog scanning mutagenesis using recombinant hybrid human/porcine fVIII molecules to a sequence bounded by Arg484-Ile508 (Healey, J. F. , Lubin, I. M., Nakai, H., Saenko, E. L., Hoyer, L. W., Scandella, D. , and Lollar, P. (1995) J. Biol. Chem. 270, 14505-14509). Within this region, human residues Arg484, Pro485, Tyr487, Ser488, Arg489, Pro492, Val495, Phe501, and Ile508 differ from porcine fVIII. We stably expressed in mammalian cells nine active B-domainless human fVIII molecules containing single alanine substitutions at these sites. Their inhibition by a murine anti-A2 monoclonal antibody, monoclonal antibody (mAb) 413, and by three A2-specific alloimmune and two A2-specific autoimmune human inhibitor plasmas was measured by the Bethesda assay. The inhibition of Arg484 --> Ala, Tyr487 --> Ala, Arg489 --> Ala, and Arg492 --> Ala by mAb413 was reduced by greater than 90% compared with wild-type, B-domainless human fVIII. mAb413 inhibited the most severely affected mutant, Arg489 --> Ala, 0.01% as well as wild-type fVIII. For all five patient plasmas, the Tyr487 --> Ala mutant displayed the greatest reduction in inhibition. The inhibition of the Tyr487 --> Ala mutant by these antibodies ranged from 10% to 20% that of wild-type fVIII. The inhibition of the Ser488 --> Ala, Arg489 --> Ala, Pro492 --> Ala, Val495 --> Ala, Phe501 --> Ala, and Ile508 --> Ala mutants by most of the plasmas also was significantly reduced. In contrast, the Arg484 --> Ala and Pro485 --> Ala mutants were relatively unaffected. Thus, although mAb413 binds to the same region as human A2 inhibitors, it recognizes a different set of amino acid side chains. The side chains recognized by human A2 inhibitors appear to be similar, despite the differing immune settings that give rise to fVIII alloantibodies and autoantibodies.     

Steady state and time-resolved fluorescence study of residual structures in an unfolded form of yeast phosphoglycerate kinase

A previous study performed using steady state fluorescence has revealed the existence of residual structures surrounding the two tryptophan residues in an unfolded form of yeast phosphoglycerate kinase [Garcia, P., et al. (1995) Biochemistry 34, 397-404]. In this paper, we present a more detailed characterization of these residual structures, through the study of two single tryptophan-containing mutants of yPGK, W333F and W308Y. Denaturation experiments have first been performed at low temperatures to assess the nature of the interactions stabilizing these residual structures. On the other hand, the compactness and dynamics of the protein matrix were probed using tryptophan fluorescence quenching by acrylamide at various denaturant concentrations. Taking into consideration the changes in sample viscosity induced by addition of guanidinium chloride made feasible the use of this technique during the denaturation process. These different approaches have shown that the residual structures around tryptophan 308 are mainly stabilized by hydrophobic interactions and are more compact and less fluctuant than the ones surrounding tryptophan 333. Native and denatured yPGK have also been studied by time-resolved fluorescence spectroscopy. In the native protein, tryptophan buried in the core, W333, is mainly associated with a lifetime close to 0.1 ns, whereas tryptophan that is partially accessible to the solvent, W308, has a lifetime close to 0. 5 ns. The time-resolved tryptophan fluorescence emission of wild-type yPGK can be accounted for quantitatively by the summed emissions of its two single tryptophan mutants. The significance of minor long lifetime components is discussed for the two tryptophan residues. This new assignment of fluorescent decay times has allowed for the detection of a folding intermediate in which the environment of tryptophan 333 is modified for denaturant concentrations lower than those for tryptophan 308.     

Four aromatic residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011: effects of replacements on substrate binding and cyclization characteristics

Three-dimensional structures of cyclodextrin glucanotransferases (CGTases) have revealed that four aromatic residues, which are highly conserved among CGTases but not found in alpha-amylases, are located in the active center. To analyze the roles of these aromatic residues, Phe-183, Tyr-195, Phe-259, and Phe-283 of Bacillus sp. 1011 CGTase were replaced by site-directed mutagenesis, and the effects of this procedure were examined. Y195L-CGTase, in which Tyr-195 was replaced by a leucine residue, underwent a drastic change in its cyclization characteristics: it produced considerably more gamma-cyclodextrin than the wild-type enzyme and virtually no alpha-cyclodextrin. Y195L-CGTase had increased Km values for cyclodextrins, whereas the values for a linear maltooligosaccharide donor were insignificantly changed. Taken together with the structural information of CGTase crystals soaked with substrates, we propose that Tyr-195 plays an important role in the spiral binding of substrate. Replacing either Phe-183 or Phe-259 with leucine induced increased Km values for acceptors. Furthermore, the double mutant F183L/F259L-CGTase had considerably decreased cyclization efficiency, but the intermolecular transglycosylation activity remained normal. These results indicated that Phe-183 and Phe-259 are cooperatively involved in acceptor binding, and that they play a critical role in cyclization when the nonreducing end of amylose binds to the active center of CGTase. Replacing Phe-283 with a leucine residue induced a decrease in kcat and in affinity for acarbose, suggesting that Phe-283 is involved in transition-state stabilization.     

Mutations in the environment of the primary quinone facilitate proton delivery to the secondary quinone in bacterial photosynthetic reaction centers

In Rhodobacter capsulatus, we constructed a quadruple mutant that reversed a structural asymmetry that contributes to the functional asymmetry of the two quinone sites. In the photosynthetically incompetent quadruple mutant RQ, two acidic residues near QB, L212Glu and L213Asp, have been mutated to Ala; conversely, in the QA pocket, the symmetry-related residues M246Ala and M247Ala have been mutated to Glu and Asp. We have selected photocompetent phenotypic revertants (designated RQrev3 and RQrev4) that carry compensatory mutations in both the QA and QB pockets. Near QA, the M246Ala --> Glu mutation remains in both revertants, but M247Asp is replaced by Tyr in RQrev3 and by Ala in RQrev4. The engineered L212Ala and L213Ala substitutions remain in the QB site of both revertants but are accompanied by an additional electrostatic-type mutation. To probe the respective influences of the mutations occurring near the QA and QB sites on electron and proton transfer, we have constructed two additional types of strains. First, "half" revertants were constructed that couple the QB site of the revertants with a wild-type QA site. Second, the QA sites of the two revertants were linked with the L212Glu-L213Asp --> Ala-Ala mutations of the QB site. We have studied the electron and proton-transfer kinetics on the first and second flashes in reaction centers from these strains by flash-induced absorption spectroscopy. Our data demonstrate that substantial improvements of the proton-transfer capabilities occur in the strains carrying the M246Ala --> Glu + M247Ala --> Tyr mutations near QA. Interestingly, this is not observed when only the M246Ala --> Glu mutation is present in the QA pocket. We suggest that the M247Ala --> Tyr mutation in the QA pocket, or possibly the coupled M246Ala --> Glu + M247Ala --> Tyr mutations, accelerates the uptake and delivery of protons to the QB anions. The M247Tyr substitution may enable additional pathways for proton transfer that are located near QA.     

Functional roles of the conserved aromatic amino acid residues at position 108 (motif IV) and position 196 (motif VIII) in base flipping and catalysis by the N6-adenine DNA methyltransferase from Thermus aquaticus

The DNA methyltransferase (Mtase) from Thermus aquaticus (M.TaqI) catalyzes the transfer of the activated methyl group of S-adenosyl-L-methionine to the N6 position of adenine within the double-stranded DNA sequence 5'-TCGA-3'. To achieve catalysis M.TaqI flips the target adenine out of the DNA helix. On the basis of the three-dimensional structure of M.TaqI in complex with the cofactor and its structural homology to the C5-cytosine DNA Mtase from Haemophilus haemolyticus, Tyr 108 and Phe 196 were suggested to interact with the extrahelical adenine. The functional roles of these two aromatic amino acid residues in M.TaqI were investigated by mutational analysis. The obtained mutant Mtases were analyzed in an improved kinetic assay, and their ability to flip the target base was studied in a fluorescence-based assay using a duplex oligodeoxynucleotide containing the fluorescent base analogue 2-aminopurine at the target position. While the mutant Mtases containing the aromatic amino acid Trp at position 108 or 196 (Y108W and F196W) showed almost wild-type catalytic activity, the mutant Mtases with the nonaromatic amino acid Ala (Y108A and F196A) had a strongly reduced catalytic constant. Y108A was still able to flip the target base, whereas F196A was strongly impaired in base flipping. These results indicate that Phe 196 is important for stabilizing the extrahelical target adenine and suggest that Tyr 108 is involved in placing the extrahelical target base in an optimal position for methyl group transfer. Since both aromatic amino acids belong to the conserved motifs IV and XIII found in N6-adenine and N4-cytosine DNA Mtases as well as in N6-adenine RNA Mtases, a similar function of aromatic amino acid residues within these motifs is expected for the different Mtases.     

Nucleotide binding sites in wild-type creatine kinase and in W227Y mutant probed by photochemical release of nucleotides and infrared difference spectroscopy

Structural changes induced by nucleotide binding to the wild-type rabbit muscle creatine kinase (CK) and to its W227Y mutant were compared and probed by reaction-induced difference spectroscopy (RIDS). The reaction was induced by the photorelease of nucleotide from the caged nucleotides ADP[Et(PhNO2)] or ATP[Et(PhNO2)], producing the RIDS of CK. The concomitant addition of a saturated concentration of nucleotide and caged nucleotide modified the RIDS of CK, permitting structural changes caused by nucleotide binding in the wild-type creatine kinase to be identified. The W227Y mutant was inactive and its nucleotide binding site was partially impaired as shown by the disappearance or decrease of several nucleotide-sensitive bands in the RIDS of W227Y mutant. The magnitude of the decrease was not the same for each band, suggesting that distinct groups of W227Y mutant were affected differently during nucleotide binding. More precisely, the binding sites for gamma-phosphate and beta-phosphate of the nucleotide were not accessible in W227Y mutant as shown by the absence of the phosphate-sensitive 1666-1667-cm(-1) and 1625-cm(-1) bands in the RIDS of W227Y mutant. However the binding site of other parts of the nucleotide was partially accessible, since the 1638-1639-cm(-1) phosphate-insensitive band did not completely vanish in the RIDS of W227Y mutant. The RIDS of W227Y mutant with ADP[Et(PhNO2)] and creatine lacked the 1613-cm(-1) and 1581-cm(-1) bands, associated with vibrational modes of creatine, suggesting that coupling between the binding sites of the nucleotide and of creatine was altered in W227Y mutant. These results are in accordance with the earlier suggestions that residue W227 in CK is essential for preventing water molecules from penetrating into the active site and for orienting nucleotide in the binding site, by forming stacking interactions between its indole group and purine of the nucleotide and its indole group.     

Isolated systolic hypertension: an important cardiovascular risk factor

BACKGROUND: The rising number of vancomycin-resistant enterococci (VREs) is a major concern to modern medicine because vancomycin is currently the 'last resort' drug for life-threatening infections. The D-alanyl-D-X ligases (where X is an hydroxy or amino acid) of bacteria catalyze a critical step in bacterial cell-wall peptidoglycan assembly. In bacteria that produce glycopeptide antibiotics and in opportunistic pathogens, including VREs, D-, D-ligases serve as switches that confer antibiotic resistance on the bacteria themselves. Peptidoglycans in vancomycin-sensitive bacteria end in D-alanyl-D-alanine, whereas in vancomycin-resistant bacteria they end in D-alanyl-D-lactate or D-alanyl-D-serine. RESULTS: We demonstrate that the selective utilization of D-serine by the Enterococcus casseliflavus VanC2 ligase can be altered by mutagenesis of one of two residues identified by homology to the X-ray structure of the Escherichia coli D-alanyl-Dalanine ligase (DdlB). The Arg322-->Met (R322M) and Phe250-->Tyr (F250Y) ligase mutants show a 36-44-fold decrease in the use of D-serine, as well as broadened specificity for utilization of other D-amino acids in place of D-serine. The F250Y R322M double mutant is effectively disabled as a D-alanyl-D-serine ligase and retains 10% of the catalytic activity of wild-type D-alanyl-D-alanine ligases, reflecting a 6,000-fold switch to the D-alanyl-D-alanine peptide. Correspondingly, the Leu282-->Arg mutant of the wild-type E. coli DdlB produced a 560-fold switch towards D-alanyl-D-serine formation. CONCLUSIONS: Single-residue changes in the active-site regions of D-, D-ligases can cause substantial changes in recognition and activation of hydroxy or amino acids that have consequences for glycopeptide antibiotic efficacy. The observations reported here should provide an approach for combatting antibiotic-resistant bacteria.     

High-level production, chemical modification and site-directed mutagenesis of a cephalosporin C acylase from Pseudomonas strain N176

A cephalosporin acylase from Pseudomonas strain N176 hydrolyses both 7-beta-(4-carboxybutanamido)-cephalosporanic acid (glutarylcephalosporanic acid) and cephalosporin C to 7-amino-cephalosporanic acid. However, its productivity in the original host was low and its activity against cephalosporin C was not sufficient for direct large-scale production of 7-amino-cephalosporanic acid. In order to overcome these problems, we established a high-level expression system for the acylase in Escherichia coli. Tyr270 in the acylase is reported to play an important role in the interaction with glutarylcephalosporanic acid, as determined from the reaction with an affinity-label reagent, 7 beta-(6-bromohexanoylamido) cephalosporanic acid [Ishii, Y., Saito, Y., Sasaki, H., Uchiyama, F., Hayashi, M., Nakamura, S. & Niwa, M. (1994) J. Ferment. Bioeng. 77, 598-603] and modification with tetranitromethane [Nobbs, T. J., Ishii, Y., Fujimura, T., Saito, Y. & Niwa, M. (1994) J. Ferment. Bioeng. 77, 604-609]. From carbamoylation with potassium cyanate and site-directed point mutagenesis of the cephalosporin C acylase, we have deduced that Tyr270 exists at a position where it can interact with a residue (possibly Ser239) corresponding to inactivation by carbamoylation. We mutated Met269 and Ala271 of the acylase and found that mutation of Met269 to Tyr or Phe caused a 1.6-fold and 1.7-fold increase, respectively, of specific activity against cephalosporin C as compared to that of the wild-type enzyme. Kinetic studies of these mutants revealed that their kcat values increased, although their Km values against cephalosporin C were not changed. These data indicate that the mutation of Met269 near Tyr270 induces a minor conformational change to increase the stability of the activated complex with the enzyme and cephalosporin C. In particular, a mutant in which Met269 was replaced by Tyr was 2.5-fold more efficient in converting cephalosporin C to 7-amino-cephalosporanic acid than the wild-type enzyme under conditions similar to those in a bio-reactor system.     

Solvent effects on horse apomyoglobin dynamics

The effects of the solvent conditions (buffer pH 9, 8, or 7 or buffer pH 6.5 alone or mixed with 3.2% ethanol or 6.2% formamide) on the protein dynamics of horse apomyoglobin were investigated through tryptophan fluorescence quenching, spectra, and decay properties. Raising the pH (which induces discontinuous protein conformation changes) increases the structural fluctuations inside the hydrophobic A, G, and H helix core. Mixed solutions containing either 3.2% ethanol or 6.2% formamide (which redistribute water molecules on the protein surface) produce protein dynamics changes in the vicinity of the two Trp residues, without inducing particular constraints on these very residues. Formamide increases, in the same way, the polarity and the protein flexibility while ethanol reduces both. The present fluorescence work also shows that, whatever the outside solvent, the two Trp residues W7 and W14, embedded in the A, G, and H helix core, are equally and statistically reached by small molecules diffusing inside the protein matrix. Hydrogen-tritium exchange measurements on the protein in mixed solvents reveal that the dynamics of the A, G, and H helix cluster and of the B and E helixes are greatly influenced by the nature of the outside medium. A small amount of formamide in the buffer increases the protein fluctuations while an ethanol-water mixture reduces them. We suggest that the hydratation state of the protein surface could be the relevant parameter of the protein dynamics.     

Tyrosine and tryptophan structure markers in hemoglobin ultraviolet resonance Raman spectra: mode assignments via subunit-specific isotope labeling of recombinant protein

Phenyl-deuterated tyrosine (Tyr-d4) and indole-deuterated tryptophan (Trp-d5) have been selectively incorporated into hemoglobin (Hb) by expressing the gene in auxotrophic strains of Escherichia coli. Ultraviolet resonance Raman (UVRR) spectra, using 229-nm excitation, show that difference features characteristic of the Hb quaternary R --> T transition are not perturbed by the incorporation of the isotopes. All the UVRR bands between 800 and 1700 cm-1 are assigned to either Tyr or Trp except for the 1511 cm-1 band, which had been thought to arise from the Trp 2 x W18 overtone. This band does not shift upon Trp or Tyr labeling but does shift 5 cm-1 in D2O, suggesting assignment to a histidine (His) residue. Its intensification in the T-state is consistent with His protonation. The alpha- and beta-subunits were selectively labeled, by reconstitution of labeled subunits with unlabeled subunits, to make isotope hybrids. Selective Tyr labeling identified the alpha subunits as the locus of the Y8a upshift observed in Hb, supporting the previous inference that this shift is associated with the T-state H-bond involving the interfacial Tyr alpha42 [Rodgers, Su, Subramaniam, & Spiro (1992) J. Am. Chem. Soc. 114, 3697]. Selective Trp labeling showed the Trp alpha14 contributions to the T - R difference spectrum to be negligible and confirmed Trp beta37 as the locus of the W3 difference signal, and probably of the remaining Trp signals as well. The observed downshift of W17 and upshift of Wd5 in the T-state are consistent with a stronger T-state H-bond between Trp beta37 and Asp alpha94; the resulting excitation profile red shift accounts for the dominance of the Trp beta37 contribution to the T - R difference UVRR spectrum.