Fumitaka Kudo

Precursor-directed biosynthesis: biochemical basis of the remarkable selectivity of the erythromycin polyketide synthase toward unsaturated triketides.

The structural basis for the striking stereochemical discrimination among triketide analogs has been investigated by incubating a series of N-acetyl cysteamine (-SNAC) esters of unsaturated triketides with DEBS module 2+TE. The triketide analogs were first screened under a standard set of short-term incubation conditions in the presence of the extender substrate methylmalonyl-CoA and NADPH. For those triketide analogs that served as substrates for module 2+TE, the relative specificity, represented by the k(cat)/K(M) values, was quantitated. Triketide diastereomers that were converted in precursor-directed biosynthesis experiments to unsaturated 16-membered ring macrolides by DEBS(KS1(0)) were good to excellent substrates for DEBS module 2+TE, whereas analogs that were converted to the 14-membered ring analogs of 10,11-dehydro-6-deoxyerythronolide B by DEBS(KS1(0)) were not turned over at all by module 2+TE.

Biosynthetic genes for aminoglycoside antibiotics

Biosynthetic studies of aminoglycoside antibiotics have progressed remarkably during the last decade. Many biosynthetic gene clusters for aminoglycoside antibiotics including streptomycin, kanamycin, butirosin, neomycin and gentamicin have been identified to date. In addition, most butirosin and neomycin biosynthetic enzymes have been functionally characterized using recombinant proteins. Herein, we reanalyze biosynthetic genes for structurally related 2-deoxystreptamine (2DOS)-containing aminoglycosides, such as kanamycin, gentamicin and istamycin, based on genetic information including characterized biosynthetic enzymes in neomycin and butirosin biosynthetic pathways. These proposed enzymatic functions for uncharacterized enzymes are expected to support investigation of the complex biosynthetic pathways for this important class of antibiotics.

Cloning of the Pactamycin Biosynthetic Gene Cluster and Characterization of a Crucial Glycosyltransferase Prior to a Unique Cyclopentane Ring Formation

The biosynthetic gene (pct) cluster for an antitumor antibiotic pactamycin was identified by use of a gene for putative radical S-adenosylmethionine methyltransferase as a probe. The pct gene cluster is localized to a 34 kb contiguous DNA from Streptomyces pactum NBRC 13433 and contains 24 open reading frames. Based on the bioinformatic analysis, a plausible biosynthetic pathway for pactamycin comprising of a unique cyclopentane ring, 3-aminoacetophenone, and 6-methylsalicylate was proposed. The pctL gene encoding a glycosyltransferase was speculated to be involved in an N-glycoside formation between 3-aminoacetophenone and UDP-N-acetyl-α-D-glucosamine prior to a unique cyclopentane ring formation. The pctL gene was then heterologously expressed in Escherichia coli and the enzymatic activity of the recombinant PctL protein was investigated. Consequently, the PctL protein was found to catalyze the expected reaction forming β-N-glycoside. The enzymatic activity of the PctL protein clearly confirmed that the present identified gene cluster is for the biosynthesis of pactamycin. Also, a glycosylation prior to cyclopentane ring formation was proposed to be a general strategy in the biosynthesis of the structurally related cyclopentane containing compounds.

A natural protecting group strategy to carry an amino acid starter unit in the biosynthesis of macrolactam polyketide antibiotics.

Macrolactam antibiotics are an important class of macrocyclic polyketides that contain a unique nitrogen-containing starter unit. In the present study, a set of starter biosynthetic enzymes in the macrolactam antibiotic vicenistatin was characterized. We found that the protection-deprotection strategy of the aminoacyl-ACP intermediate was critical in this system. On the basis of bioinformatics, the described pathway is also proposed as a common method for carrying amino acids in the biosynthesis of other macrolactam antibiotics.

Biosynthesis of 2-Deoxystreptamine by Three Crucial Enzymes in Streptomyces fradiae NBRC 12773

NeoA, B, and C encoded in the neomycin biosynthetic gene cluster have been enzymatically confirmed to be responsible to the formation of 2-deoxystreptamine (DOS) in Streptomyces fradiae. NeoC was functionally characterized as 2-deoxy-scyllo-inosose synthase, which catalyzes the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. Further, NeoA appeared to catalyze the oxidation of 2-deoxy-scyllo-inosamine (DOIA) with NAD(P)+ forming 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI). Consequently, NeoA was characterized as 2-deoxy-scyllo-inosamine dehydrogenase. Finally, amino-DOI produced by NeoA from DOIA was transformed into DOS by NeoB. Since NeoB (Neo6) was also reported to be L-glutamine:2-deoxy-scyllo-inosose aminotransferase, all the enzymes in the DOS biosynthesis were characterized for the first time.

Genome mining reveals two novel bacterial sesquiterpene cyclases: (-)-germacradien-4-ol and (-)-epi-α-bisabolol synthases from Streptomyces citricolor.

Now found in bacteria: An increasing number of genome sequences indicate that bacteria possess a variety of terpenoid cyclase genes. The characterization of two sesquiterpene cyclase (SC) genes found in the draft genome sequence of Streptomyces citricolor is described here. Our study strongly supports the idea that genome mining is a useful approach in revealing the terpenoid diversity in bacteria.

Biosynthetic enzymes for the aminoglycosides butirosin and neomycin.

Butirosin and neomycin belong to a family of clinically valuable 2-deoxystreptamine (2DOS)-containing aminoglycoside antibiotics. The biosynthetic gene clusters for butirosin and neomycin were identified in 2000 and in 2005, respectively. In recent years, most of the enzymes encoded in the gene clusters have been characterized, and thus almost all the biosynthetic steps leading to the final antibiotics have been understood. This knowledge could shed light on the complex biosynthetic pathways for other related structurally diverse aminoglycoside antibiotics. In this chapter, the enzymatic reactions in the biosynthesis of butirosin and neomycin are reviewed step by step.

An expeditious chemo-enzymatic route from glucose to catechol by the use of 2-deoxy-scyllo-inosose synthase

Abstract A potential two-step process to catechol from d -glucose comprising one-pot incubation of d -glucose with recombinant 2-deoxy- scyllo -inosose synthase (BtrC) and hexokinase, together with chemical reductive dehydration of the resulting 2-deoxy- scyllo -inosose with HI, was developed.

Involvement of two distinct N-acetylglucosaminyltransferases and a dual-function deacetylase in neomycin biosynthesis.

Neomycin is one of the clinically important 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics, which specifically interact with bacterial rRNA and inhibit protein synthesis. These compounds have attracted attention through their potential as anti-HIV and antiplasmid agents owing to their unique nucleotide recognition ability. Structural diversification of aminoglycosides is therefore a promising approach for the generation of novel bioactive compounds. Glycoside diversification by the use of glycosyltransferases is an attractive way to carry this out. 5] However, in the biosynthetic pathway of aminoglycosides, only a phosphoribosyltransferase has been characterized, even though many putative glycosyltransferase genes have been identified in the biosynthetic gene clusters. The only readily apparent glycosyltransferase gene in the neomycin biosynthetic gene cluster is neoD (neo8). 9] The deduced product (NeoD) belongs to the GT4 family, members of which catalyze retaining glycosyl transfer reactions with NDP-sugars. Comparative genetics has revealed that neoD-homologous genes are conserved among all the reported gene clusters of DOScontaining aminoglycosides, and are thus proposed to be involved in the formation of a common biosynthetic intermediate, paromamine (4, Scheme 1). Recently, 2’-N-acetylparomamine (3) was proposed to be a biosynthetic intermediate ACHTUNGTRENNUNGaccording to the deacetylase activity toward 3 of BtrD, an enzyme encoded in the butirosin biosynthetic gene cluster. Thus, NeoD was presumed to catalyze N-acetylglucosaminylation of DOS. To confirm the function of NeoD, the enzymatic activity of the recombinant NeoD protein was investigated. NeoD was co-expressed with molecular chaperone GroES and GroEL in E. coli to increase the amount of soluble protein (Figure 1A). Because NeoD showed low binding affinity for any kind of resin, even Ni affinity resin when expressed as a His-tagged protein, the cell-free extract of E. coli expressing NeoD was used for enzymatic assays. After incubating recombinant NeoD with UDP-GlcNAc and DOS, enzyme reaction products were treated with 2,4-dinitrofluorobenzene (DNFB), and the derivatives were analyzed by HPLC. As a result, about 80% of DOS was consumed, and a new product was observed at a retention time of 18 min (Figure 2A). This peak was not observed in a control reaction with the cell-free extract of E. coli harboring an empty plasmid (Figure 2B). The new peak showed m/z 696.4 by LC–ESIMS analysis, indicating that bis(2,4-dinitrophenyl)-3 ([M H] 696.3) was produced. The NeoD reaction product was further isolated from a large-scale enzyme reaction (4 mL) and its structure was confirmed by NMR and FABMS to be 3 (Supporting Information). Another possible glycosyl donor, UDP-glucose, and other possible gly[a] Prof. Dr. T. Eguchi Department of Chemistry and Materials Science Tokyo Institute of Technology O-okayama, Meguro-ku, Tokyo 152-8551 (Japan) Fax: (+81)3-5734-2631 E-mail : eguchi@cms.titech.ac.jp [b] K. Yokoyama, Y. Yamamoto, Dr. F. Kudo Department of Chemistry, Tokyo Institute of Technology O-okayama, Meguro-ku, Tokyo 152-8551 (Japan) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Scheme 1. Sequential glycosylation and deacetylation steps in neomycin biosynthesis.

Cloning and Characterization of the Biosynthetic Gene Cluster of 16-Membered Macrolide Antibiotic FD-891: Involvement of a Dual Functional Cytochrome P450 Monooxygenase Catalyzing Epoxidation and Hydroxylation

FD‐891 is a 16‐membered cytotoxic antibiotic macrolide that is especially active against human leukemia such as HL‐60 and Jurkat cells. We identified the FD‐891 biosynthetic (gfs) gene cluster from the producer Streptomyces graminofaciens A‐8890 by using typical modular type I polyketide synthase (PKS) genes as probes. The gfs gene cluster contained five typical modular type I PKS genes (gfsA, B, C, D, and E), a cytochrome P450 gene (gfsF), a methyltransferase gene (gfsG), and a regulator gene (gfsR). The gene organization of PKSs agreed well with the basic polyketide skeleton of FD‐891 including the oxidation states and α‐alkyl substituent determined by the substrate specificities of the acyltransferase (AT) domains. To clarify the involvement of the gfs genes in the FD‐891 biosynthesis, the P450 gfsF gene was inactivated; this resulted in the loss of FD‐891 production. Instead, the gfsF gene‐disrupted mutant accumulated a novel FD‐891 analogue 25‐O‐methyl‐FD‐892, which lacked the epoxide and the hydroxyl group of FD‐891. Furthermore, the recombinant GfsF enzyme coexpressed with putidaredoxin and putidaredoxin reductase converted 25‐O‐methyl‐FD‐892 into FD‐891. In the course of the GfsF reaction, 10‐deoxy‐FD‐891 was isolated as an enzymatic reaction intermediate, which was also converted into FD‐891 by GfsF. Therefore, it was clearly found that the cytochrome P450 GfsF catalyzes epoxidation and hydroxylation in a stepwise manner in the FD‐891 biosynthesis. These results clearly confirmed that the identified gfs genes are responsible for the biosynthesis of FD‐891 in S. graminofaciens.