High yield expression of human BACE constructs in Eschericia coli for refolding, purification, and high resolution diffracting crystal forms.

BACE (beta-site APP cleaving enzyme) or beta-secretase, the enzyme responsible for processing APP to give the N-terminal portion of the Abeta peptide, is a membrane bound aspartyl protease consisting of an ectodomain catalytic unit, a C-terminal transmembrane segment and a cytoplasmic domain. Three BACE constructs, pET11a-BACE, pQE80L-BACE, and pQE70-BACE were designed to terminate at a position just before the transmembrane domain (Ser(432)) and are described schematically below. (1) pET11a-T7.Tag-G-S-M-(A-8GV......QTDES(432)), (2) pQE80L-Met-R-G-S-(His)(6)-G-S-I-E-T-D-(T(1)QH...QTDES(432)), and (3) pQE70-Met-BACE (R(36)GSFVEMG....PQTDES(432) (His) (6)) Each construct was over-expressed in Escherichia coli as inclusion bodies. The inclusion body proteins were solubilized in urea and refolded by dilution in water to yield active enzyme. Maximal activity for pET11a-BACE and pQE80L-BACE was usually reached at day 3 to 4, while construct pQE70-BACE required about 21 days. Active BACE was purified to homogeneity by anion-exchange chromatography and affinity chromatography over a column of immobilized peptide inhibitor. The process, easily scalable to 60 liters of cell culture, yielded in excess of 400 mg of active enzyme for crystallographic analysis. Highly purified pET11a-BACE and pQE70-BACE formed complexes with various inhibitors, the latter protein giving crystals diffracting up to 1.45 A resolution. In addition, a crystal form that does not require the presence of an inhibitor has been obtained for pQE70-BACE. This ligand-free crystal form has proven useful for the preparation of BACE-inhibitor complexes in soaking experiments.

Susceptibility of bacteria to serum lysis or phagocytosis following growth in subinhibitory levels of lincosaminide or spectinomycin related antibiotics.

The effects of subinhibitory concentrations of antibiotics on polymorphonuclear leukocyte (PML) and serum killing of Staphylococcus aureus 502A (UC 9116) and Escherichia coli UC 9451 were studied. Exposure of these bacteria to subinhibitory levels of certain lincosaminides, spectinomycin, or 6'-n-propylspectinomycin altered their susceptibility to these host defense mechanisms, while exposure to gentamicin had no effect. However, each organism responded differently to treatment with the antibiotics. S. aureus pretreated during log phase growth with subinhibitory concentrations of clindamycin, lincomycin, or pirlimycin was more susceptible to killing by PMLs than untreated bacteria. No effect on phagocytic killing was found when S. aureus was pretreated with spectinomycin, 6'-n-propylspectinomycin, or gentamicin. The S. aureus remained resistant to serum lysis despite antibiotic treatment. In contrast, spectinomycin and 6'-n-propylspectinomycin as well as clindamycin dramatically increased the susceptibility of E. coli to serum lysis (greater than 99% destroyed). Moderate killing of E. coli by PMLs was also found.

Isolation and characterization of a new antibiotic U-62162.

A new antibiotic U-62162 has been isolated from the fermentations of Streptomyces verdensis Dietz, sp. n. (UC-8157). The compound has been characterized and its gross structure has been elucidated. The antibiotic inhibited the growth of Gram-positive bacteria (particularly Staphylococcus aureus) but was inactive in experimentally infected animals.

Augmentation of serum bactericidal activity by paldimycin.

At concentrations below the MIC, paldimycin induced changes in Staphylococcus aureus 502A (UC 9116, ATCC 28417) which increased its sensitivity to serum. The enhanced sensitivity to serum was concentration dependent with the maximal sensitivity found when bacteria were grown in approximately 1/10 MIC of paldimycin. Within an 1-hour incubation, S. aureus 502A typically grew 1.5-2-fold in serum. Following exposure to paldimycin, however, approximately 30-50% of the bacteria were killed in serum. The paldimycin treated bacteria were not more susceptible to phagocytosis and killing by polymorphonuclear leukocytes. At the concentrations utilized, the Staphylococci were enlarged and had thickened cell walls. The organisms were still viable and replicating, but irregularities in cell division were observed in transmission electron micrographs.

Microbial O-carbamylation of novobiocin.

Novobiocin was inactivated by Streptomyces niveus US 2094 in fermentation. The inactivation product was isolated and characterized by NMR and MS as 2"-O-carbamylnovobiocin. The MICs of novobiocin and 2"-O-carbamylnovobiocin were determined for S. niveus strains.

MIC values do not predict the intraphagocytic killing of Staphylococcus aureus by naphthalenic ansamycins.

Ten naphthalenic ansamycins were compared for their ability to kill extracellular or phagocytosed Staphylococcus aureus 502A. These included rifamycins, streptovaricins and tolypomycin Y. Although the compounds differed markedly in killing extracellular S. aureus, there was surprisingly little difference between them in assisting human leukocytes to kill phagocytosed S. aureus. In fact, when compared to rifampin, some ansamycins that were less effective in killing extracellular bacteria were more effective in killing phagocytosed bacteria. These data, together with an analysis of structure and activity, suggested that a specific transport mechanism might be involved. First considered was a vitamin K transport mechanism. Indeed warfarin, a vitamin K antagonist, blocked the ability of rifampin to kill phagocytosed S. aureus, as did the coumarins, novobiocin and coumarin-3-carboxylic acid. However, direct evidence for a vitamin K transport mechanism could not be obtained using vitamin K preparations. The fused phenolic, bicyclic system common to all of these ansamycins was tentatively considered to be the portion necessary for phagocyte penetration.

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.

Biosynthesis of marcfortine A.

This report describes the results of a biosynthesis study of marcfortine A (MA). We report here that MA is derived from methionine, tryptophan, lysine and two isoprene units, the latter two being derived from acetic acid. From the 13C enrichment pattern of the pipecolic acid moeity we further conclude that this unit is derived from lysine via alpha-ketoglutarate. Therefore, we have accounted for the biogenesis of every carbon atom of MA and established the biosynthetic pathway for the pipecolic acid moiety of MA.

Microbiological N-deoxygenation and C-oxygenation of pioglitazone-N-oxide in a single fermentation.

Oxygenation of pioglitazone-N-oxide by a microorganism isolated from soil was accompanied by N-deoxygenation to produce the pioglitazone metabolites 5-[4-[2-[5-(1-hydroxyethyl)-2-pyridyl]ethoxy]benzyl]-2,4-thiazolidinedio ne and 5-[4-[2-(5-acetyl-2-pyridyl)ethoxy]benzyl]2,4-thiazolidinedione. The oxygenating/deoxygenating organism has been characterized as Streptomyces hygroscopicus strain 02179 (UC 11099). The culture has been deposited with Agricultural Research Service, USDA, with accession number NRRL 18975.

Purification of lincosaminide O-nucleotidyltransferase from Streptomyces coelicolor Müller.

An enzyme (lincosaminide O-nucleotidyltransferase) that catalyzes 3-(5'-ribonucleotidylation) of pirlimycin and several other lincosaminide antibodies has been purified approximately 35-fold from cell-free extracts of Streptomyces coelicolor Müller NRRL 3532 (UC 5240). The crude enzyme was prepared using lysozyme and was treated with MnCl2 and (NH4)2SO4. Final purification was achieved by anion exchange chromatography. The pirlimycin reaction product was verified as being pirlimycin-3-(5'-adenylate) by NMR spectroscopy and MS. As a result of purification, this lincosaminide nucleotidylating and inactivating enzyme was separated from the macrolide phosphorylating enzyme also present in the cell-free extract.

The effect of neutral resins on the fermentation production of rubradirin.

Rubradirin is an antibiotic of complex chemical structure which is active vs. methicillin resistant staphylococci. Its development has been limited due to inadequate production yields. The incorporation of neutral resins into fermentations of Streptomyces achromogenes v. rubradiris, UC 8051 resulted in the enhanced production of rubradirin. Resins HP-20, HP-21, XAD-2, XAD-7 and XAD-16 were employed in flask and tank fermentations. The incorporation of these resins promoted 2- to 4-fold enhancements of the rubradirin activity produced in flask fermentations, and the incorporation of XAD-16 and HP-21 into tank fermentations promoted production titer increases greater than 5 fold.

Microbial glycosylation of erythromycin A.

Erythromycin A (compound 1) was inactivated by Streptomyces vendargensis ATCC 25507 in fermentation. The inactivation product was isolated and characterized by nuclear magnetic resonance and mass spectroscopy as 2'-(O-[beta-D-glucopyranosyl])erythromycin A (compound 2). The MICs of compounds 1 and 2 were determined. Compound 2 lacked antibiotic activity when tested against several gram-positive pathogens, as well as S. vendargensis.

Effects of trospectomycin on serum sensitivity of Escherichia coli UC 9451.

Trospectomycin sulfate, a chemically synthesized analog of spectinomycin, exhibits a broad range of activity against both aerobes and anaerobes, including the etiological agents of sexually transmitted diseases. Its activity in vitro against Escherichia coli is considered only moderate. At subinhibitory levels, however, trospectomycin induced changes in a pathogenic strain of E. coli, UC 9451, which significantly increased its sensitivity to serum lysis. This strain of E. coli shows high-level resistance to serum in vitro, typically growing twofold within a 45-min incubation period. Following exposure to one-fifth the MIC of trospectomycin, greater than 99% of the bacteria were killed in 25% serum within 15 min. Surviving bacteria were static in this level of serum for over 3 h. Killing was due to lysis mediated by both the classical and alternative complement pathways. The bacteria exposed to trospectomycin were enlarged in both diameter and length, but they still grew at rates comparable to those of untreated bacteria. No other visible morphological changes could be directly related to the increase in serum sensitivity. The profile of outer membrane proteins obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was identical for trospectomycin-treated or untreated bacteria. However, the relative proportion of four major outer membrane proteins varied considerably.

A structural variation for MurB: X-ray crystal structure of Staphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase (MurB).

The X-ray crystal structure of the substrate free form of Staphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase (MurB) has been solved to 2.3 A resolution with an R-factor of 20.3% and a free R-factor of 22.3%. While the overall fold of the S. aureus enzyme is similar to that of the homologous Escherichia coli MurB X-ray crystal structure, notable distinctions between the S. aureus and E. coli MurB protein structures occur in residues involved in substrate binding. Analysis of available MurB sequences from other bacteria suggest that the S. aureus MurB structure is representative of a distinct structural class of UDP-N-acetylenolpyruvylglucosamine reductases including Bacillus subtilis and Helicobacter pylori that are characterized by a modified mechanism for substrate binding.