Butirosin Biosynthesis Research Articles

Crystal structure of BtrK, a decarboxylase involved in the (S)-4-amino-2-hydroxybutyrate (AHBA) formation during butirosin biosynthesis

Butirosin is an aminoglycoside that has an (S)-4-amino-2-hydroxybutyrate (AHBA) moiety capable of preventing the attack of several aminoglycoside modifying enzymes. The biosynthesis and the attachment of the AHBA to the 2-deoxystreptamine (2-DOS) involve seven enzymes that use glutamate as a precursor. BtrK is a pyridoxal-5-phosphate (PLP)-dependent enzyme and performs the decarboxylation of a glutamyl moiety tethered to the peptidyl carrier protein BtrI during the AHBA biosynthetic pathway. The structure of BtrK was solved at 1.4 Å resolution and indicated a conserved folding. The PLP is covalently linked through a Schiff base to Lys49 and performs intensive hydrogen bond interactions with active site residues that are also conserved in other members of type IV PLP-dependent enzymes. Additionally, a docking simulation indicates the possible anchoring of a substrate fragment constituted of O-S-γ-Lglutamylpantetheine-4′-phosphate. The glutamyl moiety forms a number of hydrogen bonds with the putative active site residues of BtrK. The pantetheine moiety seems to perform only a few interactions and should adopt a more flexible conformation. The description of BtrK structure contributes to the understanding of the large family of PLP-dependent enzymes and also in this crucial step during the butirosin biosynthesis.

Exploration of Catalytic Selectivity for Aminotransferase (BtrR) Based on Multiple Molecular Dynamics Simulations.

The aminotransferase from Bacillus circulans (BtrR), which is involved in the biosynthesis of butirosin, catalyzes the pyridoxal phosphate (PLP)-dependent transamination reaction to convert valienone to β-valienamine (a new β-glycosidase inhibitor for the treatment of lysosomal storage diseases) with an optical purity enantiomeric excess value. To explore the stereoselective mechanism of valienamine generated by BtrR, multiple molecular dynamics (MD) simulations were performed for the BtrR/PLP/valienamine and BtrR/PLP/β-valienamine complexes. The theoretical results showed that β-valienamine could make BtrR more stable and dense than valienamine. β-valienamine could increase the hydrogen bond probability and decrease the binding free energy between coenzyme PLP and BtrR by regulating the protein structure of BtrR, which was conducive to the catalytic reaction. β-valienamine maintained the formation of cation-p interactions between basic and aromatic amino acids in BtrR, thus enhancing its stability and catalytic activity. In addition, CAVER 3.0 analysis revealed that β-valienamine could make the tunnel of BtrR wider and straight, which was propitious to the removal of products from BtrR. Steered MD simulation results showed that valienamine interacted with more residues in the tunnel during dissociation compared with β-valienamine, resulting in the need for a stronger force to be acquired from BtrR. Taken together, BtrR was more inclined to catalyze the substrates to form β-valienamine, either from the point of view of the catalytic reaction or product removal.

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.

Differences in the Roles of a Glutamine Amidotransferase Subunit of Pyridoxal 5'-Phosphate Synthase betweenBacillus circulansandBacillus subtilis

BtrC2 of the butirosin producer Bacillus circulans is a non-catalytic subunit of 2-deoxy-scyllo-inosose (DOI) synthase that is involved in butirosin biosynthesis, and also a homolog of glutamine amidotransferase subunit (PdxT) of pyridoxal 5'-phosphate (PLP) synthase of Bacillus subtilis. BtrC2 has been found to have functions in B. circulans both in primary and secondary metabolism. In this study, we investigated the properties of PdxT of B. subtilis in order to determine whether the property of enzyme stabilization is universal among PdxT homologs. Complementation with PdxT in the btrC2 disruptant of B. circulans restored the growth and short-term production of antibiotics, but long-term production of antibiotics cannot be restored. Additionally, PdxT did not bind physically with or stabilize BtrC. Our results indicate that the function of BtrC2 in secondary metabolism is specific properties, not universal among PdxT homologs.

Biosynthesis of Ribostamycin Derivatives by Reconstitution and Heterologous Expression of Required Gene Sets

Ribostamycin is a 4,5-disubstituted 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics and naturally produced by Streptomyces ribosidificus ATCC 21294. It is also an intermediate in the biosynthesis of butirosin and neomycin. In the biosynthesis of ribostamycin, DOS is glycosylated to generate paromamine which is converted to neamine by successive dehydrogenation followed by amination, and finally ribosylation of neamine gives ribostamycin. Here, we report the biosynthesis of 6'-deamino-6'-hydroxyribostamycin (a ribostamycin derivative or pseudoribostamycin) in Streptomyces venezuelae YJ003 by reconstructing gene cassettes for direct ribosylation of paromamine. A trace amount of pseudoribostamycin was detected with ribostamycin in the isolates of ribostamycin cosmid heterologously expressed in Streptomyces lividans TK24. It has also indicated that the ribosyltransferase can accept both neamine and paromamine. Thus, the present in vivo modification of ribostamycin could be useful for the production of hybrid compounds to defend against bacterial resistance to aminoglycosides.

Roles of a 20 kDa Protein Associated with a Carbocycle-Forming Enzyme Involved in Aminoglycoside Biosynthesis in Primary and Secondary Metabolism

2-Deoxy-scyllo-inosose (DOI) synthase participates in the biosynthesis of 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics. The enzyme is expected to be of industrial use, because it converts a sustainable resource (glucose 6-phosphate) into carbocycle (DOI), which easily aromatizes to yield catechol. In the present study, we clarified the physiological role of a non-catalytic 20 kDa protein, BtrC2, associated with DOI synthase from the butirosin-producer Bacillus circulans. Based on the results of complementation analysis using btrC2 disruptant and western analysis against catalytic 40 kDa protein (BtrC) and BtrC2, it is suggested that BtrC2 has two functions. It is involved in the vitamin B(6) biosynthesis in primary metabolism, like homologous protein (Pdx2) in Bacillus subtilis. Additionally, it takes part in butirosin biosynthesis as stabilizer of DOI synthase by forming a heterodimer with BtrC, and qualifies B. circulans for stable and constant production of butirosin for long periods.

Combined Chemical‐Enzymatic Assembly of Aminoglycoside Derivatives with N‐1‐AHB Side Chain

AbstractA series of unprotected pseudo‐disaccharides and pseudo‐trisaccharides of 2‐deoxystreptamine‐containing aminoglycosides have been selectively acylated at the N‐1 position with the valuable (S)‐4‐amino‐2‐hydroxybutanoyl (AHB) pharmacophore by using the recombinant BtrH and BtrG enzymes from butirosin biosynthesis in combination with a synthetic acyl donor. The process was optimized by performing two enzymatic steps in a sequential manner without purification of the intermediate product.

Characterization and Mechanistic Study of a Radical SAM Dehydrogenase in the Biosynthesis of Butirosin

BtrN encoded in the butirosin biosynthetic gene cluster possesses a CXXXCXXC motif conserved within the radical S-adenosyl methionine (SAM) superfamily. Its gene disruption in the butirosin producer Bacillus circulans caused the interruption of the biosynthetic pathway between 2-deoxy-scyllo-inosamine (DOIA) and 2-deoxystreptamine (DOS). Further, in vitro assay of the overexpressed enzyme revealed that BtrN catalyzed the oxidation of DOIA under the strictly anaerobic conditions along with consumption of an equimolar amount of SAM to produce 5'-deoxyadenosine, methionine, and 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI). Kinetic analysis showed substrate inhibition by DOIA but not by SAM, which suggests that the reaction is the Ordered Bi Ter mechanism and that SAM is the first substrate and DOIA is the second. The BtrN reaction with [3-2H]DOIA generated nonlabeled, monodeuterated and dideuterated 5'-deoxyadenosines, while no deuterium was incorporated by incubation of nonlabeled DOIA in the deuterium oxide buffer. These results indicated that the hydrogen atom at C-3 of DOIA was directly transferred to 5'-deoxyadenosine to give the radical intermediate of DOIA. Generation of nonlabeled and dideuterated 5'-deoxyadenosines proved the reversibility of the hydrogen abstraction step. The present study suggests that BtrN is an unusual radical SAM dehydrogenase catalyzing the oxidation of the hydroxyl group by a radical mechanism. This is the first report of the mechanistic study on the oxidation of a hydroxyl group by a radical SAM enzyme.

Characterization of the Enzyme BtrD from Bacillus circulans and Revision of Its Functional Assignment in the Biosynthesis of Butirosin

Neubestimmte Funktion: BtrD, ein im Butirosin-Gencluster kodiertes Protein, fungiert als Desacetylase und nicht wie früher angenommen als Nucleotidyltransferase. BtrD katalysiert selektiv die Umwandlung von 2′-N-Acetylparomamin in Paromamin – ein deutlicher Hinweis darauf, dass die Neosamineinheit von Butirosin von Uridindiphosphat(UDP)-N-acetylglucosamin stammt (siehe Bild).

Characterization of the Enzyme BtrD from Bacillus circulans and Revision of Its Functional Assignment in the Biosynthesis of Butirosin

Characterization of the Enzyme BtrD from <i>Bacillus circulans</i> and Revision of Its Functional Assignment in the Biosynthesis of Butirosin

Crystal structures of the PLP‐ and PMP‐bound forms of BtrR, a dual functional aminotransferase involved in butirosin biosynthesis

The aminotransferase (BtrR), which is involved in the biosynthesis of butirosin, a 2-deoxystreptamine (2-DOS)-containing aminoglycoside antibiotic produced by Bacillus circulans, catalyses the pyridoxal phosphate (PLP)-dependent transamination reaction both of 2-deoxy-scyllo-inosose to 2-deoxy-scyllo-inosamine and of amino-dideoxy-scyllo-inosose to 2-DOS. The high-resolution crystal structures of the PLP- and PMP-bound forms of BtrR aminotransferase from B. circulans were solved at resolutions of 2.1 A and 1.7 A with R(factor)/R(free) values of 17.4/20.6 and 19.9/21.9, respectively. BtrR has a fold characteristic of the aspartate aminotransferase family, and sequence and structure analysis categorises it as a member of SMAT (secondary metabolite aminotransferases) subfamily. It exists as a homodimer with two active sites per dimer. The active site of the BtrR protomer is located in a cleft between an alpha helical N-terminus, a central alphabetaalpha sandwich domain and an alphabeta C-terminal domain. The structures of the PLP- and PMP-bound enzymes are very similar; however BtrR-PMP lacks the covalent bond to Lys192. Furthermore, the two forms differ in the side-chain conformations of Trp92, Asp163, and Tyr342 that are likely to be important in substrate selectivity and substrate binding. This is the first three-dimensional structure of an enzyme from the butirosin biosynthesis gene cluster.

Extended Sequence and Functional Analysis of the Butirosin Biosynthetic Gene Cluster in Bacillus circulans SANK 72073

Butirosin produced by Bacillus circulans is among the clinically important 2-deoxystreptamine containing aminoglycoside antibiotics and its unique structure is found in (S)-4-amino-2-hydroxyburyric acid substituted at C-1 of 2-deoxystreptamine. Recently, the key part of the butirosin biosynthetic gene cluster has been identified from Bacillus circulans SANK 72073, however the whole gene for the biosynthesis awaited for identification. In the present study, we undertook extended analysis of the butirosin biosynthetic gene cluster and found nine additional open reading flames (ORFs), btrQ, btrR1, btrR2, btrT, btrU, btrV, btrW, btrX and orf1 in the cluster. In addition, we constructed disruption mutants of btrR1 and btrP-V, and found that the btr genes (ca. 24Kb) between btrR1 and btrP-V are at least required for the butirosin biosynthesis.

Significance of the 20-kDa subunit of heterodimeric 2-deoxy-scyllo-inosose synthase for the biosynthesis of butirosin antibiotics in Bacillus circulans.

A gene (btrC2) encoding the 20-kDa subunit of 2-deoxy-scyllo-inosose (DOI) synthase, a key enzyme in the biosynthesis of 2-deoxystreptamine, was identified from the butirosin-producer Bacillus circulans by reverse genetics. The deduced amino acid sequence of BtrC2 closely resembled that of YaaE of B. subtilis, but the function of the latter has not been known to date. Instead, BtrC2 appeared to show sequence similarity to a certain extent with HisH of B. subtilis, an amidotransferase subunit of imidazole glycerol phosphate synthase. Disruption of btrC2 reduced the growth rate compared with the wild type, and simultaneously antibiotic producing activity was lost. Addition of NH4Cl to the medium complemented only the growth rate of the disruptant, and both the growth rate and antibiotic production were restored by addition of yeast extract. In addition, a heterologous co-expression system of btrC2 with btrC was constructed in Escherichia coli. The simultaneously over-expressed BtrC2 and BtrC constituted a heterodimer, the biochemical features of which resembled those of DOI synthase from B. circulans more than those of the recombinant homodimeric BtrC. Despite the similarity of BtrC2 to HisH the heterodimer showed neither aminotransfer nor amidotransfer activity for 2-deoxy-scyllo-inosose as a substrate. All the observations suggest that BtrC2 is involved not only in the secondary metabolism, but also in the primary metabolism in B. circulans. The function of BtrC2 in the butirosin biosynthesis appears to be indirect, and may be involved in stabilization of DOI synthase and in regulation of its enzyme activity.

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.

Biosynthesis of butirosin in Bacillus circulans NRRL B3312: identification by sequence analysis and insertional mutagenesis of the butB gene involved in antibiotic production

As an approach to an analysis of the biosynthesis of the aminoglycoside antibiotic butirosin (But), we investigated the chromosomal regions flanking the But R gene ( aphA4/butA) of Bacillus circulans NRRL-B3312, and have identified, by nucleotide sequence analysis, a large open reading frame (ORF; ButB) upstream from the But R gene. Hybridization was detected between butB and chromosomal DNA from other Bacillaceae that produce But-like compounds (but not from non-producers). Interruption of this sequence by insertion of an erythromycin-resistance-encoding gene ( erm) at either of two distinct sites eliminated the production (biosynthesis or export) of But, thus indicating a role for butB in antibiotic production. Gene butB is transcribed in the same direction as but A and encodes a protein of 1616 amino acid (aa) residues with a 30-aa N-terminal signal peptide. Comparison of the sequence for the translation product (ButB) with the aa compositions and sequences of known bacterial surface proteins, such as S-layer proteins, suggests that this protein is cell-wall associated. It is proposed that ButB plays a role in the export of But from the producing organism.

Relationship between butirosin biosynthesis and sporulation in Bacillus circulans

The relationship between butirosin biosynthesis and certain biochemical characteristics related to sporulation in a strain of Bacillus circulans NRRL B-3313 was examined. The cellular content of dipicolinic acid increased while the amount of poly-beta-hydroxybutyrate decreased with changes in antibiotic productivity. Oligosporogenous mutants failed to synthesize the antibiotic and to degrade poly-beta-hydroxybutyrate. These observations suggest that spore formation may be related to antibiotic production in this strain of B. circulans.