Kosuke Oda

A molecular mechanism for copper transportation to tyrosinase that is assisted by a metallochaperone, caddie protein

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.

Molecular Cloning and Heterologous Expression of a Biosynthetic Gene Cluster for the Antitubercular Agent d-Cycloserine Produced by Streptomyces lavendulae

ABSTRACT In the present study, we successfully cloned a 21-kb DNA fragment containing a d-cycloserine (DCS) biosynthetic gene cluster from a DCS-producing Streptomyceslavendulae 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. First, l-serine is O acetylated by a dcsE-encoded enzyme homologous to homoserine O-acetyltransferase. Second, O-acetyl-l-serine accepts hydroxyurea via an O-acetylserine sulfhydrylase homolog (dcsD product) and forms O-ureido-l-serine. The hydroxyurea must be supplied by the catalysis of a dcsB-encoded arginase homolog using the l-arginine derivative, NG-hydroxy-l-arginine. The resulting O-ureido-l-serine is then racemized to O-ureido-d-serine by a homolog of diaminopimelate epimerase. Finally, O-ureido-d-serine is cyclized to form DCS with the release of ammonia and carbon dioxide. The cyclization must be done by the dcsG or dcsH product, which belongs to the ATP-grasp fold family of protein.

Catalytic Mechanism of Bleomycin N-Acetyltransferase Proposed on the Basis of Its Crystal Structure

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, BmA2, 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.

A novel transformation system using a bleomycin resistance marker with chemosensitizers for Aspergillus oryzae

Aspergillus oryzae is resistant to many kinds of antibiotics, which hampers their use to select transformants. In fact, the fungus is resistant to over 200microg/ml of bleomycin (Bm). By enhancing the susceptibility of A. oryzae to Bm using Triton X-100 as a detergent and an ATP-binding cassette (ABC) pump inhibitor, chlorpromazine, to the growing medium, we established a novel transformation system by Bm selection for A. oryzae. In a medium containing these reagents, A. oryzae showed little growth even in the presence of 30microg Bm/ml. Based on these findings, we constructed a Bm-resistance expression cassette (BmR), in which blmB encoding Bm N-acetyltransferase from Bm-producing Streptomyces verticillus was expressed under the control of a fungal promoter. We obtained a gene knockout mutant efficiently by Bm selection, i.e., the chromosomal ligD coding region was successfully replaced by BmR using ligD disruption cassette consisted of ligD flanking sequence and BmR through homologous recombination.

Establishment of an In Vitro d-Cycloserine-Synthesizing System by Using O-Ureido-l-Serine Synthase and d-Cycloserine Synthetase Found in the Biosynthetic Pathway

ABSTRACT 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.

Heme Protein and Hydroxyarginase Necessary for Biosynthesis of D-Cycloserine

ABSTRACT We have recently cloned a d-cycloserine (DCS) biosynthetic gene cluster that consists of 10 genes, designated dcsA∼dcsJ, from Streptomyces lavendulae ATCC 11924 (16). In the predicted pathway of hydroxyurea (HU) formation in DCS biosynthesis, l-arginine (L-Arg) must first be hydroxylated, prior to the hydrolysis of Nω-hydroxy-l-arginine (NHA) by DcsB, an arginase homolog. The hydroxylation of L-Arg is known to be catalyzed by nitric oxide synthase (NOS). In this study, to verify the supply route of HU, we created a dcsB-disrupted mutant, ΔdcsB. While the mutant lost DCS productivity, its productivity was restored by complementation of dcsB, and also by the addition of HU but not NHA, suggesting that HU is supplied by DcsB. A NOS-encoding gene, nos, from S. lavendulae chromosome was cloned, to create a nos-disrupted mutant. However, the mutant maintained the DCS productivity, suggesting that NOS is not necessary for DCS biosynthesis. To clarify the identity of an enzyme necessary for NHA formation, a dcsA-disrupted mutant, designated ΔdcsA, was also created. The mutant lost DCS productivity, whereas the DCS productivity was restored by complementation of dcsA. The addition of NHA to the culture medium of ΔdcsA mutant was also effective to restore DCS production. These results indicate that the dcsA gene product, DcsA, is an enzyme essential to generate NHA as a precursor in the DCS biosynthetic pathway. Spectroscopic analyses of the recombinant DcsA revealed that it is a heme protein, supporting an idea that DcsA is an enzyme catalyzing hydroxylation.

Structural Basis of the Inhibition of STAT1 Activity by Sendai Virus C Protein

ABSTRACT Sendai virus (SeV) C protein inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/β) and IFN-γ by binding to the N-terminal domain of STAT1 (STAT1ND), thereby allowing SeV to escape from host innate immunity. Here we determined the crystal structure of STAT1ND associated with the C-terminal half of the C protein (Y3 [amino acids 99 to 204]) at a resolution of 2.0 Å. This showed that two molecules of Y3 symmetrically bind to each niche created between two molecules of the STAT1ND dimer. Molecular modeling suggested that an antiparallel form of the full-length STAT1 dimer can bind only one Y3 molecule and that a parallel form can bind two Y3 molecules. Affinity analysis demonstrated anticooperative binding of two Y3 molecules with the STAT1 dimer, which is consistent with the hypothetical model that the second Y3 molecule can only target the STAT1 dimer in a parallel form. STAT1 with excess amounts of Y3 was prone to inhibit the dephosphorylation at Tyr701 by a phosphatase. In an electrophoretic mobility shift assay, tyrosine-phosphorylated STAT1 (pY-STAT1) with Y3 associated with the γ-activated sequence, probably as high-molecular-weight complexes (HMWCs), which may account for partial inhibition of a reporter assay from IFN-γ by Y3. Our study suggests that the full-length C protein interferes with the domain arrangement of the STAT1 dimer, leading to the accumulation of pY-STAT1 and the formation of HMWCs. In addition, we discuss the mechanism by which phosphorylation of STAT2 is inhibited in the presence of the C protein after stimulation by IFN-α/β. 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-α/β) and IFN-γ by binding to the transcription factor STAT1. In virus-infected 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 Tyr701, further inducing high-molecular-weight complex formation and inhibition of transcription by IFN-γ. We also discuss the possible mechanism of inhibition of the IFN-α/β 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.

Crystallographic Study To Determine the Substrate Specificity of an l-Serine-Acetylating Enzyme Found in the d-Cycloserine Biosynthetic Pathway

DcsE, one of the enzymes found in the d-cycloserine biosynthetic pathway, displays a high sequence homology to l-homoserine O-acetyltransferase (HAT), but it prefers l-serine over l-homoserine as the substrate. To clarify the substrate specificity, in the present study we determined the crystal structure of DcsE at a 1.81-Å resolution, showing that the overall structure of DcsE is similar to that of HAT, whereas a turn region to form an oxyanion hole is obviously different between DcsE and HAT: in detail, the first and last residues in the turn of DcsE are Gly(52) and Pro(55), respectively, but those of HAT are Ala and Gly, respectively. In addition, more water molecules were laid on one side of the turn region of DcsE than on that of HAT, and a robust hydrogen-bonding network was formed only in DcsE. We created a HAT-like mutant of DcsE in which Gly(52) and Pro(55) were replaced by Ala and Gly, respectively, showing that the mutant acetylates l-homoserine but scarcely acetylates l-serine. The crystal structure of the mutant DcsE shows that the active site, including the turn and its surrounding waters, is similar to that of HAT. These findings suggest that a methyl group of the first residue in the turn of HAT plays a role in excluding the binding of l-serine to the substrate-binding pocket. In contrast, the side chain of the last residue in the turn of DcsE may need to form an extensive hydrogen-bonding network on the turn, which interferes with the binding of l-homoserine.

Structural evidence that puromycin hydrolase is a new type of aminopeptidase with a prolyl oligopeptidase family fold

Puromycin [PM, Supporting Information Fig. S1(A)]1 and blasticidin S [BS, Supporting Information Fig. S1(B)]2 are nucleoside antibiotics. We have previously found that BS-producing Streptomyces morookaensis JCM4673 inactivates the own product in the presence of acetyl CoA.3 We have also found that the bacterium has an enzyme to inactivate PM without the cofactor.4 The enzyme catalyzes the hydrolysis of an amide linkage between the aminonucleoside and O-methyl-L-tyrosine moieties in the PM molecule. We have revealed that the S. morookaensis PM hydrolase (PMH) has some aminopeptidase-like properties closely related to those of proline iminopeptidases [EC 3.4.11.5].5 Proline iminopeptidases (PIPs) are among the serine peptidases catalyzing the removal of the N-terminal proline from a substrate with strict specificity.6 However, PMH exhibits broad substrate specificity toward b-naphthylamide derivatives of amino acids, although it prefers L-prolyl-bnaphthylamide in such substrates. We have cloned and sequenced a gene encoding PMH from S. morookaensis.7 The deduced amino acid sequence was similar to those of several peptidases classified into the prolyl oligopeptidase (POP) family.8 This family includes POP (EC 3.4.21.26),9 which cleaves the internal peptide bond on the C-terminal side of the prolyl residues, oligopeptidase B (EC 3.4.21.83),10 which cleaves the internal peptide bond on the C-terminal side of the lysyl or arginyl residues, dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5),11 which removes N-terminal dipeptides from the polypeptides having unsubstituted N-termini and the penultimate proline residue, prolyl tripeptidyl aminopeptidase (PTP; EC 3.4.14.12),12 which removes N-terminal tripeptides from polypeptides having a proline residue at the third position, and acyl-peptide hydrolase (ACPH; EC 3.4.19.1),13 which catalyzes the hydrolysis of the amino-terminal peptide bond of an N-acetylated peptide to generate an N-acetylated amino acid and a peptide with a free amino terminus. After we have found PMH, it has been shown that PMH homologues, which had been annotated as putative ACPH, from S. griseus and S. coelicolor display aminopeptidase activities [EC 3.4.11].14 However, the tertiary structure of aminopeptidases, classified into the POP family, is not yet determined. To understand the manner of recognition of the substrate and the catalytic

The structural and mutational analyses of O-ureido-L-serine synthase necessary for D-cycloserine biosynthesis

We have recently been successful in cloning a gene cluster necessary for the biosynthesis of d‐cycloserine (d‐CS) from d‐CS‐producing Streptomyces lavendulae ATCC11924. Although dcsD, one of the ORFs located on the gene cluster, encodes a protein homologous to O‐acetylserine sulfhydrylase that synthesizes l‐cysteine using O‐acetyl‐l‐serine together with sulfide, it functions to form O‐ureido‐l‐serine as a d‐CS biosynthetic intermediate, using O‐acetyl‐l‐serine together with hydroxyurea (HU). In the present study, using crystallographic and mutational studies, three amino acid residues in DcsD that are important for the substrate preference toward HU were determined. We showed that two of the three residues are important for the binding of HU into the substrate‐binding pocket. The other residue contributes to the formation of a loose hydrogen‐bond network during the catalytic reaction. Information regarding the amino acid residues will be very useful in the design of a new catalyst for synthesizing the β‐substituted‐l‐alanine derivatives.