Identification of L-Glutamine: 2-Deoxy-scyllo-inosose Aminotransferase Required for the Biosynthesis of Butirosin in Bacillus circulans.

Abstract

Using inverse PCR, two new genes (btrN and btrS) were identified upstream of the putative glycosyltransferase gene btrM in the butirosin-biosynthetic btr gene cluster of Bacillus circulans. The upstream gene btrS showed significant homology with stsC of Streptomyces griseus, which encodes L-glutamine: scyllo-inosose aminotransferase in the biosynthesis of streptomycin. The function of BtrS was further confirmed by heterologous expression in Escherichia coli and chemical identification of the conversion of 2-deoxy-scyllo-inosose into 2-deoxy-scyllo-inosamine.The identification of BtrS as L-glutamine: 2-deoxy-scyllo-inosose aminotransferase is the first report of the aminotransferase gene responsible for 2-deoxystreptamine biosynthesis.

Biosynthetic genes for aminoglycoside antibiotics

“…1-7 in Table 1), 2DOI synthase (BtrC, NeoC), L-glutamine:2DOI aminotransferase (BtrS, NeoS), NAD-dependent dehydrogenase (BtrE, NeoE), glycosyltransferase (BtrM, NeoM), deacetylase (BtrD, NeoD), flavin adenine dinucleotide (FAD)-dependent dehydrogenase (BtrQ, NeoQ) and another aminotransferase (BtrB, NeoB), are all conserved in butirosin, neomycin, ribostamycin, paromomycin, lividomycin, kanamycin, tobramycin, gentamicin and istamycin biosynthetic gene clusters, indicating that these are responsible for the construction of a common neamine-like structure. In fact, 2DOI formed from D-glucose-6-phosphate by 2DOI synthase is converted into 2-deoxy-scyllo-inosamine (2DOIA) by Gln:2DOI aminotransferase, 24,25 which is a homologous protein to StsC in streptomycin biosynthesis. Next, an NADdependent dehydrogenase catalyzes the dehydrogenation at C-1 of 2DOIA to give 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI).…”

“…Characterization of StsC was important, as described in the Introduction section, and thus led to the characterization of Gln:2DOI aminotransferase BtrS in the 2DOS biosynthesis. 24 This characteristic inosose aminotransferase is conserved completely in the aminocyclitol-containing aminoglycoside antibiotic biosynthetic gene clusters (Table 1). Kasugamycin lacks the amino functionality in the cyclitol moiety and therefore does not possess the aminotransferase gene in the gene cluster.…”

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

“…Two genes, pctC and pctV encoding putative aminotransferases exist in the pct cluster and either of these seems to be involved in the presumed transamination. PctC shows homology to DegT/DnrJ/EryC1/StsC family of aminotransferase, which are known to be involved in the biosynthesis of the unique aminocyclitols streptamine and 2-deoxystreptamine (DOS) of the aminoglycoside antibiotics, streptomycin, butirosin, kanamycin, and neomycin [43,44]. 3-Amino-5-hydroxybenzoic acid (AHBA) synthase, which is a pyridoxal 5Ј-phosphate (PLP) dependent enzyme catalyzing the a ,b -dehydration and stereospecific 1,4-enolization of aminoDHS to AHBA in the aminoshikimate pathway, shows homology to this family of aminotransferase [45ϳ47].…”

Extended Sequence and Functional Analysis of the Butirosin Biosynthetic Gene Cluster in Bacillus circulans SANK 72073

“…Gene walking toward the upstream of the known region was carried out by inverse PCR. Based on the previously determined sequence [12], two oligonucleotides btrS1: 5Ј-TTATCGCCCATCTGTTCG-ACTGAAA-3Ј and btrS2: 5Ј-CGCGAGGCAATGGAA-GAACAAATTA-3Ј were designed in inverse direction as primers. EcoRI-digested chromosomal DNA of B. circulans was, after the phenol-chloroform extraction, self-ligated.…”

“…BtrD is a novel enzyme involved in the biosynthesis of dTDP-glucosamine from dTTP and glucosamine-1-phosphate prior to the glycosylation in the paromamine synthesis [21]. Although seventeen open reading frames (ORFs) have been reported so far [11,12], all the required genes for the butirosin biosynthesis have not yet been determined. Thus, in order to address this important issue, we have pursued extended analysis of the butirosin biosynthetic gene cluster toward upstream and downstream, and nine additional open reading flames (ORFs), btrQ, btrR1, btrR2, btrT, btrU, btrV, btrW, btrX and orf1 have been supplemented to the cluster.…”