Epimerization at C-3'' in butirosin biosynthesis by an NAD(+) -dependent dehydrogenase BtrE and an NADPH-dependent reductase BtrF.

Butirosin is an aminoglycoside antibiotic consisting two epimers at C-3'' of ribostamycin/xylostasin with a unique 4-amino-2-hydroxybutyrate moiety at C-1 of the aminocyclitol 2-deoxystreptamine (2DOS). To date, most of the enzymes encoded in the biosynthetic gene cluster for butirosin, from the producing strain Bacillus circulans, have been characterized. A few unknown functional proteins, including nicotinamide adenine dinucleotide cofactor-dependent dehydrogenase/reductase (BtrE and BtrF), are supposed to be involved in the epimerization at C-3'' of butirosin B/ribostamycin but remain to be characterized. Herein, the conversion of ribostamycin to xylsostasin by BtrE and BtrF in the presence of NAD(+) and NADPH was demonstrated. BtrE oxidized the C-3'' of ribostamycin with NAD(+) to yield 3''-oxoribostamycin. BtrF then reduced the generated 3''-oxoribostamycin with NADPH to produce xylostasin. This reaction step was the last piece of butirosin biosynthesis to be described.

Structural basis of APH(3')-IIIa-mediated resistance to N1-substituted aminoglycoside antibiotics.

Butirosin is unique among the naturally occurring aminoglycosides, having a substituted amino group at position 1 (N1) of the 2-deoxystreptamine ring with an (S)-4-amino-2-hydroxybutyrate (AHB) group. While bacterial resistance to aminoglycosides can be ascribed chiefly to drug inactivation by plasmid-encoded aminoglycoside-modifying enzymes, the presence of an AHB group protects the aminoglycoside from binding to many resistance enzymes, and hence, the antibiotic retains its bactericidal properties. Consequently, several semisynthetic N1-substituted aminoglycosides, such as amikacin, isepamicin, and netilmicin, were developed. Unfortunately, butirosin, amikacin, and isepamicin are not resistant to inactivation by 3'-aminoglycoside O-phosphotransferase type IIIa [APH(3')-IIIa]. We report here the crystal structure of APH(3')-IIIa in complex with an ATP analog, AMPPNP [adenosine 5'-(beta,gamma-imido)triphosphate], and butirosin A to 2.4-A resolution. The structure shows that butirosin A binds to the enzyme in a manner analogous to other 4,5-disubstituted aminoglycosides, and the flexible antibiotic-binding loop is key to the accommodation of structurally diverse substrates. Based on the crystal structure, we have also constructed a model of APH(3')-IIIa in complex with amikacin, a commonly used semisynthetic N1-substituted 4,6-disubstituted aminoglycoside. Together, these results suggest a strategy to further derivatize the AHB group in order to generate new aminoglycoside derivatives that can elude inactivation by resistance enzymes while maintaining their ability to bind to the ribosomal A site.

Biosynthesis of butirosin: transfer and deprotection of the unique amino acid side chain.

Butirosin, an aminoglycoside antibiotic produced by Bacillus circulans, bears the unique (S)-4-amino-2-hydroxybutyrate (AHBA) side chain, which protects the antibiotic from several common resistance mechanisms. The AHBA side chain is advantageously incorporated into clinically valuable antibiotics such as amikacin and arbekacin by synthetic methods. Therefore, it is of significant interest to explore the biosynthetic origins of this useful moiety. We report here that the AHBA side chain of butirosin is transferred from the acyl carrier protein (ACP) BtrI to the parent aminoglycoside ribostamycin as a gamma-glutamylated dipeptide by the ACP:aminoglycoside acyltransferase BtrH. The protective gamma-glutamyl group is then cleaved by BtrG via an uncommon gamma-glutamyl cyclotransferase mechanism. The application of this pathway to the in vitro enzymatic production of novel AHBA-bearing aminoglycosides is explored with encouraging implications for the preparation of unnatural antibiotics via directed biosynthesis.

Biosynthesis of the unique amino acid side chain of butirosin: possible protective-group chemistry in an acyl carrier protein-mediated pathway.

Butirosins A and B are naturally occurring aminoglycoside antibiotics that have a (2S)-4-amino-2-hydroxybutyrate (AHBA) side chain. Semisynthetic addition of AHBA to clinically valuable aminoglycoside antibiotics has been shown both to improve their pharmacological properties and to prevent their deactivation by a number of aminoglycoside-modifying enzymes involved in bacterial resistance. We report here that the biosynthesis of AHBA from L-glutamate, encoded within a previously identified butirosin biosynthetic gene cluster, proceeds via intermediates tethered to a specific acyl carrier protein (ACP). Five components of the pathway have been purified and characterized, including the ACP (BtrI), an ATP-dependent ligase (BtrJ), a pyridoxal phosphate-dependent decarboxylase (BtrK), and a two-component flavin-dependent monooxygenase system (BtrO and the previously unreported BtrV). The proposed biosynthetic pathway includes a gamma-glutamylation of an ACP-derived gamma-aminobutyrate intermediate, possibly a rare example of protective group chemistry in biosynthesis.

The complete 1H NMR assignments of aminoglycoside antibiotics and conformational studies of butirosin A through the use of 2D NMR spectroscopy.

The complete proton assignments of the aminoglycoside antibiotics, butirosin A, kanamycin A and kanamycin B, at pH 6.5 have been made through the use of various homonuclear and heteronuclear 2D NMR methods. Butirosin A NOESY experiments suggest a stacking arrangement between the xylose and 2,6-diamino-2,6-dideoxyglucose rings, while the 2-deoxystreptamine ring and its substituent, the (S)-4-amino-2-hydroxybutyryl group, extend away from the stacked rings. Informative long-range NOEs were observed for butirosin A but not with kanamycin A or kanamycin B. Many intra-ring NOEs were observed with all three aminoglycosides that confirm the proton assignments made in this study.

Butirosin-biosynthetic gene cluster from Bacillus circulans.

Butirosin is an interesting 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotic produced by non-actinomycete Bacilli. Recently we were successful in purification of 2-deoxy-scyllo-inosose synthase from butirosin-producer Bacillus circulans as the key enzyme for the biosynthesis of DOS, in cloning of the responsible gene (btrC), and in its overexpression in Escherichia coli. The present study involved gene-walking approach, which allowed us to find a gene cluster around btrC. The function of each gene was further investigated by gene disruption, and the disruptants of btrB, btrC, btrD and btrM showed no antibiotic producing activity. Therefore, the gene cluster found so far was determined to be a part of the butirosin biosynthetic gene cluster. Functions of some ORFs are also discussed in terms of butirosin biosynthesis on the basis of database search.

Synthesis and activity of butirosin derivatives with 5''-amidino and 5''-guanidino substituents.

The preparation and antibacterial activity of the 5''-guanidino (6) and 5''-amidino (7) derivatives of 4'-deoxybutirosin A (1) as well as the 5''-guanidino derivative (8) of butirosin A are described. The key intermediates, tetra-N-benzyloxycarbonyl-5''-azido derivatives were selectively reduced with NiCl2-NaBH4 to give the corresponding 5'-amino derivatives. Subsequent guanidination or amidination followed by deblocking afforded the final compounds 6, 7 and 8. The 5''-guanidino derivatives (6 and 8) were more active against Gram-positive and Gram-negative bacteria than the corresponding 5''-hydroxy derivatives (1 and butirosin A). Compound 6 was also active against a variety of methicillin-resistant Staphylococcus aureus (MRSA).

Biologically important conformations of aminoglycoside antibiotics bound to an aminoglycoside 3'-phosphotransferase as determined by transferred nuclear Overhauser effect spectroscopy.

NMR spectroscopy has been used to study the interaction of aminoglycoside antibiotics with an aminoglycoside antibiotic 3'-phosphotransferase [APH(3')-IIIa]. APH(3')-IIIa is an enterococcal enzyme that is responsible for the ATP-dependent O-phosphorylation of a broad range of aminoglycoside antibiotics. The NMR method of transferred nuclear Overhauser effect spectroscopy (TRNOESY) was used to detect intra- and inter-ring NOEs for butirosin A and amikacin in their respective ternary complexes with APH(3')-IIIa and ATP. NOE-derived distance constraints were used in energy minimization and dynamics routines to yield enzyme-bound structures for butirosin A. These structures suggest that the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings have restricted motions and are in a stacking arrangement. The TRNOE spectra for amikacin suggest that the 6-amino-6-deoxy-D-glucose ring is flexible when the antibiotic is bound to APH(3')-IIIa. The 15N resonances of butirosin A were assigned and the pKa values of the amino groups of butirosin A and amikacin were determined by 15N NMR spectroscopy. The N3 amino groups of butirosin A and amikacin have lowered pKa values, which is attributed to the (S)-4-amino-2-hydroxybutyryl (AHB) group of the antibiotics. This work provides an insight into the geometrical and electrostatic nature of aminoglycoside antibiotics bound to a modifying enzyme and will provide a basis for the design of inhibitors of APH(3')-IIIa.

Solution studies of isepamicin and conformational comparisons between isepamicin and butirosin A when bound to an aminoglycoside 6'-N-acetyltransferase determined by NMR spectroscopy.

NMR spectroscopy, combined with molecular modeling, was used to determine the conformations of isepamicin and butirosin A in the active site of aminoglycoside 6'-N-acetyltransferase-Ii [AAC-(6')-Ii]. The results suggest two enzyme-bound conformers for isepamicin and one for butirosin A. The dihedral angles that describe the glycosidic linkage between the A and B rings for the two conformers of AAC(6')-Ii-bound isepamicin were phi AB = -7.9 +/- 2.0 degrees and psi AB = -46.2 +/- 0.6 degrees for conformer 1 and phi AB = -69.4 +/- 2.0 degrees and psi AB = -57.7 +/- 0.5 degrees for conformer 2. Unrestrained molecular dynamics calculations showed that these distinct conformers are capable of interconversion at 300 K. When superimposed at the 2-deoxystreptamine ring, one enzyme-bound conformer of isepamicin (conformer 1) places the reactive 6' nitrogen in a similar position as that of butirosin A. Conformer 2 of AAC(6')-Ii-bound isepamicin may represent an unproductive binding mode. Unproductive binding modes (to aminoglycoside modifying enzymes) could provide one reason isepamicin remains one of the more effective aminoglycoside antibiotics. The enzyme-bound conformation of butirosin A yielded an orthogonal arrangement of the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings, as opposed to the parallel arrangement which was observed for this aminoglycoside in the active site of an aminoglycoside 3'-O-phosphotransferase [Cox, J. R., and Serpersu, E. H. (1997) Biochemistry 36, 2353-2359]. The complete proton and carbon NMR assignments of the aminoglycoside antibiotic isepamicin at pH 6.8 as well as the pKa values for it's amino groups are also reported.

New antibiotic produced by bacteria, 5-beta-D-xylofuranosylneamine.

A new aminoglycoside antibiotic was isolated from the fermentation broths of two strains of Bacillus species. The antibiotic is active against gram-positive and some gram-negative bacteria, and its antimicrobial spectrum is similar to that of ribostamycin. The chemical structure was determined to be 5-beta-d-xylofuranosylneamine, which is identical to the deacylated product obtained from butirosin A.