游动放线菌中orf20基因敲除对雷莫拉宁合成的影响

雷莫拉宁生物合成基因簇中orf20与已知的卤化酶基因高度同源.本研究在大肠杆菌中构建同框敲除质粒pSM-3,将其转入游动放线菌Actinoplanes sp.ATCC 33076,通过同源重组双交换的方法将orf20基因内部编码64个氨基酸的序列敲除,筛选得到双交换阻断突变株SIPI-A.2001 dorf20(Δor...     

Mischarging Escherichia coli tRNAPhe with L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine, a photoactivatable analogue of phenylalanine

The Boc-protected derivative of a photoactivatable, carbene-generating analogue of phenylalanine, L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine [(Tmd)Phe], was used to acylate 5'-O-phosphorylcytidylyl(3'-5')adenosine (pCpA). A diacyl species was isolated which upon successive treatments with trifluoroacetic acid and 0.01 M HCl yielded a 1:1 mixture of 2'(3')-O-(Tmd)phenylalanyl-pCpA and of its 2'-5'-phosphodiester isomeric form. Adapting a procedure introduced by Hecht's group [Heckler, T.G., Chang, L.H., Zama, Y., Naka, T., Chorghade, M.S., & Hecht, S.M. (1984) Biochemistry 23, 1468-1473], brief incubation of a 15 molar excess of this material with Escherichia coli tRNAPhe, missing at the acceptor stem the last two nucleotides (pCpA), in the presence of T4 RNA ligase and ATP afforded "chemically misaminoacylated" tRNAPhe in approximately 50% yield. Following chromatographic purification on DEAE-Sephadex A-25, benzoylated DEAE-cellulose, and Bio-Gel P-6, the misaminoacylated tRNAPhe was characterized by (i) urea-polyacrylamide gel electrophoresis, (ii) enzymatic reaminoacylation under homologous conditions following chemical deacylation, and (iii) its ability to stimulate protein synthesis in an in vitro translation system which, through the addition of the phenylalanyl-tRNA synthetase inhibitor phenylalaninyl-AMP, was unable to charge its endogenous tRNAPhe. The data demonstrate that we have prepared a biologically active misaminoacylated tRNAPhe.     

A new bacterial mannosidase for the selective modification of ramoplanin and its derivatives

Ramoplanin is a lipoglycodepsipeptide produced by Actinoplanes sp. ATCC 33076 and active on bacterial cell wall biosynthesis by binding to Lipid II. A screening of an actinomycetes collection was performed to select enzymatic activities able to introduce specific modifications in the ramoplanin molecule. An extracellular mannosidase from Streptomyces GE 91081 was found to selectively remove one mannose unit from ramoplanin and tetrahydroramoplanin to give the corresponding mannosyl aglycones. These molecules show an improved microbiological activity versus some resistant staphylococci and streptococci, and are useful intermediates in the synthesis of novel ramoplanin-like antibiotics. The biotransformation of ramoplanin has been optimised to improve molar conversion and the transformation reaction rate.     

A new bacterial mannosidase for the selective modification of ramoplanin and its derivatives

Ramoplanin is a lipoglycodepsipeptide produced by Actinoplanes sp. ATCC 33076 and active on bacterial cell wall biosynthesis by binding to Lipid II. A screening of an actinomycetes collection was performed to select enzymatic activities able to introduce specific modifications in the ramoplanin molecule. An extracellular mannosidase from Streptomyces GE 91081 was found to selectively remove one mannose unit from ramoplanin and tetrahydroramoplanin to give the corresponding mannosyl aglycones. These molecules show an improved microbiological activity versus some resistant staphylococci and streptococci, and are useful intermediates in the synthesis of novel ramoplanin-like antibiotics. The biotransformation of ramoplanin has been optimised to improve molar conversion and the transformation reaction rate.     

Increase of the deacylation rate of PBP2x from Streptococcus pneumoniae by single point mutations mimicking the class A beta-lactamases.

The class A beta-lactamases and the transpeptidase domain of the penicillin-binding proteins (PBPs) share the same topology and conserved active-site residues. They both react with beta-lactams to form acylenzymes. The stability of the PBP acylenzymes results in the inhibition of the transpeptidase function and the antibiotic activity of the beta-lactams. In contrast, the deacylation of the beta-lactamases is extremely fast, resulting in a high turnover of beta-lactam hydrolysis, which confers resistance to these antibiotics. In TEM-1 beta-lactamase from Escherichia coli, Glu166 is required for the fast deacylation and occupies the same spatial location as Phe450 in PBP2x from Streptococcus pneumoniae. To gain insight into the deacylation mechanism of both enzymes, Phe450 of PBP2x was replaced by various residues. The introduction of ionizable side chains increased the deacylation rate, in a pH-dependent manner, for the acidic residues. The aspartic acid-containing variant had a 110-fold faster deacylation at pH 8. The magnitude of this effect is similar to that observed in a naturally occurring variant of PBP2x, which confers increased resistance to cephalosporins.     

Novel synthesis and pharmacological evaluation as alpha2-adrenoceptor ligands of O-phenylisouronium salts

The synthesis of nine new mono- and bis-O-phenylisouronium compounds (2, 6b-10b and 12b-14b) and their Boc-protected isourea precursors (2a, 6a-10a and 12a-14a) is described. The carbodiimide 4, which was formed, had been suggested as the reactive intermediate species and driving force of the reaction. All final substrates were tested as potential alpha(2)-ARs ligands in human brain tissue by means of radioligand binding experiments and were compared to the potential antidepressant 1, as well as other related guanidine containing derivatives.     

A chemoenzymatic route to mannosamine derivatives bearing different N-acyl groups

The chemoenzymatic route to 2-deoxy-2-propionamido-D-mannose (1b), 2-butyramido-2-deoxy-D-mannose (2b) and 2-deoxy-2-phenylacetamido-D-mannose (3b) involved N-acylation of 2-amino-2-deoxy-D-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido-D-glucose (1a), 2-butyramido-2-deoxy-D-glucose (2a) and 2-deoxy-2-phenylacetamido-D-glucose (3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy-D-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 degrees C and 8.0-9.0, respectively. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose and 2-acetamido-2-deoxy-6-O-sulfo-D-glucose but not 2-acetamido-2-deoxy-1-O-phospho-D-glucose or 2-acetamido-2-deoxy-D-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al3+, Ca2+, Co2+, Cu2+, Mn2+ or Zn2+ and activated by 1 mM Mg2+. The concentrated enzyme is highly stable at 4 degrees C in the presence of 0.1 M ammonium sulfate.     

The mechanism of action of ramoplanin and enduracidin

The lipoglycodepsipeptide antibiotic ramoplanin is proposed to inhibit bacterial cell wall biosynthesis by binding to intermediates along the pathway to mature peptidoglycan, which interferes with further enzymatic processing. Two sequential enzymatic steps can be blocked by ramoplanin, but there is no definitive information about whether one step is inhibited preferentially. Here we use inhibition kinetics and binding assays to assess whether ramoplanin and the related compound enduracidin have an intrinsic preference for one step over the other. Both ramoplanin and enduracidin preferentially inhibit the transglycosylation step of peptidoglycan biosynthesis compared with the MurG step. The basis for stronger inhibition is a greater affinity for the transglycosylase substrate Lipid II over the MurG substrate Lipid I. These results provide compelling evidence that ramoplanin's and enduracidin's primary cellular target is the transglycosylation step of peptidoglycan biosynthesis.     

Production of ramoplanin analogues by genetic engineering of Actinoplanes sp.

Ramoplanins are lipopeptides effective against a wide range of Gram-positive pathogens. Ramoplanin A2 is in Phase III clinical trials. The structure–activity relationship of the unique 2Z,4E-fatty acid side-chain of ramoplanins indicates a significant contribution to the antimicrobial activities but ramoplanin derivatives with longer 2Z,4E-fatty acid side-chains are not easy to obtain by semi-synthetic approaches. To construct a strain that produces such analogues, an acyl-CoA ligase gene in a ramoplanin-producing Actinoplanes was inactivated and a heterologous gene from an enduracidin producer (Streptomyces fungicidicus) was introduced into the mutant. The resulting strain produced three ramoplanin analogues with longer alkyl chains, in which X1 was purified. The MIC value of X1 was ~0.12 μg/ml against Entrococcus sp. and was also active against vancomycin-resistant Staphylococcus aureus (MIC = 2 μg/ml).     

Preparation of lyso-GM1 (II3Neu5AcGgOse4-long chain bases) by a one-pot reaction.

A simple procedure is described for preparing lyso-GM1, a GM1 derivative that lacks the fatty acid moiety, starting from GM1 ganglioside using a one-pot reaction. Ganglioside deacylation was carried out in KOH/propan-1-ol in the absence of oxygen. The yield of lyso-GM1 under optimal conditions (6 h, 90 degrees C, 0.2 N KOH, 1 mM GM1) was 54%. The chemical structure of lyso-GM1 was determined by 1H-NMR and FAB-MS analyses, thus proving that the acetamide groups of galactosamine and sialic acid units were not affected during the deacylation reaction.     

Chemistry and biology of the ramoplanin family of peptide antibiotics

The peptide antibiotic ramoplanin factor A2 is a promising clinical candidate for treatment of Gram-positive bacterial infections that are resistant to antibiotics such as glycopeptides, macrolides, and penicillins. Since its discovery in 1984, no clinical or laboratory-generated resistance to this antibiotic has been reported. The mechanism of action of ramoplanin involves sequestration of peptidoglycan biosynthesis Lipid intermediates, thus physically occluding these substrates from proper utilization by the late-stage peptidoglycan biosynthesis enzymes MurG and the transglycosylases (TGases). Ramoplanin is structurally related to two cell wall active lipodepsipeptide antibiotics, janiemycin, and enduracidin, and is functionally related to members of the lantibiotic class of antimicrobial peptides (mersacidin, actagardine, nisin, and epidermin) and glycopeptide antibiotics (vancomycin and teicoplanin). Peptidomimetic chemotherapeutics derived from the ramoplanin sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and related pathogens. Here we review the chemistry and biology of the ramoplanins including its discovery, structure elucidation, biosynthesis, antimicrobial activity, mechanism of action, and total synthesis.     

Efficient chemoenzymatic synthesis of (S)- and (R)-5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide: key intermediate for Src-SH2 inhibitor

A facile chemoenzymatic synthesis of both the S and R forms of 5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide a key intermediate of non-peptidic Src SH2 inhibitors is described. Both the enantiomers were synthesized in high optical purity (>99% ee) by reduction followed by lipase-mediated acylation of the precursor 6 in one-pot. Immobilized Pseudomonas cepacia lipase offered high degree of enantioselectivity with spontaneity.     

Photolysis and deacylation of inhibited chymotrypsin

Inhibited chymotrypsin was reactivated through the photolysis of the covalently bound light-reversible cinnamates described in our previous paper [Stoddard, B.L., Bruhnke, J., Porter, N.A., Ringe, D., & Petsko, G. (1990) Biochemistry 29, 4871-4879]. The light-induced deacylation was accomplished both in solution and in protein crystals, with the release of inhibitor from the crystal monitored and confirmed by X-ray diffraction. The product of photolysis has been characterized as a 3-methylcoumarin, leading to a mechanism for light-driven deacylation of an internal lactonization that is dependent on the presence of an internal hydroxyl nucleophile. The acyl enzyme formed from cinnamate A is not suitable for photochemical studies, as the complex has a short half-life in solution and does not have a chromophore that is well separated from protein absorbance. Cinnamate B, with a p-diethylamino substituent, shows an enzyme deacylation rate enhancement of 10(9) for the cis photoisomer relative to the trans starting material. The half-life and deacylation rate of this compound in the E-I complex after photon absorption have been directly measured by subsecond UV absorption studies. X-ray diffraction studies of photoactivation using a flow cell show that the cinnamate B acyl enzyme complex is fully capable of light-induced isomerization and regeneration of native enzyme in the crystalline state. The E-I complex formed upon binding of cinnamate A, however, shows little if any effect from irradiation due to competitive absorbance by the highly concentrated protein at the shorter UV wavelengths. Photolysis of cinnamate B appears to occur on a time scale fast enough for applications in crystallographic studies of enzymatic intermediate-state structures.     

Regulation of arachidonic acid metabolism in Madin-Darby canine kidney cells. Comparison of A23187 and 12-O-tetradecanoyl-phorbol-13-acetate

Challenge of Madin-Darby canine kidney (MDCK) cells with the divalent cation ionophore A23187 caused a marked increase in the deacylation of [3H]arachidonic acid but not of [14C]palmitic acid. When the cells were treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and A23187, there was an additional increase in the deacylation of [3H]arachidonic acid compared to that observed with either agent alone. In contrast to deacylation, the stimulation of prostaglandin production by A23187 was small compared to the stimulation by TPA. Cycloheximide inhibited synthesis of prostaglandins in TPA-treated cells, but did not block the stimulated deacylation caused by either TPA or A23187. These data indicate that, while both TPA and A23187 stimulated the deacylation of [3H]arachidonic acid, TPA had an additional, cycloheximide-sensitive effect that was required for efficient conversion of the release fatty acids to prostaglandins. Thus, although required, deacylation appeared to be independent of and insufficient to stimulate maximum prostaglandin synthesis in these cells.     

Conversion of 1-O-[3H]alkyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine to lyso platelet-activating factor by the CoA-independent transacylase in membrane fractions of human neutrophils

The first step in the synthesis of platelet-activating factor (PAF) in stimulated neutrophils is generally accepted to be hydrolysis of 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (1-O-alkyl-2-acyl-GPC), with 1-O-alkyl-2-arachidonoyl-GPC being the preferred precursor. Characterization of the enzymatic activity responsible for the hydrolysis of 1-O-alkyl-2-arachidonoyl-GPC has been hampered by lack of an active and reliable cell-free system for study. In the present studies, membrane preparations containing 1-O-[3H]alkyl-2-arachidonoyl-GPC were prepared from intact human neutrophils that had been labeled using 1-O-[3H]hexadecyl-2-lyso-GPC. When the labeled membrane preparations were incubated in the presence of unlabeled 1-O-alkyl-2-lyso-GPC (5 microM), rapid deacylation (up to 25% of the label in 10 min) of the 1-O-[3H]alkyl-2-arachidonoyl-GPC to 1-O-[3H]alkyl-2-lyso-GPC (lyso-PAF) was observed. The deacylation activity appeared to be the same in preparations from resting or stimulated cells. No requirement for Ca2+, various nucleotides, or protein kinase activation could be demonstrated. A number of observations indicated that [3H]lyso-PAF is formed in the system by the action of the CoA-independent transacylase present in the cells rather than by phospholipase A2. Both 1-O-alkyl-2-lyso-GPC and 1-acyl-2-lyso-GPC elicited deacylation of 1-O-[3H]alkyl-2-arachidonoyl-GPC, whereas neither 3-O-alkyl-2-lyso-GPC nor 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphorylcholine, which should act as detergents but are not transacylase substrates, effected deacylation. The deacylation activity and CoA-independent transacylase activities were blocked in parallel by a number of inhibitors and by heat inactivation. In preparations containing 1-O-alkyl-2-[3H]arachidonoyl-GPC, no release of free [3H]arachidonic acid was observed. However, a shift of the [3H]arachidonate into exogenous 1-O-tetradecyl-2-lyso-GPC was observed in the system. These findings are consistent with the generation of [3H]lyso-PAF by the CoA-independent transacylase activity.     

Chemoenzymatic synthesis of 1-deaza-pyridoxal 5′-phosphate

The first synthesis of 1-deaza-pyridoxal 5′-phosphate (2-formyl-3-hydroxy-4-methylbenzyl phosphate) is described. The chemoenzymatic approach described here is a reliable route to this important isosteric pyridoxal phosphate analogue. This work enables elucidation of the role of the pyridine nitrogen in pyridoxal 5′-phosphate dependent enzymes.     

Functional identification of the gene encoding the enzyme involved in mannosylation in ramoplanin biosynthesis in Actinoplanes sp.

Ramoplanin is a lipopeptide antibiotic active against multi-drug-resistant, Gram-positive pathogens. Structurally, it contains a di-mannose moiety attached to the peptide core at Hpg¹¹. The biosynthetic gene cluster of ramoplanin has already been reported and the assembly of the depsipeptide has been elucidated but the mechanism of transferring sugar moiety to the peptide core remains unclear. Sequence analysis of the biosynthetic gene cluster indicated ramo-orf29 was a mannosyltransferase candidate. To investigate the involvement of ramo-orf29 in ramoplanin biosynthesis, gene inactivation and complementation have been conducted in Actinoplanes sp. ATCC 33076 by homologous recombination. Metabolite analysis revealed that the ramo-orf29 inactivated mutant produced no ramoplanin but the ramoplanin aglycone. Thus, ramo-orf29 codes for the mannosyltransferase in the ramoplanin biosynthesis pathway. This lays the foundation for further exploitation of the ramoplanin mannosyltransferase and aglycone in combinatorial biosynthesis.     

Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans

Nonphosphorylating nicotinamide adenine dinucleotide (phosphate)- [NAD(P)-] dependent aldehyde dehydrogenases share a number of conserved amino acid residues, several of which are directly implicated in catalysis. In the present study, the role of Glu-268 from nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans was investigated. Its substitution by Ala resulted in a k(cat) decrease by 3 orders of magnitude. Pre-steady-state analysis showed that, for both the wild-type and E268A GAPNs, the rate-limiting step of the reaction is associated with deacylation. The pH dependence of the rate of acylation of wild-type GAPN is characterized by the contributions of distinct enzyme protonic species with two pK(a)s of 6.2 and 7.5. Substitution of Glu-268 by Ala resulted in a monosigmoidal pH dependence of the rate constant of acylation with a pK(a) of 6.2, which suggested the assignment of pK(a) 7.5 to Glu-268. Moreover, the E268A substitution did not significantly affect the efficiency of acylation of GAPN, showing that Glu-268 is not critically involved in the acylation, which includes Cys-302 nucleophilic activation and hydride transfer. On the contrary, the drastic decrease of the steady-state rate constant for the E268A GAPN demonstrated the essential role of Glu-268 in the deacylation. At basic pH, the solvent isotope effect of 2.3, characterized by a unique pK(a) of 7.7, and the linearity of the proton inventory showed that the rate-limiting process for deacylation is associated with the hydrolysis step and suggested that the glutamate form of Glu-268 acts as a base catalyst in this process. Surprisingly, the double-sigmoidal form of the pH-steady-state rate constant profile, characterized by pK(a) values of 6.1 and 7.4, revealed the high efficiency of the deacylation even at pH lower than 7.4. Therefore, we propose that the major role of Glu-268 is to promote deacylation through activation and orientation of the attacking water molecule, and in addition to act as a base catalyst at basic pH. From these results in relation to those recently described [Marchal, S., and Branlant, G. (1999) Biochemistry 38, 12950-12958], a scenario for the chemical catalysis of GAPN is proposed.     

In vitro activity of ramoplanin and comparator drugs against anaerobic intestinal bacteria from the perspective of potential utility in pathology involving bowel flora

Susceptibility of intestinal bacteria to various antimicrobial agents in vitro, together with levels of those agents achieved in the gut, provides information on the likely impact of the agents on the intestinal flora. Orally administered drugs that are poorly absorbed may be useful for treatment of intestinal infections and for certain other situations in which intestinal bacteria may play a role. The antimicrobial activity of ramoplanin (MDL 62,198) against 928 strains of intestinal anaerobic bacteria was determined using the NCCLS-approved Wadsworth brucella laked-blood agar dilution method. The activity of ramoplanin was compared with that of ampicillin, bacitracin, metronidazole, trimethoprim/sulfamethoxazole (TMP/SMX), and vancomycin. The organisms tested included Bacteroides fragilis group (n=89), other Bacteroides species (n=16), other anaerobic Gram-negative rods (n=56) anaerobic cocci (n=114), Clostridium species (n=426), and non-sporeforming anaerobic Gram-positive rods (n=227). The overall MIC(90)s of ramoplanin, ampicillin, bacitracin, metronidazole, and vancomycin were 256, 32, 128, 16, and 128 mcg/ml, respectively. Ramoplanin was almost always highly active vs. Gram-positive organisms and relatively poor in activity against Gram-negative organisms, particularly Bacteroides, Bilophila, Prevotella, and Veillonella. Vancomycin was quite similar to ramoplanin in its activity. Ampicillin was relatively poor in activity vs. organisms that often produce beta-lactamase, including most of the Gram-negative rods as well as Clostridium bolteae and C. clostridioforme. Bacitracin was relatively poor in activity against most anaerobic Gram-negative rods, but better vs. most Gram-positive organisms. Metronidazole was very active against all groups other than bifidobacteria and some strains of other types of non-sporeforming Gram-positive bacilli. TMP/SMX was very poorly active, with an MIC(90) of >2048 mcg/ml.     

Two distinct pathways of platelet-activating factor-induced hydrolysis of phosphoinositides in primary cultures of rat Kupffer cells

Stimulation of rat Kupffer cells in primary culture with platelet-activating factor (PAF) caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate with a concomitant increase in the levels of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,4-bisphosphate. This phospholipase C-mediated hydrolysis of polyphosphoinositides was independent of extracellular Ca2+ but was inhibited by the intracellular Ca2+ antagonist TMB-8. A second slower response to PAF was characterized by deacylation of PI leading to the accumulation of glycerophosphoinositol (GPI). PAF-induced GPI synthesis was not inhibited by TMB-8. These effects of PAF were accompanied by initial transient mobilization of Ca2+ from intracellular stores followed by a rather slow influx of Ca2+ from the extracellular medium. PAF-stimulated deacylation and phosphodiesteric hydrolysis of inositol lipids were differentially affected by cholera toxin and pertussis toxin. Pretreatment of the Kupffer cells with either of these toxins caused inhibition of phospholipase C activity. Pertussis toxin also inhibited PAF-stimulated deacylation. However, cholera toxin itself stimulated GPI release and addition of PAF to the cholera toxin-treated cells caused a further increase in GPI release. Phorbol ester inhibited PAF-induced phosphodiesteric hydrolysis of phosphoinositides, but not deacylation. PAF-induced metabolism of phosphoinositides was inhibited by the PAF antagonist, U66985. These results suggest that PAF-induced phosphodiesteric hydrolysis and deacylation of inositol phospholipids are regulated via distinct mechanisms involving activation of separate G-proteins in rat Kupffer cells. Also the regulation of phosphoinositide metabolism by Ca2+ mobilization from two separate Ca2+ pools is indicated by this study.