Molecular Cloning of the Gene for the Key Carbocycle-forming Enzyme in the Biosynthesis of 2-Deoxystreptamine-containing Aminocyclitol Antibiotics and Its Comparison with Dehydroquinate Synthase.

Abstract

The 2-deoxystreptamine aglycon is a commonstructural feature found in aminocyclitol antibiotics including neomycin, kanamycin, tobramycin, gentamicin, sisomicin, butirosin and ribostamycin. A key enzyme involved in the biosynthesis of the 2-deoxystreptamine moiety is 2-deoxy-1sic>'//<9-inosose (DOI) synthase which catalyses the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scy//<9-inosose. The recent success of isolating the 2-deoxy-,s'cy//6>-inosose synthase from Bacillus circulans prompted us to clone the gene responsible for this important enzyme by the use of reverse genetics approach. With the aid of DNAprobes constructed on the basis of the amino-terminal sequence of the purified 42kDa subunit of the enzyme, the responsible gene btrC was successfully cloned. Subsequently the btrC gene was heterologously expressed in Escherichia coll, and the 2-deoxy-scy//oinosose synthase activity of the recombinant polypeptide was confirmed by chemical analysis. The btrC gene encodes a protein composed of 368 amino acids with a molecular mass of 40.7 kDa. Our previous proposal for the similarity of 2-deoxy-scylloinosose synthase to dehydroquinate synthase has been confirmed on the basis of their amino acid sequences. Significant differences in the sequences can also be observed however, particularly in the crucial substrate recognition regions. Comparison of the BtrC sequence with those ofbiosynthetic enzymes for other related microbial products is also discussed.

Biosynthetic genes for aminoglycoside antibiotics

“…19 Its corresponding gene, btrC, was then cloned and the recombinant enzyme was found to have 2DOI synthase activity. 20 In addition, a butirosin biosynthetic gene cluster (btr) was identified by chromosomal gene walking from the DOI synthase gene (Table 1). 21 The majority of aminoglycosides contain 2DOS as a core unique aminocyclitol aglycon.…”

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

“…2-Deoxy-scyllo-inosose synthase (DOIS), which catalyzes the first crucial carbocycle formation from D-glucose-6-phosphate in the DOS biosynthesis, has been purified from the butirosin producer Bacillus circulans and its gene btrC was identified based on the N-terminal sequence [9,10]. Subsequently, the butirosin biosynthetic gene (btr) cluster was identified [11,12].…”

Biosynthesis of 2-Deoxystreptamine-containing Antibiotics in Streptoalloteichus hindustanus JCM 3268: Characterization of 2-Deoxy-scyllo-inosose Synthase

“…2-Deoxy-scylloinosose (DOI) synthase catalyzes the first crucial carbocyclization in the most abundant aminocyclitol 2-deoxystreptamine (DOS). Our identification of this gene in the butirosin producer Bacillus circulans [3], is an important key to the identification of many DOS-containing antibiotic gene clusters. So far, in addition to the butirosin biosynthetic gene cluster [4,5], the biosynthetic genes for tobramycin [6], kanamycin [7,8], neomycin [9,10], ribostamycin [11], hygromycin B (only sequence data available; AJ628642), istamycin (AJ845083), lividomycin (AJ748832), paromomycin (AJ628955), and apramycin (AJ629123) from Streptomyces, and gentamicin [12,13] from Micromonospora have been identified.…”