Molecular Basis for Enzymatic Aziridine Formation via Sulfate Elimination.

Natural products containing an aziridine ring, such as mitomycin C and azinomycin B, exhibit antitumor activities by alkylating DNA via their aziridine rings; however, the biosynthetic mechanisms underlying the formation of these rings have not yet been elucidated. We herein investigated the biosynthesis of vazabitide A, the structure of which is similar to that of azinomycin B, and demonstrated that Vzb10/11, with no similarities to known enzymes, catalyzed the formation of the aziridine ring via sulfate elimination. To elucidate the detailed reaction mechanism, crystallization of Vzb10/11 and the homologous enzyme, AziU3/U2, in the biosynthesis of azinomycin B was attempted, and the structure of AziU3/U2, which had a new protein fold overall, was successfully determined. The structural analysis revealed that these enzymes adjusted the dihedral angle between the amino group and the adjacent sulfate group of the substrate to almost 180° and enhanced the nucleophilicity of the C6-amino group temporarily, facilitating the SN2-like reaction to form the aziridine ring. The present study reports for the first time the molecular basis for aziridine ring formation.

Guanidyl modification of the 1-azabicyclo[3.1.0]hexane ring in ficellomycin essential for its biological activity.

The 1-azabicyclo[3.1.0]hexane ring is a key moiety in natural products for biological activities against bacteria, fungi, and tumor through DNA alkylation. Ficellomycin is a dipeptide that consists of l-valine and a non-proteinogenic amino acid with the 1-azabicyclo[3.1.0]hexane ring structure. Although the biosynthetic gene cluster of ficellomycin has been identified, the biosynthetic pathway currently remains unclear. We herein report the final stage of ficellomycin biosynthesis involving ring modifications and successive dipeptide formation. After the ring is formed, the hydroxy group of the ring is converted into the guanidyl unit by three enzymes, which include an aminotransferase with a novel inter ω-ω amino-transferring activity. In the last step, the resulting 1-azabicyclo[3.1.0]hexane ring-containing amino acid is connected with l-valine by an amino acid ligase to yield ficellomycin. The present study revealed a new machinery that expands the structural and biological diversities of natural products.

Immunosuppressive effect of a non-proteinogenic amino acid from Streptomyces through inhibiting allogeneic T cell proliferation.

The immunosuppressive activity of myriocin (ISP-1), a lead compound of fingolimod (FTY720), is derived from its 2-amino-1,3-propandiol structure. A non-proteinogenic amino acid, (2S,6R)-diamino-(5R,7)-dihydroxy-heptanoic acid (DADH), that contains this structure, was recently identified as a biosynthetic intermediate of a dipeptide secondary metabolite, vazabitide A, in Streptmyces sp. SANK 60404; however its effect on adaptive immunity has not yet been examined. In this study, we examined whether DADH suppresses mixed lymphocyte reaction using mouse bone marrow-derived dendritic cells (BMDCs) and allogeneic splenic T cells. Although T cell proliferation induced by cross-linking CD3 and CD28 were not suppressed by DADH unlike ISP-1, the pre-incubation of BMDCs with DADH but not ISP-1 significantly decreased allogeneic CD8+ T cell expansion. Based on these results, we concluded that DADH suppresses DC-mediated T cell activation by targeting DCs.

Mechanisms of Sugar Aminotransferase-like Enzymes to Synthesize Stereoisomers of Non-proteinogenic Amino Acids in Natural Product Biosynthesis.

(2,6)-Diamino-(5,7)-dihydroxyheptanoic acid (DADH), a non-proteinogenic amino acid, is converted to 1-azabicyclo[3.1.0]hexane ring-containing amino acids that are subsequently incorporated into ficellomycin and vazabitide A. The present study revealed that the sugar aminotransferase-like enzymes Fic25 and Vzb9, with a high amino acid sequence identity (56%) to each other, synthesized stereoisomers of DADH with (6S) and (6R) configurations, respectively. The crystal structure of the Fic25 complex with a PLP-(6S)-N2-acetyl-DADH adduct indicated that Asn45 and Gln197 (Asn205 and Ala53 in Vzb9) were located at positions that affected the stereochemistry of DADH being synthesized. A modeling study suggested that amino acid substitutions between Fic25 and Vzb9 allowed the enzymes to bind to the substrate with almost 180° rotation in the C5-C7 portions of the DADH molecules, accompanied by a concomitant shift in their C1-C4 portions. In support of this result, the replacement of two corresponding residues in Fic25 and Vzb9 increased (6R) and (6S) stereoselectivities, respectively. The different stereochemistry at C6 of DADH resulted in a different stereochemistry/orientation of the aziridine portion of the 1-azabicyclo[3.1.0]hexane ring, which plays a crucial role in biological activity, between ficellomycin and vazabitide A. A phylogenic analysis suggested that Fic25 and Vzb9 evolved from sugar aminotransferases to produce unusual building blocks for expanding the structural diversity of secondary metabolites.

Major intraoperative bleeding and drastic change in circulatory dynamics in a pregnant patient with metastatic pheochromocytoma: a case report.

BACKGROUND: Metastatic pheochromocytoma in the spine is a very rare complication during pregnancy. We report anesthesia in a pregnant woman for resection of an undiagnosed spinal tumor, accompanied by remarkable hemodynamic changes and massive bleeding. CASE PRESENTATION: A 33-year-old woman at 17 weeks of gestation presented with the rapid progress of bilateral lower leg paralysis. A diagnosis of spinal tumor was made, and surgical resection was planned. Although the surgery was suspended because of remarkable hemodynamic changes and massive bleeding, fetal heart rate was stable. Postoperative examination revealed pheochromocytoma in the urinary bladder as a primary lesion with spinal metastasis. CONCLUSION: Although spinal pheochromocytoma is extremely rare in pregnant women, it should be suspected when abnormal hypertension is observed with accompanying neurological deficits. Preservation of maternal circulation and uteroplacental blood flow should be the first priority during anesthesia.

Structural basis for potent inhibition of d-amino acid oxidase by thiophene carboxylic acids.

A series of thiophene-2-carboxylic acids and thiophene-3-carboxylic acids were identified as a new class of DAO inhibitors. Structure-activity relationship (SAR) studies revealed that small substituents are well-tolerated on the thiophene ring of both the 2-carboxylic acid and 3-carboxylic acid scaffolds. Crystal structures of human DAO in complex with potent thiophene carboxylic acids revealed that Tyr224 was tightly stacked with the thiophene ring of the inhibitors, resulting in the disappearance of the secondary pocket observed with other DAO inhibitors. Molecular dynamics simulations of the complex revealed that Tyr224 preferred the stacked conformation irrespective of whether Tyr224 was stacked or not in the initial state of the simulations. MM/GBSA indicated a substantial hydrophobic interaction between Tyr244 and the thiophene-based inhibitor. In addition, the active site was tightly closed with an extensive network of hydrogen bonds including those from Tyr224 in the stacked conformation. The introduction of a large branched side chain to the thiophene ring markedly decreased potency. These results are in marked contrast to other DAO inhibitors that can gain potency with a branched side chain extending to the secondary pocket due to Tyr224 repositioning. These insights should be of particular importance in future efforts to optimize DAO inhibitors with novel scaffolds.