Enzymatic activity of a glycosyltransferase KanM2 encoded in the kanamycin biosynthetic gene cluster

The biosynthetic gene cluster for kanamycin, comprising 24 open reading frames, was identified in 2004.⁵ Two other research groups also deposited independently identical kanamycin biosynthetic genes by different symbols in the public DNA databases simultaneously.^{6, 7} To prevent confusion, herein we use the btr gene-based names, which were used systematically by the Piepersberg group and also in our recent review.^{8, 9} The entire kanamycin biosynthetic gene cluster was expressed in Streptomyces venezuelae and the resultant recombinant strain was reported to produce kanamycin A, which indicates that the kan genes are responsible for the biosynthesis of kanamycins.¹⁰ The kanC, kanS1, kanE, kanM1 and kanN genes expressing Streptomyces lividans reportedly produce paromamine, confirming that these genes, respectively, encode 2DOI synthase, Gln:2DOI aminotransferase, 2-deoxy-scyllo-inosamine dehydrogenase, UDP-GlcNAc:2DOS N-acetylglucosaminyltransferase and N-acetylparomamine deacetylase (Figure 1).¹¹ Among these, the kanC gene was expressed in Escherichia coli and the recombinant KanC protein was confirmed to be 2DOI synthase.⁶ Recombinant KanM1 protein was also reported to have weak glycosyltransferase activity with 2DOS as a glycosyl acceptor, and TDP-glucose or GDP-mannose as a glycosyl donor.¹² However, this KanM1 activity is contradictory to the presumed enzymatic function as suggested by the above-mentioned heterologous expression of UDP-GlcNAc:2DOS N-acetylglucosaminyltransferase in S. lividans. Detailed enzymatic analysis is thus necessary to elucidate the substrate specificity of KanM1 on using a pure enzyme.

Next, we attempted to isolate the KanM2 reaction product from paromamine and UDP-Glc, and determine the chemical structure. Ammonium sulfate fractionation between 10 and 40% saturation was effective in recovering most of the enzymatic activity from the cell-free extract of KanM2-expressing E. coli, although its activity was significantly decreased after a standard dialysis probably because of the instability of the enzyme. We then used the 10–40% (NH₄)₂SO₄ fractionated solution just after the fractionation, paromamine (3 mM, 10 mg), and UDP-Glc (4.7 mM, 29 mg) in 50 mM HEPES buffer (pH 8.0) containing 1 mM MgCl₂ and 10% glycerol (total 10 ml) at 28 °C for 24 h for a large-scale enzymatic reaction. To ensure maximum consumption of the starting material, the reaction mixture was further treated under identical enzymatic conditions using freshly prepared enzyme solution (0.25 μl) and UDP-Glc (5–10 mg) nine times. Consequently, 84% of paromamine was converted to the new glycosylated product, which was obtained as the sulfate form of aminoglycoside (7.9 mg) by ion exchange chromatography. HR-FAB-MS (positive mode, glycerol) of the compound gave an m/z of 486.2307, which corresponds to the calculated mass for C₁₈H₃₆O₁₂N₃ (486.2299), [M+H]⁺ of glucosylated paromamine.¹ H-NMR analysis of the product showed two anomeric protons and a methylene of the 2DOS moiety, although the other signals were very complex for assignment (see Supplementary information). Spectra of the compound obtained by total correlation spectroscopy (TOCSY) indicated that the signals for H-1 and H-3 of 2DOS at 3.3 p.p.m., those for H-6 at 3.6 p.p.m., and those for H-4 and H-5 at 3.7 p.p.m. are correlated with the signals for H-2ax and H-2eq at 1.7 and 2.3 p.p.m., respectively (data not shown). The use of ¹H–¹H-COSY, heteronuclear multiple quantum coherence and heteronuclear multiple bond coherence facilitated assignment of ¹³C chemical shifts for C1″, C1′, C-2′, C-1 or C-3, C-4, C-5 and C-6. Consequently, a glycosidic linkage between C1″ and C-6 was confirmed in addition to the C1′–O–C4 bond of the paromamine moiety. Therefore, the KanM2 reaction product from paromamine and UDP-Glc appeared to be 3″-deamino-3″-hydroxykanamycin C.

Log in or create a free account to read this content

Gain free access to this article, as well as selected content from this journal and more on nature.com

Access through your institution

or

Sign in or create an account

Continue with Google

Continue with ORCiD