Biosynthetic Origin of the Octose Core and Its Mechanism of Assembly during Apramycin Biosynthesis

Abstract

Apramycin is an aminoglycoside antibiotic isolated from Streptoalloteichus tenebrarius and S. hindustanus that has found clinical use in veterinary medicine. The apramycin structure is notable for its atypical eight-carbon bicyclic dialdose (octose) moiety. While the apramycin biosynthetic gene cluster (apr) has been identified and several of the encoded genes functionally characterized, how the octose core itself is assembled has remained elusive. Nevertheless, recent gene deletion studies have hinted at an N-acetyl aminosugar being a key precursor to the octose, and this hypothesis is consistent with the additional feeding experiments described in the present report. Moreover, bioinformatic analysis indicates that AprG may be structurally similar to GlcNAc-2-epimerase and hence recognize GlcNAc or a structurally similar substrate suggesting a potential role in octose formation. AprG with an extended N-terminal sequence was therefore expressed, purified, and assayed in vitro demonstrating that it does indeed catalyze a transaldolation reaction between GlcNAc or GalNAc and 6'-oxo-lividamine to afford 7'-N-acetyldemethylaprosamine with the same 6'-R and 7'-S stereochemistry as those observed in the apramycin product. Biosynthesis of the octose core in apramycin thus proceeds in the [6 + 2] manner with GlcNAc or GalNAc as the two-carbon donor, which has not been previously reported for biological octose formation, as well as novel inverting stereochemistry of the transferred fragment. Consequently, AprG appears to be a new transaldolase that lacks any apparent sequence similarity to the currently known aldolases and catalyzes a transaldolation for which there is no established biological precedent.

Figure 1.

Biosynthetic pathway for apramycin (1).^,,, Conversion of paromamine (4) to lividamine (5) is catalyzed by AprD4 and AprD3.^– Subsequent AprQ mediated C6′ oxidation of 5 to oxolividamine (6) sets the stage for octose assembly. The final steps include AprI catalyzed N-7′ methylation of 9 to give 10, AprH mediated attachment of a 4-aminosugar, and AprZ catalyzed dephosphorylation of 11 to yield 1. The annotation of apr genes is listed in Table S1.

Figure 2.

Proposed precursors providing the C7′, C8′, and 7′-N centers of the octose moiety in 7.

Figure 3.

Screening of potential C₂ donor substrates for the AprG-catalyzed transaldolation reaction. (A) Structures of possible C₂ donors. (B) Extracted ion chromatograms (EICs) at m/z 903.2035 corresponding to [M – H]⁻ for the tridinitrophenyl (DNP)-derivatives of 7, which was obtained after workup of the BtrQ/AprG reactions and then treated with 1-fluoro-2,4-dinitrobenzene (DNFB). Each EIC trace (a–f) is labeled with the compound number of the C₂ donor candidate used in the assay.

Figure 4.

BtrQ/AprG/AprU reaction with lividamine (5) and GalNAc (16). (A) Scheme of the one-pot reaction with BtrQ, AprG, and AprU. (B) Electrospray ionization (ESI) mass spectra of the one-pot reaction with (a) BtrQ, (b) BtrQ + AprG, (c) BtrQ + AprG + AprU, (d) BtrQ + AprU, and (e) lividamine (5) standard. All spectra were acquired in positive ion mode. Compound 5: [M+1]⁺ m/z 308.1816; compound 7: [M+1]⁺ m/z 407.2136; compound 8: [M+1]⁺ m/z 487.1800; and compound 18: [M+1]⁺ m/z 322.1609.

Figure 5.

(A) Possible mechanisms of octose formation catalyzed by AprG. (B) Proposed stereochemical course of the C–C bond formation is catalyzed by AprG. (C) ESI mass spectra of the octose product (8) formed in the BtrQ/AprG/AprU reaction using (a) unlabeled 12 or (b) [2-²H]-12.

Figure 6.

Proposed mechanism of retro-aldol bond cleavage catalyzed by AprG to form the C₂-donor. In the absence of an acceptor compound (e.g., 6), 25 along with 21 were detected as reaction products.

Figure 7.

(A) Octose formation involving [5 + 3] coupling: (a) three-carbon donor is PEP (33) in the reaction catalyzed by KDO synthase; (b) three-carbon donor is derived from F6P (36) or S7P (37) via the retro-aldol reaction catalyzed by LmbR. (B) Octose formation involving [6 + 2] coupling: (a) two-carbon donor is pyruvate (39) or hydroxypyruvate (41); (b) GlcNAc (12) or GalNAc (16) serves as the two-carbon donor in the AprG-catalyzed reaction.