In the present study, we successfully cloned a 21-kb DNA fragment containing a D-cycloserine (DCS) biosynthetic gene cluster from a DCS-producing Streptomyces lavendulae strain, ATCC 11924. The putative gene cluster consists of 10 open reading frames (ORFs), designated dcsA to dcsJ. This cluster includes two ORFs encoding D-alanyl-D-alanine ligase (dcsI) and a putative membrane protein (dcsJ) as the self-resistance determinants of the producer organism, indicated by our previous work. When the 10 ORFs were introduced into DCS-nonproducing Streptomyces lividans 66 as a heterologous host cell, the transformant acquired DCS productivity. This reveals that the introduced genes are responsible for the biosynthesis of DCS. As anticipated, the disruption of dcsG, seen in the DCS biosynthetic gene cluster, made it possible for the strain ATCC 11924 to lose its DCS production. We here propose the DCS biosynthetic pathway. The soil-dwelling genus Streptomyces undergoes a complex morphological differentiation and produces an enormous variety of bioactive secondary metabolites. Because they include clinically useful antibiotics and immunosuppressants, the genus Streptomyces occupies an important position as an industrial microorganism.D-Cycloserine (DCS), a cyclic structural analogue of D-alanine, is produced by "Streptomyces garyphalus" and Streptomyces lavendulae. This antibiotic is used as an antitubercular agent (21). Since DCS is similar to D-alanine, it prevents the action of both alanine racemase and D-alanyl-D-alanine ligase, which are necessary for the biosynthesis of a bacterial cell wall. Thus, DCS functions as an inhibitor of bacterial cell wall biosynthesis (16,19). Although the structure of DCS is very simple, the biosynthetic genes for DCS have never been cloned until now.In general, antibiotic biosynthetic genes form a cluster and are adjacent to their self-resistance genes. We have previously cloned a gene (orfB) that confers resistance to DCS on Streptomyces lividans and Escherichia coli from DCS-producing S. garyphalus (CSH) 5-12 (17). The sequence analysis suggests that the gene may encode a membrane protein that is necessary for the excretion of the DCS outside the cell. We have found that the same gene is also present in S. lavendulae ATCC 25233 (20). We have also previously demonstrated that Dalanyl-D-alanine ligase, which is a target enzyme of DCS, functions as a self-resistance determinant in S. lavendulae ATCC 25233 (20). Interestingly, the gene encoding D-alanyl-D-alanine ligase, designated ddlS, was located just upstream of the putative membrane protein gene, orfB (20). Although the selfresistance genes in the DCS producers have been thoroughly analyzed, no attempt to clone the DCS biosynthetic genes has been carried out yet.To clone the biosynthetic genes for DCS from another DCSproducing S. lavendulae strain, ATCC 11924, we first investigated whether this strain harbors orfB and ddlS. Since both genes were found to be conserved, the flanking region was cloned from the chromosomal DNA. Here, we ...

The Cu(II)-soaked crystal structure of tyrosinase that is present in a complex with a protein, designated "caddie," which we previously determined, possesses two copper ions at its catalytic center. We had identified two copper-binding sites in the caddie protein and speculated that copper bound to caddie may be transported to the tyrosinase catalytic center. In our present study, at a 1.16 -1.58 Å resolution, we determined the crystal structures of tyrosinase complexed with caddie prepared by altering the soaking time of the copper ion and the structures of tyrosinase complexed with different caddie mutants that display little or no capacity to activate tyrosinase. Based on these structures, we propose a molecular mechanism by which two copper ions are transported to the tyrosinase catalytic center with the assistance of caddie acting as a metallochaperone.Among the many biological systems that employ transition metal ions for their function, it is a requirement that specific metal co-factors be transported into the correct metalloenzyme. Proteins known as "metallochaperones" play key roles in this process. Although a few structures of the metallochaperone have now been determined (1-6), the molecular mechanisms underlying specific metal transfers remain unclear with

Sendai virus (SeV IMPORTANCE Sendai virus, a paramyxovirus that causes respiratory diseases in rodents, possesses the C protein, which inhibits the signal transduction pathways of interferon alpha/beta (IFN-␣/␤) and IFN-␥ by binding to the transcription factor STAT1. In virusinfected cells, phosphorylation of STAT1 at the Tyr701 residue is potently enhanced, although transcription by STAT1 is inert. Here, we determined the crystal structure of the N-terminal domain of STAT1 associated with the C-terminal half of the C protein. Molecular modeling and experiments suggested that the two C proteins bind to and stabilize the parallel form of the STAT1 dimer, which are likely to be phosphorylated at Tyr 701 , further inducing high-molecular-weight complex formation and inhibition of transcription by IFN-␥. We also discuss the possible mechanism of inhibition of the IFN-␣/␤ pathways by the C protein. This is the first structural report of the C protein, suggesting a mechanism of evasion of the paramyxovirus from innate immunity.

We have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic, D-cycloserine. The gene cluster is composed of 10 open reading frames, designated dcsA to dcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization of O-ureidoserine. DcsD is similar to O-acetylserine sulfhydrylase, which generates L-cysteine using O-acetyl-L-serine with sulfide, and therefore, DcsD may be a synthase to generate O-ureido-L-serine using O-acetyl-L-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase converting O-ureido-D-serine into D-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed in Escherichia coli and purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substrates O-acetyl-L-serine and hydroxyurea, synthesis of D-cycloserine was successfully attained. These in vitro studies yield the conclusion that DcsD and DcsG are necessary for the syntheses of O-ureido-L-serine and D-cycloserine, respectively. DcsD was also able to catalyze the synthesis of L-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclic D-amino acid analogs, such as D-homocysteine thiolactone.

Bleomycin (Bm) N-acetyltransferase, BAT, is a self-resistance determinant in Bm-producing Streptomyces verticillus ATCC15003. In our present study, we crystallized BAT under both a terrestrial and a microgravity environment in the International Space Station. In addition to substrate-free BAT, the crystal structures of BAT in a binary complex with CoA and in a ternary complex with Bm and CoA were determined. BAT forms a dimer structure via interaction of its C-terminal domains in the monomers. However, each N-terminal domain in the dimer is positioned without mutual interaction. The tunnel observed in the N-terminal domain of BAT has two entrances: one that adopts a wide funnel-like structure necessary to accommodate the metal-binding domain of Bm, and another narrow entrance that accommodates acetyl-CoA (AcCoA). A groove formed on the dimer interface of two BAT C-terminal domains accommodates the DNA-binding domain of Bm. In a ternary complex of BAT, BmA 2 , and CoA, a thiol group of CoA is positioned near the primary amine of Bm at the midpoint of the tunnel. This proximity ensures efficient transfer of an acetyl group from AcCoA to the primary amine of Bm. Based on the BAT crystal structure and the enzymatic kinetic study, we propose that the catalytic mode of BAT takes an ordered-like mechanism.