The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter.

Escherichia coli MacAB-TolC is a tripartite macrolide efflux transporter driven by hydrolysis of ATP. In this complex, MacA is the periplasmic membrane fusion protein that stimulates the activity of MacB transporter and establishes the link with the outer membrane channel TolC. The molecular mechanism by which MacA stimulates MacB remains unknown. Here, we report that the periplasmic membrane proximal domain of MacA plays a critical role in functional MacA-MacB interactions and stimulation of MacB ATPase activity. Binding of MacA to MacB stabilizes the ATP-bound conformation of MacB, whereas interactions with both MacB and TolC affect the conformation of MacA. A single G353A substitution in the C-terminus of MacA inactivates MacAB-TolC function by changing the conformation of the membrane proximal domain of MacA and disrupting the proper assembly of the MacA-MacB complex. We propose that MacA acts in transport by promoting MacB transition into the closed ATP-bound conformation and in this respect, is similar to the periplasmic solute-binding proteins.

Unsaturated fatty acids augment protein transport via the SecA:SecYEG translocon.

The translocon SecYEG and the associated ATPase SecA form the primary protein secretion system in the cytoplasmic membrane of bacteria. The secretion is essentially dependent on the surrounding lipids, but the mechanistic understanding of their role in SecA : SecYEG activity is sparse. Here, we reveal that the unsaturated fatty acids (UFAs) of the membrane phospholipids, including tetraoleoyl-cardiolipin, stimulate SecA : SecYEG-mediated protein translocation up to ten-fold. Biophysical analysis and molecular dynamics simulations show that UFAs increase the area per lipid and cause loose packing of lipid head groups, where the N-terminal amphipathic helix of SecA docks. While UFAs do not affect the translocon folding, they promote SecA binding to the membrane, and the effect is enhanced up to fivefold at elevated ionic strength. Tight SecA : lipid interactions convert into the augmented translocation. Our results identify the fatty acid structure as a notable factor in SecA : SecYEG activity, which may be crucial for protein secretion in bacteria, which actively change their membrane composition in response to their habitat.

Sweetly expanding enzymatic glycodiversification.

Directed evolution is a powerful tool to modify substrate specificity for a wide array of enzyme catalysts. In this issue of Chemistry & Biology, Thorson and coworkers use directed evolution to increase the catalytic proficiency of a model glycosyltransferase, OleD, 300-fold for a nonphysiological substrate (Williams et al., 2008).

Optimizing glycosyltransferase specificity via "hot spot" saturation mutagenesis presents a catalyst for novobiocin glycorandomization.

A comprehensive two-phase "hot spot" saturation mutagenesis strategy for the rapid evolution of glycosyltransferase (GT) specificity for nonnatural acceptors is described. Specifically, the application of a high-throughput screen (based on the fluorescent acceptor umbelliferone) was used to identify key amino acid hot spots that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower-throughput screen to provide OleD prodigy capable of efficiently glycosylating the nonnatural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold.

Inversion of the anomeric configuration of the transferred sugar during inactivation of the macrolide antibiotic oleandomycin catalyzed by a macrolide glycosyltransferase.

Macrolides are a group of antibiotics structurally characterized by a macrocyclic lactone to which one or several deoxy-sugar moieties are attached. The sugar moieties are transferred to the different aglycones by glycosyltransferases (GTF). The OleI GTF of an oleandomycin producer, Streptomyces antibioticus, catalyzes the inactivation of this macrolide by glycosylation. The product of this reaction was isolated and its structure elucidated. The donor substrate of the reaction was UDP-alpha-D-glucose, but the reaction product showed a beta-glycosidic linkage. The inversion of the anomeric configuration of the transferred sugar and other data about the kinetics of the reaction and primary structure analysis of several GTFs are compatible with a reaction mechanism involving a single nucleophilic substitution at the sugar anomeric carbon in the catalytic center of the enzyme.

In vitro interaction of rat liver cytochromes P-450 with erythromycin, oleandomycin and erythralosamine derivatives. Importance of structural factors.

Several derivatives of the erythromycin, erythralosamine and oleandomycin series have been prepared. Their abilities to bind to rat liver microsomal cytochrome P-450 and to lead to the formation of stable 456 nm absorbing cytochrome P-450-metabolite complexes after their oxidative microsomal metabolism in vitro have been compared. The obtained data confirmed that cytochrome P-450 induced in rats either by macrolides or by 16 alpha-pregnenolone carbonitrile were the major isozymes involved in the binding of macrolides to liver microsomes and in metabolite-complex formation. They showed that (i) hydrophobicity was in general a beneficial factor for these two properties, (ii) the presence of a bulky substituent in position 3 of erythromycin dramatically decreased their affinity for these isozymes, and (iii) the simultaneous presence of bulky substituents in position 2' and 3 prevented iron-metabolite complex formation. These results led to the selection of two compounds, erythralosamine-2'-benzoate and erythralosamine-2',3-diacetate, which exhibited a particularly high affinity for macrolide inducible cytochrome P-450 and were very good precursors of cytochrome P-450-iron-metabolite complex formation.

Purification and characterization of an erythromycin esterase from an erythromycin-resistant Pseudomonas sp.

An erythromycin esterase (molecular mass 51200 Da) was purified from Pseudomonas sp. GD100, which was isolated from a salmon hatchery sediment sample from Washington State. The pI of the protein was 4.5-4.8. The enzyme was inhibited by 1 mM mercuric acid, and had the substrate specificity for structurally related 14-membered macrolides, which decreased in the order of oleandomycin, erythromycin A and erythromycin A enol ether. The activity for erythromycin A varied with temperature, but the effect of pH was minimal at pH 6.0-9.0. The half-life of the enzyme was estimated to be 8.9 h at 35 degrees C and 0.23 h at 55 degrees C, and the activation energy of the catalytic reaction of erythromycin A was estimated at 16.2 kJ mol(-1).

Characterization of the ATPase activity of the N-terminal nucleotide binding domain of an ABC transporter involved in oleandomycin secretion by Streptomyces antibioticus.

The oleB gene of Streptomyces antibioticus, oleandomycin producer, encodes an ABC transporter containing two putative ATP-binding domains and is involved in oleandomycin resistance and secretion in this organism. We have overexpressed in Escherichia coli the N-terminal nucleotide-binding domain of OleB (OleB') as a fusion protein and purified the fusion protein by affinity chromatography. The fusion protein showed ATPase activity dependent on the presence of Mg2+ ions. ATPase activity was resistant to specific inhibitors of P-, F-, and V-type ATPase whereas sodium azide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) were strong inhibitors. The change of Lys71, located within the Walker A motif of the OleB' protein, to Gln or Glu caused a loss of ATPase activity, whereas changing to Gly did not impair the activity. The results suggest that the intrinsic ATPase activity of purified fusion protein can be clearly distinguished from other ATP-hydrolysing enzymes, including ion-translocating ATPases or ABC-traffic ATPases, both on the basis of inhibition by different agents and since it hydrolyzes ATP without interacting with a hydrophobic membrane component.

Interspecies correlation of the pharmacokinetics of erythromycin, oleandomycin, and tylosin.

Knowledge of the disposition of macrolides in a single animal species has been insufficient for the prediction of the pharmacokinetics of macrolides in humans. To better understand the species differences in the pharmacokinetics of macrolide antibiotics, the disposition of erythromycin, oleandomycin, and tylosin in several mammalian species was examined. Generally, the serum concentration versus time profiles of these drugs after intravenous administration were described by two-compartment kinetic models and were similar within each species. These drugs were rapidly cleared, resulting in terminal half-lives of less than 2 h. Comparison of their pharmacokinetics showed greater variation in antibiotic disposition among animal species than noted for the differences within a species. When the pharmacokinetic data was fitted to an allometric model, the logarithms of volume of distribution, clearance, and half-life were linearly related to the logarithms of body weight. From these relationships, the human pharmacokinetics of erythromycin and oleandomycin were extrapolated and found to approximate observed human pharmacokinetics.

Enzymatic phosphorylation of macrolide antibiotics.

Five macrolide antibiotics (erythromycin A, 1; oleandomycin, 3a; tylosin, 4a; spiramycins, 5a; leucomycin A3, 6a) have been phosphorylated enzymatically using cell-free extracts derived from Streptomyces coelicolor UC 5240. The necessary cofactors and the rates of the conversion have been determined.

Cytochrome P450 (CYP105F2) from Streptomyces peucetius and its activity with oleandomycin.

The cytochrome P450 enzyme is one of the most versatile redox proteins and it is responsible for the oxidative metabolism of a wide variety of endogenous and exogenous compounds. The cytochrome P450 gene, CYP105F2, from Streptomyces peucetius was subcloned into the pET-32a(+) vector to overexpress the protein in E. coli BL21 (DE3) pLysS. The expressed enzyme was purified by fast protein liquid chromatography with a DEAE and UNO Q column. A 3D model was constructed based on the known crystallographic structures of cytochrome P450, and comparison with PikC and MoxA signified broad substrate specificity toward structurally diverse compounds. In addition, the in vitro hydroxylation of oleandomycin by purified CYP105F2 observed in liquid chromatography/mass spectrometry and mass/mass spectrometry indicated its flexibility towards alternative polyketides for the structural diversification of the macrolide by post-polyketide synthase hydroxylation.

Probing the breadth of macrolide glycosyltransferases: in vitro remodeling of a polyketide antibiotic creates active bacterial uptake and enhances potency.

The glycan portion of macrolide antibiotics modulates their efficacy. High-level expression of three macrolide GTs and kinetic analysis has revealed a highly selective synthetic "tool kit" with such plasticity that 12 glycan-modified macrolide antibiotics have been readily created. One of these (1-Gal) is enhanced over its parent oleandomycin (1) by "glycotargeting", allowing higher uptake through active internalization by virtue of the attachment of a glycan (Gal) not normally found on 1. Subsequent release of the targeting glycan by endogenous galactosidase activity releases 1.

Intracellular glycosylation and active efflux as mechanisms for resistance to oleandomycin in Streptomyces antibioticus, the producer organism.

Resistance to macrolides in producing organisms can be achieved by target site modification, intracellular inactivation of the antibiotic or active efflux mechanisms for the excretion of the antibiotic. The oleandomycin producer, Streptomyces antibioticus, possesses oleandomycin-sensitive ribosomes all along the cell cycle. However, it contains an intracellular glycosyltransferase capable of inactivating oleandomycin in the presence of UDP-glucose as cofactor. The correspondent gene (oleD) has been cloned and sequenced and the glycosyltransferase purified. Two other genes (oleB and oleC) that confer oleandomycin resistance have been cloned and characterized and both encode ABC (ATP-Binding Cassette) transporters. These may constitute the excretion mechanism throughout which the glycosylated oleandomycin is excreted. A second enzyme activity has been purified from culture supernatants of the oleandomycin producer that releases the glucose from the inactive glycosylated oleandomycin generating active antibiotic. This enzyme would probably catalyse the last step in the biosynthesis of oleandomycin.

[Oleandomycin content of mycelium and fermentation solution during cultivation of Streptomyces antibioticus]. / Dinamika soderzhaniia oleandomitsina v mitselii i nativnom rastvore pri kul'tivirovanii Streptomyces antibioticus.

The time-course of the oleandomycin content in the mycelium and fermentation broth-filtrate was studied by the microbiological assay at different periods of cultivation of strains 471 and 961 in fermenters and flasks containing a rich soybean-corn medium. It was shown that centrifugation of the mycelium over the sucrose density gradient induced a 25-80 per cent decrease in its moist weight at the expense of removal of the admixture components of the rich medium. Addition of glucose (2 per cent) to the culture-grown in a lactose medium by the 72nd hour of fermentation had no effect on further increase of the cell biomass. However, it lowered the content of the mycelium-fixed and excreted antibiotic at all the subsequent fermentation periods. The content of oleandomycin in the untreated mycelium was only 0.36 per cent of its content in the fermentation broth filtrate. After centrifugation of the mycelium over the sucrose density gradient and its intensive washing with distilled water the content of the mycelium-fixed antibiotic decreased still more. The time-course of the content of the mycelium-fixed and excreted oleandomycin was characterized by the presence of two activity peaks; by the 80-110th and by the 140-170th hour of cultivation.

[Pharmacokinetics of a new antibiotic preparation, novoolean]. / Farmakokinetika na noviia antibiotichen preparat novoolean.

We studied the resorption, the distribution, the duration of retention, as well as the routes of elimination by the organism of the complex antibiotic medicine (novobiocin salt of the oleandomycin). It was proved that after a single muscular injection of doses 20 and 50 mg/kg t the medicine, unlike its constituting components, possesses a retained action and keeps up bacteriostatic concentrations of blood in sheep, rabbits and chickens for a period of 12, respectively 24 hours. No substantial differences were observed between the blood levels created of the different species after the use of equal doses. No resorption was observed (or practically an insignificant one) in the digestive tract of rabbits. After its resorption from the place of its application the 'novoolean' penetrates into all organs studied, tissues and liquids of the organisms of guinea pigs, except for the brain. The longest period of retention is that in the lungs and in the kidneys. It is eliminated by the organism mainly through the urine and the bile secretion, and in the case of the lactating mammals-through the milk. The organism of the treated animals is practically free from 'novoolean' after the fourth day and as to the milk--after the second day of the last intramuscular injection.

Purification and characterization of an extracellular enzyme from Streptomyces antibioticus that converts inactive glycosylated oleandomycin into the active antibiotic.

Cell-free extracts from the oleandomycin producer, Streptomyces antibioticus, possess an intracellular glycosyltransferase capable of inactivating oleandomycin by glycosylation of the 2'-hydroxyl group in the desosamine moiety of the molecule [Vilches, C., Hernández, C., Méndez, C. & Salas, J. A. (1992) J. Bacteriol. 174, 161-165]. Using a four-step purification procedure, we have purified an enzyme activity from the culture supernatants from this organism which is able to release glucose from the inactive glycosylated molecule thus reactivating the antibiotic activity. This enzyme activity appeared in the culture supernatants immediately before oleandomycin is detected. The enzyme (molecular mass 87 kDa) showed a high degree of substrate specificity, not acting on other glycosylated macrolides such as methymycin, lankamycin and rosaramicin which are substrates for the glycosyltransferase. A second activity was detected corresponding to a 34-kDa polypeptide which probably originates from proteolytic cleavage of the larger polypeptide. The 87-kDa polypeptide possibly catalyses the last biosynthetic step in oleandomycin biosynthesis by S. antibioticus.

Application of nuclear magnetic resonance spectrometry to measure the activity of bacterial macrolide esterase.

A new method of measuring the activity of macrolide antibiotic esterase in whole bacteria and in crude enzyme extracts was determined through the application of nuclear magnetic resonance spectrometry. The structure of the enzymatically esterolytic cleavage compound of oleandomycin was clearly shown to be the cyclic hemiacetal between the C-9 keto and the hydroxy group of C-13 in the open macrolide nucleus by physicochemical techniques.