The biosynthetic gene cluster for lariatins A and B, anti-mycobacterial peptide antibiotics with a unique "lasso" structure, was cloned from Gram-positive bacterium Rhodococcus jostii K01-B0171. Random transposition mutagenesis using IS1415 derivative was carried out to identify a chromosomal locus involved in lariatin biosynthesis and six independent lariatin non-producing variants were obtained. Arbitrary PCR revealed that one insertion was located near the region involved in lariatin biosynthesis. Using the lariatin gene as a probe, a genomic library of R. jostii K01-B0171 was screened by colony hybridization, and two clones were obtained. Sequence analysis of these clones revealed that the gene cluster for lariatin biosynthesis spanning about 4.5 kb consisted of five open reading frames (larA to larE). We proposed that the linear precursor LarA is processed by LarB, LarC, and LarD, and the mature lariatin is exported by LarE.

Various lectins have attracted attention as potential microbicides to prevent HIV transmission. Their capacity to bind glycoproteins has been suggested as a means to block HIV binding and entry into susceptible cells. The previously undescribed lectin actinohivin (AH), isolated by us from an actinomycete, exhibits potent in vitro anti-HIV activity by binding to high-mannose (Man) type glycans (HMTGs) of gp120, an envelope glycoprotein of HIV. AH contains 114 aa and consists of three segments, all of which need to show high affinity to gp120 for the anti-HIV characteristic. To generate the needed mechanistic understanding of AH binding to HIV in anticipation of seeking approval for human testing as a microbicide, we have used multiple molecular tools to characterize it. AH showed a weak affinity to Man␣(1-2)Man, Man␣(1-2)Man␣(1-2)Man, of HMTG (Man8 or Man9) or RNase B (which has a single HMTG), but exhibited a strong and highly specific affinity (K d ‫؍‬ 3.4 ؋ 10 ؊8 M) to gp120 of HIV, which contains multiple Man8 and/or Man9 units. We have compared AH to an alternative lectin, cyanovirin-N, which did not display similar levels of discrimination between high-and low-density HMTGs. X-ray crystal analysis of AH revealed a 3D structure containing three sugar-binding pockets. Thus, the strong specific affinity of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the ''cluster effect'' of lectin. Thus, AH is a good candidate for investigation as a safe microbicide to help prevent HIV transmission.action mechanism ͉ anti-HIV ͉ gp120 ͉ high-mannose type glycan ͉ cyanovirin-N

Novel brominated and halogen-less azaphilone (oxoisochromane) derivatives, 5-bromoochrephilone and dechloroisochromophilone IV, and known derivatives, dechloroisochromophilone III and isorotiorin, were isolated from the culture broth of a producing organism of isochromophilones I and II (azaphilones inhibiting gp120-CD4 binding), Penicillium multicolor FO-2338, fermented in a medium containing potassium bromide. Nineteen azaphilone-related compounds isolated from the above strain and from other fungi were tested for the inhibition of gp120-CD4 binding and the structure-activity relationship is discussed. Consequently, 5-bromoochrephilone is the strongest inhibitor chain of 6-oxoisochromane ring are necessary for gp120-CD4 binding.Fungal metabolites having an oxoisochromane ring system, which are called "azaphilones" after high affinity to ammonia1), have been found to possess various bioactivities. In the course of screening of microbial metabolites for the inhibition of gp120-CD4 binding, we discovered the inhibitors isochromophilones I (1) and II (2) having a 6-oxoisochromane ring, which were isolated from a culture broth of Penicillium multicolor FOphilone (3)5), sclerotiorin (4)6) and rubrorotiorin (5)6).

An enzyme assay for bacterial undecaprenyl pyrophosphate (UPP) synthase was performed to screen microbial culture broths for inhibitors of UPP synthase. During the course of this screening program, an EtOH extract of a rice culture of Penicillium brasilianum FKI-3368 was found to inhibit UPP synthase activity. From activity-guided purification, a new compound-designated spirohexaline was isolated together with the structurally related and known viridicatumtoxin by ethyl acetate extraction silica gel and octadecylsilane column chromatographies and high-performance liquid chromatography. The structure of spirohexaline was elucidated by spectroscopic analysis, including NMR. Spirohexaline and viridicatumtoxin have a common hexacycline structure produced by fusion of a tetracycline-type ring with a spiro-type ring. They inhibited UPP synthase activity with IC₅₀ values of 9.0 and 4.0 μM, respectively.

The structures of newprotein farnesyltransferase inhibitors, andrastins A~C, were elucidated. Thecyclopentane ring of andrastins exhibited keto-enol tautomerism, whichmadethe structure hard to elucidate. Therefore, the structure of andrastin A was elucidated by INADEQUATE and 13C-1 3C couplings using 1 3C-labeled andrastin A. The absolute configuration of the />-bromobenzoyl derivative of andrastin A was elucidated by X-ray crystallographic analysis and its skeleton was shownto be enr-5a,14/?-androstane. The biosynthesis of andrastin A was also studied by the incorporation of 13C-labeled acetates. Though the andrastins had a commonandrostane skeleton, they were biosynthesized from a sesquiterpene and a tetraketide.