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.

Complete In Vitro Reconstitution of the Apramycin Biosynthetic Pathway Demonstrates the Unusual Incorporation of a β-d-Sugar Nucleotide in the Final Glycosylation Step

“…In this study, the functions of the remaining uncharacterized enzymes in the apr cluster were elucidated, thereby establishing the complete biosynthetic pathway for apramycin (see Figures and ). The 4-amino-4-deoxy- d -glucose moiety is derived from G6P ( 4 ), which is consistent with previous results indicating the incorporation of intact glucose into the second sugar moiety of apramycin . Equilibration of G6P ( 4 ) with β-G1P ( 13 ) is catalyzed by AprJ and biased toward G6P ( K eq = 0.031).…”

“…d -Glucose ( 14 ), G6P ( 4 ), α- d -glucose 1-phosphate (α-G1P, 16 ), UDP-α- d -glucose ( 17 ), and α,α-trehalose ( 18 ) were tested as candidate sugar donors (Figure A). After overnight incubation, the 5-phosphate group was removed by treatment with AprZ prior to derivatization with 1-fluoro-2,4-dinitrobenzene (DNFB) under basic conditions . Only G6P ( 4 ) was almost completely consumed, yielding a product with m / z 1203.2633 in negative mode upon liquid chromatography with mass spectrometric analysis (LC–MS), which was consistent with the pseudomolecular ion of the tetra-dinitrophenyl (DNP) derivative of 2 (calcd.…”

“…After overnight incubation, the 5-phosphate group was removed by treatment with AprZ prior to derivatization with 1-fluoro-2,4-dinitrobenzene (DNFB) under basic conditions. 19 Only G6P (4) was almost completely consumed, yielding a product with m/z 1203.2633 in negative mode upon liquid chromatography with mass spectrometric analysis (LC−MS), which was consistent with the pseudomolecular ion of the tetra-dinitrophenyl 3B). To clarify which enzymes are required for product formation, the enzymes AprH, J, K, and O were sequentially removed from the aforementioned onepot reaction with ATP-Mg 2+ , G6P (4), and aprosamine 5phosphate (11).…”

“…The proteins encoded show high sequence homology (over 70% identity) with their counterparts in each cluster. Most of the enzymes encoded by the apr cluster have been characterized, and the biosynthetic pathway to aprosamine 5-phosphate ( 11 ) has been established (Figure B). − A key recent discovery is that the formation of the octose unit is catalyzed by AprG in a transaldol reaction using N -acetylglucosamine (GlcNAc) or N -acetylgalactosamine (GalNAc) as the two-carbon donor and 6′-oxo-lividamine ( 8 ) as the aldol acceptor ( 8 → 9 ) . While the periplasm-localized phosphatase AprZ has been demonstrated to catalyze the final hydrolysis of the phosphate group at the 5-position ( 12 → 1 ) to give apramycin, a step proposed to facilitate the efflux of apramycin ( 1 ) from the cell, it remains unclear how the 4-amino-4-deoxy -d -glucose moiety is assembled ( 11 → 12 ).…”

“…10−20 A key recent discovery is that the formation of the octose unit is catalyzed by AprG in a transaldol reaction using N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) as the two-carbon donor and 6′-oxo-lividamine (8) as the aldol acceptor (8 → 9). 19 While the periplasm-localized phosphatase AprZ has been demonstrated to catalyze the final hydrolysis of the phosphate group at the 5-position (12 → 1) to give apramycin, a step proposed to facilitate the efflux of apramycin (1) from the cell, 20 it remains unclear how the 4-amino-4-deoxy-D-glucose moiety is assembled (11 → 12). As mentioned earlier, the 4-aminosugar moiety plays a crucial role in the biological activity of apramycin (1), thus investigation of the construction of the 4-aminosugar moiety and its attachment to the disaccharide unit is important to fully comprehend the biosynthesis of apramycin.…”