When Synthesis Gets It Wrong: Unexpected Epimerization Using PyBOP in the Synthesis of the Cyclic Peptide Thermoactinoamide A.

Chemical synthesis is commonly seen as the final proof of the structure of complex natural products, but even a seemingly easy and well-established synthetic procedure may lead to an unexpected result. This is what happened with the synthesis of thermoactinoamide A (1a), an antimicrobial and antitumor nonribosomal cyclic hexapeptide produced by the thermophilic bacterium Thermoactinomyces vulgaris. The synthetic thermoactinoamide A outsourced to a company and the one described in a synthetic paper showed spectroscopic data identical to each other but different from those of the natural product. After a detailed spectroscopic, degradative, and synthetic study, the synthetic compound was shown to be an epimer (1b) of the intended target compound, originating during the cyclization reaction by extensive epimerization at the activated C-terminal amino acid. This allowed confirmation of the structure of the natural product.

Structure of the microbial carboxypeptidase T complexed with the transition state analog N-sulfamoyl-l-lysine.

Carboxypeptidase T (CPT) from Thermoactinomyces vulgaris (EC 3.4.17.18) has a broad substrate specificity, the mechanism of which remains unclear. It cleaves off arginine residues by 10, and lysine residues by 100 times worse than hydrophobic leucine residues despite the presence of negatively charged Asp260 at the bottom of the primary specificity pocket. To study the relationship between the structure and specificity the 3D structure of CPT in complex with the stable transition state analog N-sulfamoyl-l-lysine (SLys) was determined in which the S-atom imitates the sp3-hybridized carbon in the scissile-bond. Crystals grown in microgravity has the symmetry of space group P6322. The present complex structure was compared with the previously reported complex structure of CPT and N-sulfamoyl-L-arginine (SArg). The location/binding of SLys in the active site of CPT very closely resembled that of SArg, and the positively charged N-atom of SLys was at the same position as the corresponding positively charged N-atom of SArg. The SLys complex is stabilized by the hydrogen bond between the nitrogen atom and OH-group of Thr257. The contact areas of the residues Tyr255, Leu211, and Thr262 with SLys were reduced in comparison with the same of SArg. This difference in bonding of SArg and SLys side chains in the primary specificity pocket induces shifts differences within the catalytic center (especially Tyr255-O20 and S18-Arg129 N1 gap) that may influence the enzyme's catalytic reaction. Therefore, this information may be useful for the design of carboxypeptidases with improved selectivity towards Arg/Lys for biotechnological applications.

Crystal structure of mutant carboxypeptidase T from Thermoactinomyces vulgaris with an implanted S1' subsite from pancreatic carboxypeptidase B.

A site-directed mutagenesis method has been used to obtain the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT), in which the amino-acid residues of the S1' subsite are substituted by the corresponding residues from pancreatic carboxypeptidase B (CPB). It was shown that the mutant enzyme retained the broad, mainly hydrophobic selectivity of wild-type CPT. The mutant containing the implanted CPB S1' subsite was crystallized and its three-dimensional structure was determined at 1.29 Šresolution by X-ray crystallography. A comparison of the three-dimensional structures of CPT, the G215S/A251G/T257A/D260G/T262D CPT mutant and CPB showed that the S1' subsite of CPT has not been distorted by the mutagenesis and adequately reproduces the structure of the CPB S1' subsite. The CPB-like mutant differs from CPB in substrate selectivity owing to differences between the two enzymes outside the S1' subsite. Moreover, the difference in substrate specificity between the enzymes was shown to be affected by residues other than those that directly contact the substrate.

Thermoactinoamide A, an Antibiotic Lipophilic Cyclopeptide from the Icelandic Thermophilic Bacterium Thermoactinomyces vulgaris.

The thermophilic bacterium Thermoactinomyces vulgaris strain ISCAR 2354, isolated from a coastal hydrothermal vent in Iceland, was shown to contain thermoactinoamide A (1), a new cyclic hexapeptide composed of mixed d and l amino acids, along with five minor analogues (2-6). The structure of 1 was determined by one- and two-dimensional NMR spectroscopy, high-resolution tandem mass spectrometry, and advanced Marfey's analysis of 1 and of the products of its partial hydrolysis. Thermoactinoamide A inhibited the growth of Staphylococcus aureus ATCC 6538 with an MIC value of 35 µM. On the basis of literature data and this work, cyclic hexapeptides with mixed d/l configurations, one aromatic amino acid residue, and a prevalence of lipophilic residues can be seen as a starting point to define a new, easily accessible scaffold in the search for new antibiotic agents.

Antagonistic Properties of Some Halophilic Thermoactinomycetes Isolated from Superficial Sediment of a Solar Saltern and Production of Cyclic Antimicrobial Peptides by the Novel Isolate Paludifilum halophilum.

This study has focused on the isolation of twenty-three halophilic actinomycetes from two ponds of different salinity and the evaluation of their ability to exert an antimicrobial activity against both their competitors and several other pathogens. From the 23 isolates, 18 strains showed antagonistic activity, while 19 showed activities against one or more of the seven pathogen strains tested. Six strains exhibited consistent antibacterial activity against Gram-negative and Gram-positive pathogens characterized at the physiological and molecular levels. These strains shared only 94-95% 16S rRNA sequence identity with the closely related species of the Thermoactinomycetaceae family. Among them, the potent strain SMBg3 was further characterized and assigned to a new genus in the family for which the name Paludifilum halophilum (DSM 102817T) is proposed. Sequential extraction of the antimicrobial compounds with ethyl acetate revealed that the crude extract from SMBg3 strain had inhibitory effect on the growth of the plant pathogen Agrobacterium tumefaciens and the human pathogens Staphylococcus aureus, Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa. Based on the HRESI-MS spectral data, the cyclic lipopeptide Gramicidin S and four cyclic dipeptides (CDPs) named cyclo(L-4-OH-Pro-L-Leu), cyclo(L-Tyr-L-Pro), cyclo(L-Phe-L-Pro), and cyclo(L-Leu-L-Pro) were detected in the fermentation broth of Paludifilum halophilum. To our knowledge, this is the first report on the isolation of these compounds from members of the Thermoactinomycetaceae family.

Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine.

The 3D structure of recombinant bacterial carboxypeptidase T (CPT) in complex with N-BOC-L-leucine was determined at 1.38 Å resolution. Crystals for the X-ray study were grown in microgravity using the counter-diffusion technique. N-BOC-L-leucine and SO4(2-) ion bound in the enzyme active site were localized in the electron density map. Location of the leucine side chain in CPT-N-BOC-L-leucine complex allowed identification of the S1 subsite of the enzyme, and its structure was determined. Superposition of the structures of CPT-N-BOC-L-leucine complex and complexes of pancreatic carboxypeptidases A and B with substrate and inhibitors was carried out, and similarity of the S1 subsites in these three carboxypeptidases was revealed. It was found that SO4(2-) ion occupies the same position in the S1' subsite as the C-terminal carboxy group of the substrate.

Purification and characterization of a thermolysin like protease from Thermoactinomyces thalpophilus MCMB-380.

The extracellular thermolysin like protease (TLP) was purified and characterized from Thermoactinomyces thalpophilus MCMB-380 (Genbank Accession No. EF397000). The enzyme was purified to homogeneity by successive ultra filtration steps using 50 kDa and 10 kDa membrane filters followed by anion exchange chromatography. The molecular mass and isoelectric point of the enzyme were found to be 34.4 kDa and 9.5, respectively. The proteolytic activity was inhibited by EDTA and the enzyme required Ca2+ to show the full activity as well as thermostability. The T50 of the enzyme at 80 °C was 1 h and the activation energy was estimated to be 11.02 Kcal / mol. Atomic absorption spectrophotometric analysis revealed the presence of Zn2+ ion in the protein core indicating that it is a metalloprotease. This protease has commercial potential in catalyzing the condensation reaction of two amino acids for production of the dipeptide aspartame, an artificial sweetener. The one hour time-frame is significantly faster than that of the enzyme thermolysin from Bacillus thermoproteolyticus. Moreover the TLP was stable at 80°C for one hour which makes it industrially robust. The Zn2+ ion in the T. thalpophilus protease appears to be necessary for maintaining the active conformation of the enzyme molecule.