Generation of incednine derivatives by mutasynthesis.

The macrolactam antibiotic incednine, isolated from Streptomyces sp. ML694-90F3, contains a (S)-3-aminobutyric acid moiety in its polyketide aglycon. In this study, we performed mutasynthesis to generate incednine derivatives. We successfully obtained 28-methylincednine by feeding 3-aminopentanoic acid into culture of a strain in which the glutamate 2,3-aminomutase gene idnL4, whose product is responsible for supplying 3-aminobutyric acid, was disrupted. 28-Methylincednine showed similar suppressive activity of the antiapoptotic function of oncoprotein Bcl-xL to that of incednine. Thus, this study highlights the applicability of the mutasynthesis approach in generation of novel ß-amino acid-containing macrolactam polyketide derivatives.

Functional and structural characterization of IdnL7, an adenylation enzyme involved in incednine biosynthesis.

Adenylation enzymes play an important role in the selective incorporation of the cognate carboxylate substrates in natural product biosynthesis. Here, the biochemical and structural characterization of the adenylation enzyme IdnL7, which is involved in the biosynthesis of the macrolactam polyketide antibiotic incednine, is reported. Biochemical analysis showed that IdnL7 selects and activates several small amino acids. The structure of IdnL7 in complex with an L-alanyl-adenylate intermediate mimic, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine, was determined at 2.1 Šresolution. The structure of IdnL7 explains the broad substrate specificity of IdnL7 towards small L-amino acids.

Biochemical characterization and structural insight into aliphatic ß-amino acid adenylation enzymes IdnL1 and CmiS6.

Macrolactam antibiotics such as incednine and cremimycin possess an aliphatic ß-amino acid as a starter unit of their polyketide chain. In the biosynthesis of incednine and cremimycin, unique stand-alone adenylation enzymes IdnL1 and CmiS6 select and activate the proper aliphatic ß-amino acid as a starter unit. In this study, we describe the enzymatic characterization and the structural basis of substrate specificity of IdnL1 and CmiS6. Functional analysis revealed that IdnL1 and CmiS6 recognize 3-aminobutanoic acid and 3-aminononanoic acid, respectively. We solved the X-ray crystal structures of IdnL1 and CmiS6 to understand the recognition mechanism of these aliphatic ß-amino acids. These structures revealed that IdnL1 and CmiS6 share a common recognition motif that interacts with the ß-amino group of the substrates. However, the hydrophobic side-chains of the substrates are accommodated differently in the two enzymes. IdnL1 has a bulky Leu220 located close to the terminal methyl group of 3-aminobutanoate of the trapped acyl-adenylate intermediate to construct a shallow substrate-binding pocket. In contrast, CmiS6 possesses Gly220 at the corresponding position to accommodate 3-aminononanoic acid. This structural observation was supported by a mutational study. Thus, the size of amino acid residue at the 220 position is critical for the selection of an aliphatic ß-amino acid substrate in these adenylation enzymes. Proteins 2017; 85:1238-1247. © 2017 Wiley Periodicals, Inc.

Identification of the incednine biosynthetic gene cluster: characterization of novel ß-glutamate-ß-decarboxylase IdnL3.

A biosynthetic gene cluster for the 24-membered macrolactam antibiotic incednine was identified from the producer strain, Streptomyces sp. ML694-90F3. Among the putative incednine biosynthetic enzymes, a novel pyridoxal 5'-phosphate (PLP)-dependent ß-glutamate-ß-decarboxylase, IdnL3, was functionally characterized in vitro by demonstrating its (S)-3-aminobutyrate-forming activity with ß-glutamate in the presence of PLP. Because (S)-3-aminobutyrate is known for the direct precursor of incednine, this enzyme supplies the unique ß-amino acid starter unit. The identified gene cluster encodes five characteristic ß-amino acid carrying enzymes, consisting of a pathway-specific ATP-dependent ligase, a discrete acyl carrier protein (ACP), ß-aminoacyl-ACP ß-amino group-protecting ATP-dependent ligase, dipeptidyl-ACP:PKS-loading ACP dipeptidyltransferase and a terminal amino acid peptidase, which are completely conserved in ß-amino acid-containing macrolactam biosynthetic gene clusters. Overall, a plausible biosynthetic pathway for incednine was proposed.

A unique pathway for the 3-aminobutyrate starter unit from L-glutamate through ß-glutamate during biosynthesis of the 24-membered macrolactam antibiotic, incednine.

Incednine is a 24-membered macrolactam antibiotic produced by Streptomyces sp. ML694-90F3. A previous study demonstrated that its unique nitrogen-containing starter unit was derived from L-glutamate. To elucidate the missing link between L-glutamate and the starter unit, deuterium labeled amino acid feeding experiments were conducted. These experiments revealed that 3-[3-(2)H]aminobutyrate and ß-[2,2,4,4-(2)H(4)]glutamate were incorporated into the starter moiety. The results indicate that a novel decarboxylation of ß-glutamate to give 3-aminobutyrate is involved in incednine biosynthesis.

Effects of postoperative radiotherapy for temporomandibular joint ankylosis after gap arthroplasty: an animal study using sheep.

PURPOSE: The aim of the present study was to examine the effects of postoperative irradiation on reducing heterotopic bone formation after gap arthroplasty release of temporomandibular joint ankylosis. MATERIALS AND METHODS: Five sheep underwent induction of right temporomandibular joint ankylosis. After 3 months, the ankylosis was released by gap arthroplasty. At 24 hours after the release, they received a single radiation dose of 10 Gy. All sheep were sacrificed at 3 months after gap arthroplasty release. The body weight, jaw opening amount, and radiographs were measured at key intervals, with histologic assessment after death. The findings were compared with those in a control group treated with gap arthroplasty without irradiation. RESULTS: The clinical measurements, radiographs, and histologic findings all revealed less evidence of reankylosis in the irradiated sheep. CONCLUSION: The results of the present study have shown that a single radiation dose at 24 hours after gap arthroplasty for temporomandibular joint ankylosis inhibits heterotopic ossification.

Effect of auricular cartilage graft in the surgical treatment of temporomandibular joint ankylosis: An animal study using sheep.

PURPOSE: The purpose of this study was to test the functional and histologic fate of an auricular cartilage graft used in reconstruction of an ankylosed sheep temporomandibular joint (TMJ). MATERIALS AND METHODS: Five sheep were used in this study. TMJ ankylosis was induced in the right joints and the left joints were used as controls. The ankylosed TMJ was released by gap arthroplasty with an interposed auricular cartilage graft at 3 months. The sheep were sacrificed at 3 months after the arthroplasty. The maximal mouth opening was measured pre- and postoperatively. The joints were evaluated radiologically and histologically. RESULTS: Maximal mouth opening was maintained after placement of an auricular graft into the gap arthroplasty. Radiographically the surfaces of the temporal bone and ramus stumps were irregular, but radiolucent gaps were formed between them. Histologically, the auricular cartilage graft was alive and well attached to the mandibular ramus stump. In all operated joints, there was joint space between the grafted cartilage and temporal bone, with the space filled with fibrous connective tissue, which was oriented parallel to the temporal surface. CONCLUSION: Auricular cartilage graft with gap arthroplasty is useful in preventing reankylosis after TMJ gap arthroplasty for ankylosis.