The conformational flexibility of the antibiotic virginiamycin M1

The antibiotic virginiamycin is a combination of two molecules, virginiamycin M₁ (VM1) and virginiamycin S₁ (VS1) or analogues, which function synergistically by binding to bacterial ribosomes and inhibiting bacterial protein synthesis. Both VM1 and VS1 dissolve poorly in water and are soluble in more hydrophobic solvents. We have recently reported that the 3D conformation of VM1 in CDCl₃ solution (Aust. J. Chem. 57:415, 2004; Org. Biomol. Chem. 2:2919, 2004) differs markedly from the conformation bound to a VM1 binding enzyme (Sugantino and Roderick in Biochemistry 41:2209, 2002) and to 50S ribosomes (Hansen et al. in J. Mol. Biol. 330:1061, 2003) as found by X-ray crystallographic studies. We now report the results of further NMR studies and subsequent molecular modeling of VM1 dissolved in CD₃CN/H₂O and compare the structure with that in CD₃OD and CDCl₃. The conformations of VM1 in CD₃CN/H₂O, CD₃OD and CDCl₃ differ substantially from one another and from the bound form, with the aqueous form most like the bound structure. We propose that the flexibility of the VM1 molecule in response to environmental conditions contributes to its effectiveness as an antibiotic.     

Analysis of the reversible binding of virginiamycin M to ribosome and particle functions after removal of the antibiotic

Type A synergimycins (VM) were shown to act catalytically and to induce two ribosomal alterations: (a) inability to promote polypeptide synthesis; (b) high-affinity binding of type B synergimycins (VS). A claim for irreversible binding of type A synergimycins to ribosomes has promoted the present reinvestigation. Submission of ribosomes from VM-treated bacteria to a purification procedure (supposed to remove the drug, according to a low association constant previously reported) yielded particles still holding residual VM. The formation of VM.ribosome complexes, more stable than previously inferred but without covalent linkage, was deduced from the extractability of complexed VM by organic solvents. Moreover, incubation of these complexes with increasing amounts of anti-VM immunoglobulins progressively restored ribosome activity in protein synthesis. Binding of VS to ribosomes, by fluorimetric titrations in the presence of substoichiometric concentrations of VM, was incompatible with catalytic action of type A synergimycins. Ribosomes from VM-treated bacteria displayed also a higher affinity for VS than did control ribosomes. This property did not disappear when ribosome.VM complexes were incubated with anti-VM IgG, nor when VM-IgG complexes were withdrawn from the reaction mixture by protein A-agarose binding. We can conclude that VM binding produces: (1) an inhibition of ribosome-promoted peptide bond formation, which occurs only in the presence of the drug; and (2) an increase of ribosome affinity for VS, which lasts after VM removal. The linkage of this drug with ribosomes is tight but reversible and its action is stoichiometric.     

The in vitro binding of virginiamycin M to bacterial ribosomes and ribosomal subunits

Summary Virginiamycin M (VM), an antibiotic of type A synergimycin group of antibiotics, binds to bacterial ribosomes and subunits in vitro: the amount of linked drug is linearly dependent on ribosome and VM concentrations. The technique used to measure the association reaction is based on the finding that the unbound drug is adsorbed by norite A: this procedure is twice as sensitive as the sedimentation and filtration methods (for technical reasons, column chromatography and equilibrium dialysis are unsuitable for this study). Saturation curves with 70S and 50S particles overlap, thus indicating a comparable affinity of the inhibitor for ribosomes and large subunits; instead, very small amount of VM, if any, attaches to 30S particles. Kinetics of binding is influenced by the temperature; the 4° C and 25° C saturation curves overlap, however, upon pre-incubation of ribosomes in 10 mM Mg buffer at 37° C (reactivation). This suggests that binding of VM depends on the configura tion of the 50S particles, which is altered at low temperature. Differences in Mg⁺⁺ concentration in the range 1 to 20 mM do not modify the binding curve, nor does the replacement of K⁺ by either NH ₄ ⁺ or Na⁺. Previously bound labelled VM is slowly displaced by an excess of unlabeled VM, and the associa tion curve remains unchanged in the presence of VS. Binding of VM is inhibited (10 to 60%) in the presence of an excess (tenfold to hundredfold) of one of the 50S inhibitors: chloramphenicol, oleandomycin and erythromycin. From the Scatchard plot, an as sociation constant of 3.2 × 10⁵M^–1 has been calculated: this value is about 1/8 of that reported for VS, a component of type B synergimycin group of antibiotics. The v value is 0.85 for both ribosomes and large subunits, indicating a monomolecular association of VM with ribonucleoprotein particles.     

Fluorescence stopped flow analysis of the interaction of virginiamycin components and erythromycin with bacterial ribosomes

The kinetics of the interaction between the 50 S subunits (R) of bacterial ribosomes and the antibiotics virginiamycin S (VS), virginiamycin M (VM), and erythromycin have been studied by stopped flow fluorimetric analysis, based on the enhancement of VS fluorescence upon its binding to the 50 S ribosomal subunit. Virginiamycin components M and S exhibit a synergistic effect in vivo, which is characterized in vitro by a 5- to 10-fold increase of the affinity of ribosomes for VS, and by the loss of the ability of erythromycin to displace VS subsequent to the conformational change (from R to R*) produced by transient contact of ribosomes with VM. Our kinetic studies show that the VM-induced increase of the ribosomal affinity for VS (K*VS = 25 X 10(6) M-1 instead of KVS = 5.5 X 10(6) M-1) is due to a decrease of the dissociation rate constant (k*-VS = 0.008 s-1 instead of 0.04 s-1). The association rate constant remains practically the same (k+VS approximately k*+VS = 2.8 X 10(5) M-1 s-1), irrespective of the presence of VM. VS and erythromycin bind competitively to ribosomes. This effect has been exploited to determine the dissociation rate constant of VS directly by displacement experiments from VS . 50 S complexes, and the association rate constant of erythromycin (k+Ery = 3.2 X 10(5) M-1 S-1) on the basis of competition experiments for binding of free erythromycin and VS to ribosomes. By making use of the change in competition behavior of erythromycin and VS, after interaction of ribosomes with VM, the conformational change induced by VM has been explored. Within the experimentally available concentration region, the catalytic effect of VM has been shown to be coupled to its binding kinetics, and the association rate constant of VM has been determined (k+VM = 1.4 X 10(4) M-1 S-1). Evidence is presented for a low affinity binding of erythromycin (K*Ery approximately 3.3 X 10(4) M-1) to ribosomes altered by contact with VM. A model involving a sequence of 5 reactions has been proposed to explain the replacement of ribosome-bound erythromycin by VS upon contact of 50 S subunits with VM.     

Ribosome protection by tRNA derivatives against inactivation by virginiamycin M: evidence for two types of interaction of tRNA with the donor site of peptidyl transferase

Virginiamycin M (VM) was previously shown to interfere with the function of both the A and P sites of ribosomes and to inactivate tRNA-free ribosomes but not particles bearing peptidyl-tRNA. To explain these findings, the shielding ability afforded by tRNA derivatives positioned at the A and P sites against VM-produced inactivation was explored. Unacylated tRNA(Phe) was ineffective, irrespective of its position on the ribosome. Phe-tRNA and Ac-Phe-tRNA provided little protection when bound directly to the P site but were active when present at the A site. Protection by these tRNA derivatives was markedly enhanced by the formation of the first peptide bond and increased further upon elongation of peptide chains. Most of the shielding ability of Ac-Phe-tRNA and Phe-tRNA positioned at the A site was conserved when these tRNAs were translocated to the P site by the action of elongation factor G and GTP. Thus, a 5-10-fold difference in the protection afforded by these tRNAs was observed, depending on their mode of entry to the P site. This indicates the occurrence of two types of interaction of tRNA derivatives with the donor site of peptidyl transferase: one shared by acylated tRNAs directly bound to the ribosomal P site (no protection against VM) and the other characteristic of aminoacyl- or peptidyl-tRNA translocated from the A site (protection of peptidyl transferase against VM). To explain these data and previous observations with other protein synthesis inhibitors, a new model of peptidyl transferase is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity labeling of the virginiamycin S binding site on bacterial ribosome

Virginiamycin S (VS, a type B synergimycin) inhibits peptide bond synthesis in vitro and in vivo. The attachment of virginiamycin S to the large ribosomal subunit (50S) is competitively inhibited by erythromycin (Ery, a macrolide) and enhanced by virginiamycin M (VM, a type A synergimycin). We have previously shown, by fluorescence energy transfer measurements, that virginiamycin S binds at the base of the central protuberance of 50S, the putative location of peptidyltransferase domain [Di Giambattista et al. (1986) Biochemistry 25, 3540-3547]. In the present work, the ribosomal protein components at the virginiamycin S binding site were affinity labeled by the N-hydroxysuccinimide ester derivative (HSE) of this antibiotic. Evidence has been provided for (a) the association constant of HSE-ribosome complex formation being similar to that of native virginiamycin S, (b) HSE binding to ribosomes being antagonized by erythromycin and enhanced by virginiamycin M, and (c) a specific linkage of HSE with a single region of 50S, with virtually no fixation to 30S. After dissociation of covalent ribosome-HSE complexes, the resulting ribosomal proteins have been fractionated by electrophoresis and blotted to nitrocellulose, and the HSE-binding proteins have been detected by an immunoenzymometric procedure. More than 80% of label was present within a double spot corresponding to proteins L18 and L22, whose Rfs were modified by the affinity-labeling reagent. It is concluded that these proteins are components of the peptidyltransferase domain of bacterial ribosomes, for which a topographical model, including the available literature data, is proposed.     

Localization of virginiamycin S binding site on bacterial ribosome by fluorescence energy transfer

Virginiamycin S, a type B synergimycin inhibiting protein synthesis in bacteria, competes with erythromycin for binding to the 50S ribosomal subunits; the mechanism of action of the two antibiotics is unclear. Energy-transfer experiments between virginiamycin S (which is endowed with inherent fluorescence due to its hydroxypicolinyl moiety) and fluorescent coumarinyl derivatives of ribosomal proteins L7 and L10 have been carried out to locate the binding site of this antibiotic on the ribosome. Previous studies have indicated that two L7/L12 dimers can attach respectively to a strong binding site located on the central protuberance and to a weak binding site located on the stalk of the 50S subunits and that protein L10 is located at the base of the stalk. The distance between ribosome-bound virginiamycin S and a fluorophore located at the strong binding site of proteins L7/L12 (Lys-51 of L7) was found to be 56 (+/- 15) A. Virginiamycin S, on the other hand, was located at a distance exceeding 67 A from the weak binding site of L7/L12 dimers. A fluorophore positioned on the unique cysteine (Cys-70) of protein L10 and ribosome-bound virginiamycin S proved to be more than 60 A apart. From data available on the location of proteins L7/L12 and L10, a model is proposed, whereby the virginiamycin S binding site is placed at the base of the central protuberance of the 50S subunits, in proximity of the presumptive peptidyl transferase center. The binding sites of macrolides and lincosamides (related antibiotics of the MLS group) are expected to be very close to that of virginiamycin S.     

Crystal structure of Vat(D): an acetyltransferase that inactivates streptogramin group A antibiotics

The streptogramin class of antibiotics act to inhibit bacterial protein synthesis, and their semisynthetic derivatives, such as dalfopristin-quinupristin (Synercid), are used to treat serious or life-threatening infections due to multiply antibiotic resistant bacteria. Acquired resistance of the nosocomial pathogen Enterococcus faecium to the group A component of natural and semisynthetic streptogramin mixtures is a prerequisite for the streptogramin resistance phenotype and is mediated by a streptogramin acetyltransferase. The crystal structure of Vat(D), a streptogramin acetyltransferase from a human urinary isolate of E. faecium, has been determined as an apoenzyme and in complex with either acetyl-CoA or virginiamycin M1 and CoA. These structures illustrate the location and arrangement of residues at the active site, and point to His 82 as a residue that may function as a general base. The structural similarity of Vat(D) to the xenobiotic acetyltransferase from Pseudomonas aeruginosa indicates similarities in the catalytic mechanism for these enzymes as well as several shared and distinctive antibiotic binding interactions between these enzymes and their respective substrates. These results reveal the molecular basis for a reaction by which Gram-positive cocci acquire resistance to a last resort antibiotic.     

The action of virginiamycin M on the acceptor, donor, and catalytic sites of peptidyltransferase

Virginiamycin M inhibits both peptide bond formation and binding of aminoacyl-tRNA to bacterial ribosomes, and induces a lasting inactivation of the 50 S subunit (50 S). In the present work, the effects of this antibiotic on the acceptor and donor sites of peptidyltransferase have been explored, in the presence of virginiamycin M as well as after its removal. Virginiamycin M inhibited the binding of puromycin to ribosomes and reduced both the enzymatic and nonenzymatic binding of Phe-tRNA to the A site by inducing its release from the ribosomes (similar effects were observed with 50 S), whereas the antibiotic had no effect on the binding of unacylated tRNAPhe to the same site. Moreover, virginiamycin M caused Ac-Phe-tRNA or Phe-tRNA to be released from the ribosomal P site, when complexes were incubated with unacylated tRNA, elongation factor G, and GTP (similar finding with 50 S). Instead, peptide bond formation between Ac-Phe-tRNA positioned at the P site and Phe-tRNA at the A site was found to take place, albeit at a very low rate, in the presence of the antibiotic. The overall conclusion is that both the acceptor and donor substrate binding sites of the peptidyltransferase, which interact with the aminoacyl moiety of tRNA, are permanently altered upon transient contact of ribosomes with virginiamycin M.     

How do organic solvents affect peroxidase structure and function?

The effect of organic solvents on horseradish peroxidase structure and function has been studied. Some, but not complete, enzyme denaturation occurs even in low volumes of water-miscible organic solvents (e.g., greater than 30% v/v dioxane, greater than 50% v/v methanol, and greater than 20% v/v acetonitrile) as determined by the decreased difference between the fluorescence of peroxidase's sole tryptophan residue and free L-tryptophan in solution. Absorbance and electron paramagnetic resonance spectroscopies indicate exposure of peroxidase's active site to the organic solvent. This reduces the local polarity in the enzyme's active site and results in stronger hydrogen bonding of phenolic substrates to the enzyme. In extreme cases (e.g., 95% v/v dioxane, 90% v/v acetonitrile, and ethyl and butyl acetate containing 2 and 1% v/v aqueous buffer, respectively), the transition state of the enzymic reaction is sufficiently perturbed so as to alter the magnitude of the Hammett rho value. This is most likely the result of the increased strength of hydrogen bonding between electron-donating alkoxyphenols (negative sigma values) and an electrophilic group in the enzyme's active site, thereby reducing catalytic efficiencies for such substrates relative to alkyl- and chlorophenols. Perhaps the most important effect of the organic solvent, however, is the significant ground-state stabilization of phenolic substrates in organic media as opposed to aqueous buffer. This stabilization can account for nearly 4 orders of magnitude in reduction of catalytic efficiency and is manifested in increased Km's. This study indicates that enzymes can maintain much of their native active-site structure in organic media and that the effect of solvent on substrate thermodynamics must be considered.     

Inhibitory action of virginiamycin components on cell-free systems for polypeptide formation from Bacillus subtilis

Although virginiamycin components VM and VS are known to exert in vivo a synergistic inhibition of bacterial growth and viability, in cell-free systems only VM has proven active. In the present work, the in vivo and in vitro activities of VM and VS on Bacillus subtilis have been compared.Peptide formation in homogenates of bacteria previously incubated with either VM or VS was found strongly repressed; the 2 components acted synergistically. Ribosomes were fully responsible for this effect, as shown by mixed reconstitution experiments. On the other hand, cytoplasm from control bacteria disrupted in 10 mM Mg²⁺ buffer was refractory to in vitro inhibition by virginiamycin, whereas ribosomes prepared in 1 mM Mg²⁺ were sensitive to VM. VS was inactive on poly(U)-directed poly(phenylalanine) formation, and displayed some activity on the poly(A)-poly(lysine) system. In a cell-free system from Bacillus subtilis infected with phage 2C, both VM and VS were active and blocked synergistically protein synthesis in vitro. When the host cells were incubated with VS and the corresponding homogenate was then treated with VM, a complete inhibition of protein synthesis was observed. The present work, thus, describes the techniques for investigating the in vivo and in vitro action of synergimycins on the same organism, and for reproducing in vitro the synergistic interaction of type A and B components previously observed only in vivo.Abbreviations poly(U) poly(uridylic acid) - poly(A) poly(adenylic acid) - VM and VS the M and S components of virginiamycin - pfu plaque forming units     

Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution

For the first time, it is demonstrated that exposure of an enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes, namely, proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin were exposed to acetonitrile at 70 degrees C for three hr. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, proteinase K was analyzed in detail using X-ray diffraction method. The overall structure of the enzyme was found to be similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad remained intact after the treatment. However, the water structure in the substrate binding site underwent some rearrangement as some of the water molecules were either displaced or completely absent. The most striking observation concerning the water structure was the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules were located in the recognition site. Interlinked through water molecules, the sites occupied by acetonitrile molecules were independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu 96, Ile 107 and Leu 133. The development of such a hydrophobic environment at the recognition site introduced a striking conformation change in Ile 107 by rotating its side chain about C alpha-C beta bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change had earlier been observed in proteinase K when it was complexed to a substrate analogue, lactoferrin fragment.     

Complex Structure of Engineered Modular Domains Defining Molecular Interaction between ICAM-1 and Integrin LFA-1

Intermolecular contacts between integrin LFA-1 (α(L)β(2)) and ICAM-1 derive solely from the integrin α(L) I domain and the first domain (D1) of ICAM-1. This study presents a crystal structure of the engineered complex of the α(L) I domain and ICAM-1 D1. Previously, we engineered the I domain for high affinity by point mutations that were identified by a directed evolution approach. In order to examine α(L) I domain allostery between the C-terminal α7-helix (allosteric site) and the metal-ion dependent adhesion site (active site), we have chosen a high affinity variant without mutations directly influencing either the position of the α7-helix or the active sites. In our crystal, the α(L) I domain was found to have a high affinity conformation to D1 with its α7-helix displaced downward away from the binding interface, recapitulating a current understanding of the allostery in the I domain and its linkage to neighboring domains of integrins in signaling. To enable soluble D1 of ICAM-1 to fold on its own, we also engineered D1 to be functional by mutations, which were found to be those that would convert hydrogen bond networks in the solvent-excluded core into vdW contacts. The backbone structure of the β-sandwich fold and the epitope for I domain binding of the engineered D1 were essentially identical to those of wild-type D1. Most deviations in engineered D1 were found in the loops at the N-terminal region that interacts with human rhinovirus (HRV). Structural deviation found in engineered D1 was overall in agreement with the function of engineered D1 observed previously, i.e., full capacity binding to α(L) I domain but reduced interaction with HRV.     

Hydroxyl-terminated peptidomimetic inhibitors of neuronal nitric oxide synthase

The X-ray structure of previously studied dipeptidomimetic inhibitors bound in the active site of neuronal nitric oxide synthase (nNOS) presented a possibility for optimizing the strength of enzyme-inhibitor interactions as well as for enhancing bioavailability. These desirable properties may be attainable by replacement of the terminal amino group of the parent compounds (1-6) with a hydroxyl group (11-13, and 18-20). The hypothesized effect would be twofold: first, a change from a positively charged amino group to a neutral hydroxyl group might afford more drug-like character and blood-brain barrier permeability to the inhibitors; second, as suggested by docking studies, the incorporated hydroxyl group might displace an active site water molecule with which the terminal amino group of the original compounds indirectly hydrogen bonds. In vitro activity assays of the hydroxyl-terminated analogs (11-13 and 18-20) showed greater than an order of magnitude increase in K(i) values (decreased potency) relative to the amino-terminated compounds. These experimental data support the importance to enzyme binding of a potential electrostatic interaction relative to a hydrogen bonding interaction.     

A combined proton and phosphorus-31 nuclear magnetic resonance investigation of the combining site of M603, a phosphocholine-binding myeloma protein

Phosphorus-31 nuclear magnetic resonance (NMR) studies on the two phosphorus nuclei of the phosphonium analogue (Me3P+CH2CH2OPO3(2-)) of phosphocholine are used to monitor the charged subsites in the phosphocholine-binding immunoglobulin A mouse myeloma M603. Comparison of the 270-MHz 1H NMR difference spectrum on addition of either this analogue or phosphocholine to M603 and the almost identical changes in the pKa values of the phosphate groups on binding to M603 confirm that the analogue is a good model for phosphocholine. The pKa of the phosphate groups is decreased by 0.5 unit on binding to M603, which is consistent with the phosphate group being hydrogen bonding to Tyr-33H and Arg-95L, as suggested from the X-ray structure, and also implies that the binding energies for the mono- and dianion are similar. The P+Me3 moiety is used to probe the electrostatic interactions in the choline subsite. Titration of the chemical shift of the phosphonium phosphorus reflects a group on the protein that has a pKa value of less than or equal to 5, which from the refined X-ray structure (D.R. Davies, personal communication) of the site is assigned to Asp-97L. The choline subsite is monitored by using 1H NMR difference spectra, which indicates that the subsite is highly aromatic as expected from the crystal structure that places Trp-107H and Tyr-100L in this subsite. The ring current interactions from these rings can account for the 1H NMR chemical shift data on choline.     

Enhancement of catalytic efficiency of enzymes through exposure to anhydrous organic solvent at 70 degrees C. Three-dimensional structure of a treated serine proteinase at 2.2 A resolution

The enzyme behavior in anhydrous media has important applications in biotechnology. So far chemical modifications and protein engineering have been used to alter the catalytic power of the enzymes. For the first time, it is demonstrated that an exposure of enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes: proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin have been exposed to acetonitrile at 70 degrees C for three hours. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, the structure of one of these treated enzymes, proteinase K has been analyzed in detail using X-ray diffraction method. The overall structure of the enzyme is similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad is intact after the treatment. However, the water structure in the substrate binding site undergoes some rearrangement as some of the water molecules are either displaced or completely absent. The most striking observation concerning the water structure pertains to the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules are located in the recognition site. The sites occupied by acetonitrile molecules are independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. All of them are interlinked through water molecules. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu-96, Ile-107, and Leu-133. The development of such a hydrophobic environment at the recognition site introduces a striking conformation change in Ile-107 by rotating its side chain about C(alpha)--C(beta) bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change has earlier been observed in proteinase K when it is complexed to a substrate analog lactoferrin fragment.     

A metal ion-binding site in the kringle region of bovine prothrombin fragment 1.

45Ca(II) binding studies (equilibrium dialysis) on the kringle domain of bovine prothrombin fragment 1 were conducted using a mixture of peptides (residues 43-156 and 46-156) resulting from limited alpha-chymotryptic hydrolysis of fragment 1. Analysis of the Scatchard plot of these data indicates a single, low affinity Ca(II)-binding site to be present. Similar results were obtained from studies on the decarboxylated fragment 1 derivative, 10-gamma-MGlu-fragment 1. Acetylation of bovine fragment 1 in the absence of Ca(II) or Mg(II) ions results in the loss of the metal ion-promoted quenching of the intrinsic Trp fluorescence of the protein and the Ca(II)-mediated binding to phosphatidylserine/phosphatidylcholine (PS/PC) vesicles. The acetylation of the NH2 alpha-group of Ala-1 has been shown (Welsch, D. J., and Nelsestuen, G. L. (1988) Biochemistry 27, 4946-4952) to abolish the PS/PC binding property of fragment 1. The present study demonstrates that acetylation of a second site possibly Ser-79 or Thr-81 using the conditions described in the preceding paper results in loss of both the fluorescence transition and the Ca(II)-mediated PS/PC binding of the resulting protein derivative. Removal of the O-acetyl group at the Ser-79/Thr-81 site is accomplished by aminolysis with 0.2 M hydroxylamine, pH 10, 50 degrees C; the fluorescence transition is partially restored. PS/PC binding is partially restored if the NH2 alpha-group of Ala-1 is trinitrophenylated but is not restored if the NH2 alpha-group of Ala-1 is acetylated. We conclude that the Ser-79/Thr-81 site may represent a portion of the metal ion-binding site within the kringle domain of fragment 1. Occupancy of this site by a Ca(II) ion appears to be important in the binding of the protein to PS/PC vesicles.     

Characterization of biosynthetic gene cluster for the production of virginiamycin M, a streptogramin type A antibiotic, in Streptomyces virginiae

Virginiamycin M (VM) of Streptomyces virginiae is a hybrid polyketide-peptide antibiotic with peptide antibiotic virginiamycin S (VS) as its synergistic counterpart. VM and VS belong to the Streptogramin family, which is characterized by strong synergistic antibacterial activity, and their water-soluble derivatives are a new therapeutic option for combating vancomycin-resistant Gram-positive bacteria. Here, the VM biosynthetic gene cluster was isolated from S. virginiae in the 62-kb region located in the vicinity of the regulatory island for virginiamycin production. Sequence analysis revealed that the region consists of 19 complete open reading frames (ORFs) and one C-terminally truncated ORF, encoding hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS), typical PKS, enzymes synthesizing precursors for VM, transporters for resistance, regulatory proteins, and auxiliary enzymes. The involvement of the cloned gene cluster in VM biosynthesis was confirmed by gene disruption of virA encoding a hybrid PKS-NRPS megasynthetase, which resulted in complete loss of VM production without any effect on VS production. To assemble the VM core structure, VirA, VirF, VirG, and VirH consisting, as a whole, of 24 domains in 8 PKS modules and 7 domains in 2 NRPS modules were predicted to act as an acyltransferase (AT)-less hybrid PKS-NRPS, whereas VirB, VirC, VirD, and VirE are likely to be essential for the incorporation of the methyl group into the VM framework by a HMG-CoA synthase-based reaction. Among several uncommon features of gene organization in the VM gene cluster, the lack of AT domain in every PKS module and the presence of a discrete AT encoded by virI are notable. AT-overexpression by an additional copy of virI driven by ermEp() resulted in 1.5-fold increase of VM production, suggesting that the amount of VirI is partly limiting VM biosynthesis.     

Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis

The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.