Cyclooctatin, a diterpene characterized by a 5-8-5 fused ring system, is a potent inhibitor of lysophospholipase. Here we report the cloning and characterization of a complete cyclooctatin biosynthetic gene cluster from Streptomyces melanosporofaciens MI614-43F2 and heterologous production of cyclooctatin in S. albus. Sequence analysis coupled with subcloning and gene deletion revealed that the minimal cyclooctatin biosynthetic gene cluster consists of four genes, cotB1 to cotB4, encoding geranylgeranyl diphosphate (GGDP) synthase, terpene cyclase (CotB2), and two cytochromes P450, respectively. Incubation of the recombinant CotB2 with GGDP resulted in the formation of cyclooctat-9-en-7-ol, an unprecedented tricyclic diterpene alcohol. The present study establishes the complete biosynthetic pathway of cyclooctatin and provides insights into both the stereospecific diterpene cyclization mechanism of the GGDP cyclase and the molecular bases for the stereospecific and regiospecific hydroxylation.

A methylcobalamin (MeCbl)-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase Fom3 was found to catalyze the C-methylation of cytidylyl-2-hydroxyethylphosphonate (HEP-CMP) to give cytidylyl-2-hydroxypropylphosphonate (HPP-CMP), although it was originally proposed to catalyze the C-methylation of 2-hydroxyethylphosphonate to give 2-hydroxypropylphosphonate in the biosynthesis of a unique C-P bond containing antibiotic fosfomycin in Streptomyces. Unexpectedly, the Fom3 reaction product from HEP-CMP was almost a 1:1 diastereomeric mixture of HPP-CMP, indicating that the C-methylation is not stereoselective. Presumably, only the CMP moiety of HEP-CMP is critical for substrate recognition; on the other hand, the enzyme does not fix the 2-hydroxy group of the substrate and either of the prochiral hydrogen atoms at the C2 position can be abstracted by the 5'-deoxyadenosyl radical generated from SAM to form the substrate radical intermediates, which react with MeCbl to afford the corresponding products. This strict substrate recognition mechanism with no stereoselectivity of a MeCbl-dependent radical SAM methyltransferase is remarkable in natural product biosynthetic chemistry, because such a hidden clue for selective substrate recognition is likely to be found in the other biosynthetic pathways.

Terpene cyclization reactions are fascinating owing to the precise control of connectivity and stereochemistry during the catalytic process. Cyclooctat-9-en-7-ol synthase (CotB2) synthesizes an unusual 5-8-5 fused-ring structure with six chiral centers from the universal diterpene precursor, the achiral C20 geranylgeranyl diphosphate substrate. An unusual new mechanism for the exquisite CotB2-catalyzed cyclization that involves a carbon-carbon backbone rearrangement and three long-range hydride shifts is proposed, based on a powerful combination of in vivo studies using uniformly (13)C-labeled glucose and in vitro reactions of regiospecifically deuterium-substituted geranylgeranyl diphosphate substrates. This study shows that CotB2 elegantly demonstrates the synthetic virtuosity and stereochemical control that evolution has conferred on terpene synthases.

Terpene cyclization reactions are fascinating owing to the precise control of connectivity and stereochemistry during the catalytic process. Cyclooctat-9-en-7-ol synthase (CotB2) synthesizes an unusual 5-8-5 fused-ring structure with six chiral centers from the universal diterpene precursor, the achiral C 20 geranylgeranyl diphosphate substrate. An unusual new mechanism for the exquisite CotB2-catalyzed cyclization that involves a carbon-carbon backbone rearrangement and three long-range hydride shifts is proposed, based on a powerful combination of in vivo studies using uniformly 13 C-labeled glucose and in vitro reactions of regiospecifically deuteriumsubstituted geranylgeranyl diphosphate substrates. This study shows that CotB2 elegantly demonstrates the synthetic virtuosity and stereochemical control that evolution has conferred on terpene synthases.

Background/aims-Among the various weakening techniques of inferior oblique muscle overaction, the most commonly used techniques include myectomy, recession, and anterior transposition. Anterior transposition and myectomy were compared to evaluate the surgical results in inferior oblique overaction. Methods-20 children with bilateral +3 overacting inferior oblique muscles underwent a prospective randomised study by which the anterior transposition procedure in one eye was compared with the myectomy procedure in the other eye. Results-Postoperative follow up averaged 2 years. The success rates in two surgical procedures were 85% for the anterior transposition and 25% for the myectomy (standard of success was based on zero inferior oblique overaction). In only one case did the anterior transposition tend to limit the elevation of the eye in the midline, adduction, and abduction. Anterior transposition produced hypotropia at the primary position in only one case. Most eyes that underwent myectomy (75%) showed apparent residual overaction. Conclusion-The anterior transposition appeared to be more eVective in eliminating the overaction of inferior oblique muscle than the myectomy. (Br J Ophthalmol 1999;83:206-208) Various weakening techniques for an overactive inferior oblique muscle have been suggested since Jones et al 1 described myotomy for the first time. These include disinsertion, myotomy, recession, denervation-extirpation, myectomy, and anterior transposition. The latter two were mainly performed when inferior oblique muscle overaction is +3.For recession, although the persistence of inferior oblique muscle overaction and recurrence appeared less than for myectomy, recessed inferior oblique muscle adhered well to the sclera and rarely moved to the insertion. Also, the operating time is longer than for myectomy and the techniques of approach are diYcult in recession of inferior oblique muscle overaction. To overcome such diYculty a simple technique has been developed and used safely. 4 However, for myectomy, the operation time is short and the technique is simple, but the muscle end which is removed reattaches so that the recurrence appears and the inferior oblique underaction frequently occurs.Some surgeons approach the management of inferior oblique muscle overaction, by attaching the muscle anterior to the equator of the globe. Gobin 5 popularised a technique, known as the anterior transposition and recession of the inferior oblique muscle. Scott 6 suggested through his computer model that there were advantages of the inferior oblique anterior transposition, compared with the more classic recession technique. Elliott and Nankin 7 first reported the specific attachment of the inferior oblique muscle immediately adjacent to the temporal margin of the insertion of the inferior rectus. They showed that it was eVective for treating severe and recurrent inferior oblique muscle overaction. A subsequent report by Bremer et al 8 emphasised the risk of this procedure producing hypotropia at the primary pos...