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.     

Nature's protection racket

(2S)-4-amino-2-hydroxybutyrate (AHBA) is a side chain that is important for the antibiotic activities of aminoglycosides. The elucidation of the biosynthetic pathway to AHBA, by Spencer et al. in this issue of Chemistry & Biology [1], reveals several surprises and will facilitate biosynthetic engineering of new improved aminoglycoside antibiotics.     

Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin

Mitomycin has a unique chemical structure and contains densely assembled functionalities with extraordinary antitumor activity. The previously proposed mitomycin C biosynthetic pathway has caused great attention to decipher the enzymatic mechanisms for assembling the pharmaceutically unprecedented chemical scaffold. Herein, we focused on the determination of acyl carrier protein (ACP)-dependent modification steps and identification of the protein–protein interactions between MmcB (ACP) with the partners in the early-stage biosynthesis of mitomycin C. Based on the initial genetic manipulation consisting of gene disruption and complementation experiments, genes mitE, mmcB, mitB, and mitF were identified as the essential functional genes in the mitomycin C biosynthesis, respectively. Further integration of biochemical analysis elucidated that MitE catalyzed CoA ligation of 3-amino-5-hydroxy-bezonic acid (AHBA), MmcB-tethered AHBA triggered the biosynthesis of mitomycin C, and both MitB and MitF were MmcB-dependent tailoring enzymes involved in the assembly of mitosane. Aiming at understanding the poorly characterized protein–protein interactions, the in vitro pull-down assay was carried out by monitoring MmcB individually with MitB and MitF. The observed results displayed the clear interactions between MmcB and MitB and MitF. The surface plasmon resonance (SPR) biosensor analysis further confirmed the protein–protein interactions of MmcB with MitB and MitF, respectively. Taken together, the current genetic and biochemical analysis will facilitate the investigations of the unusual enzymatic mechanisms for the structurally unique compound assembly and inspire attempts to modify the chemical scaffold of mitomycin family antibiotics.     

Characterization of the early stage aminoshikimate pathway in the formation of 3-amino-5-hydroxybenzoic acid: the RifN protein specifically converts kanosamine into kanosamine 6-phosphate

The biosynthesis of 3-amino-5-hydroxybenzoic acid (AHBA), precursor of the ansamycin and mitomycin antibiotics, proceeds by the aminoshikimate pathway from 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP). Identification of RifN, product of one of three genes from the rifamycin biosynthetic gene cluster known to be essential for aminoDAHP formation, as a specific kanosamine (3-deoxy-3-amino-D-glucose) 6-kinase establishes kanosamine and its 6-phosphate as specific intermediates in AHBA formation. This suggests a hypothetical reaction sequence for aminoDAHP formation, and thus for the early steps of AHBA biosynthesis, starting from UDP-D-glucose and introducing the nitrogen by oxidation and transamination at C-3.     

Molecular characterization and analysis of the biosynthetic gene cluster for the antitumor antibiotic mitomycin C from Streptomyces lavendulae NRRL 2564

BACKGROUND: The mitomycins are natural products that contain a variety of functional groups, including aminobenzoquinone- and aziridine-ring systems. Mitomycin C (MC) was the first recognized bioreductive alkylating agent, and has been widely used clinically for antitumor therapy. Precursor-feeding studies showed that MC is derived from 3-amino-5-hydroxybenzoic acid (AHBA), D-glucosamine, L-methionine and carbamoyl phosphate. A genetically linked AHBA biosynthetic gene and MC resistance genes were identified previously in the MC producer Streptomyces lavendulae NRRL 2564. We set out to identify other genes involved in MC biosynthesis. RESULTS: A cluster of 47 genes spanning 55 kilobases of S. lavendulae DNA governs MC biosynthesis. Fourteen of 22 disruption mutants did not express or overexpressed MC. Seven gene products probably assemble the AHBA intermediate through a variant of the shikimate pathway. The gene encoding the first presumed enzyme in AHBA biosynthesis is not, however, linked within the MC cluster. Candidate genes for mitosane nucleus formation and functionalization were identified. A putative MC translocase was identified that comprises a novel drug-binding and export system, which confers cellular self-protection on S. lavendulae. Two regulatory genes were also identified. CONCLUSIONS: The overall architecture of the MC biosynthetic gene cluster in S. lavendulae has been determined. Targeted manipulation of a putative MC pathway regulator led to a substantial increase in drug production. The cloned genes should help elucidate the molecular basis for creation of the mitosane ring system, as well efforts to engineer the biosynthesis of novel natural products.     

Biosynthesis of tetrapetalones

The biosynthesis of tetrapetalones (tetrapetalones A, B, C, and D) in Streptomyces sp. USF-4727 was studied by feeding experiments with [1-13C] sodium propanoate, [1-13C] sodium butanoate, [carbonyl-13C] 3-amino-5-hydroxybenzoic acid (AHBA) hydrochloride, and [1-13C] glucose, followed by analysis of the 13C-NMR spectra. These feeding experiments revealed that the four tetrapetalones were polyketide compounds constructed from propanoate, butanoate, AHBA, and glucose. The tetrapetalone biosynthetic pathway was also suggested in this study. In this pathway, tetrapetalone A (1) is synthesized by polyketide synthase (PKS) using AHBA as a starter unit, then the side chain of 1 is subjected to acetoxylation to produce tetrapetalone B (2). Additionally, 1 is oxidized and transformed into tetrapetalone C (3). In a similar way, 2 is converted to tetrapetalone D (4). Therefore, the biosynthetic relationship of the four tetrapetalones was indicated.     

Crystal structure of a complex between the aminoglycoside tobramycin and an oligonucleotide containing the ribosomal decoding a site

Aminoglycoside antibiotics target the decoding aminoacyl site (A site) on the 16S ribosomal RNA and induce miscoding during translation. Here, we present the crystal structure, at 2.54 A resolution, of an RNA oligonucleotide containing the A site sequence complexed to the 4,6-disubstituted 2-deoxystreptamine aminoglycoside tobramycin. The three aminosugar rings making up tobramycin interact with the deep-groove atoms directly or via water molecules and stabilize a fully bulged-out conformation of adenines A(1492) and A(1493). The comparison between this structure and the one previously solved in the presence of paromomycin confirms the importance of the functional groups on the common neamine part of these two antibiotics for binding to RNA. Furthermore, the analysis of the present structure provides a molecular explanation to some of the resistance mechanisms that have spread among bacteria and rendered aminoglycoside antibiotics inefficient.     

Polyketide synthase acyl carrier protein (ACP) as a substrate and a catalyst for malonyl ACP biosynthesis.

BACKGROUND: Using an acyl-acyl carrier protein (ACP) as a starter unit, type II polyketide synthases (PKSs) generate a wide range of polyketide products by successive decarboxylative condensations with the two-carbon donor malonyl (ACP). In vitro experiments have demonstrated that polyketide biosynthesis in reconstituted PKS systems requires the fatty acid synthase (FAS) enzyme malonyl CoA:ACP acyltransferase (FabD) from streptomycetes. It has also been shown that holo-ACPs from a type II PKS can catalyze self-malonylation in the presence of malonyl CoA and negate this FabD requirement. The relative roles of FabD and ACP self-malonylation in PKS biosynthesis in vivo are still not known. RESULTS: We have examined the ACP specificity of the Streptomyces glaucescens FabD and shown that it reacts specifically with monomeric forms of ACP, with comparable k(cat)/K(M) values for ACPs from both type II PKS and FAS systems. Incubations of tetracenomycin ACP (TcmM) with the Escherichia coli FAS ACP (AcpP) unexpectedly revealed that, in addition to the self-malonylation process, TcmM can catalyze the malonylation of AcpP. The k(cat)/K(M) value for the TcmM-catalyzed malonylation of S. glaucescens FAS ACP is two orders of magnitude smaller than that observed for the FabD-catalyzed process. CONCLUSIONS: The ability of a PKS ACP to catalyze malonylation of a FAS ACP is a surprising finding and demonstrates for the first time that PKS ACPs and FabD can catalyze the same reaction. The differences in the catalytic efficiency of these two proteins rationalizes in vitro observations that FabD-independent polyketide biosynthesis proceeds only at high concentrations of a PKS ACP.     

The neomycin biosynthetic gene cluster of Streptomyces fradiae NCIMB 8233: characterisation of an aminotransferase involved in the formation of 2-deoxystreptamine

The biosynthetic gene cluster of the 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotic neomycin has been cloned for the first time by screening of a cosmid library of Streptomyces fradiae NCIMB 8233. Sequence analysis has identified 21 putative open reading frames (ORFs) in the neomycin gene cluster (neo) with significant protein sequence similarity to gene products involved in the biosynthesis of other DOS-containing aminoglycosides, namely butirosin (btr), gentamycin (gnt), tobramycin (tbm) and kanamycin (kan). Located at the 5'-end of the neo gene cluster is the previously-characterised neomycin phosphotransferase gene (apH). Three genes unique to the neo and btr clusters have been revealed by comparison of the neo cluster to btr, gnt, tbm and kan clusters. This suggests that these three genes may be involved in the transfer of a ribose moiety to the DOS ring during the antibiotic biosynthesis. The product of the neo-6 gene is characterised here as the L-glutamine : 2-deoxy-scyllo-inosose aminotransferase responsible for the first transamination in DOS biosynthesis, which supports the assignment of the gene cluster.     

An Unusual Dehydratase Acting on Glycerate and a Ketoreducatse Stereoselectively Reducing α‐Ketone in Polyketide Starter Unit Biosynthesis

Polyketide synthases (PKSs) usually employ a ketoreductase (KR) to catalyze the reduction of a β‐keto group, followed by a dehydratase (DH) that drives the dehydration to form a double bond between the α‐ and β‐carbon atoms. Herein, a DH*‐KR* involved in FR901464 biosynthesis was characterized: DH* acts on glyceryl‐S‐acyl carrier protein (ACP) to yield ACP‐linked pyruvate; subsequently KR* reduces α‐ketone that yields L ‐lactyl‐S‐ACP as starter unit for polyketide biosynthesis. Genetic and biochemical evidence was found to support a similar pathway that is involved in the biosynthesis of lankacidins. These results not only identified new PKS domains acting on different substrates, but also provided additional options for engineering the PKS starter pathway or biocatalysis.     

The neomycin biosynthetic gene cluster of Streptomyces fradiae NCIMB 8233: genetic and biochemical evidence for the roles of two glycosyltransferases and a deacetylase

An efficient protocol has been developed for the genetic manipulation of Streptomyces fradiae NCIMB 8233, which produces the 2-deoxystreptamine (2-DOS)-containing aminoglycoside antibiotic neomycin. This has allowed the in vivo analysis of the respective roles of the glycosyltransferases Neo8 and Neo15, and of the deacetylase Neo16 in neomycin biosynthesis. Specific deletion of each of the neo8, neo15 and neo16 genes confirmed that they are all essential for neomycin biosynthesis. The pattern of metabolites produced by feeding putative pathway intermediates to these mutants provided unambiguous support for a scheme in which Neo8 and Neo15, whose three-dimensional structures are predicted to be highly similar, have distinct roles: Neo8 catalyses transfer of N-acetylglucosamine to 2-DOS early in the pathway, while Neo15 catalyses transfer of the same aminosugar to ribostamycin later in the pathway. The in vitro substrate specificity of Neo15, purified from recombinant Escherichia coli, was fully consistent with these findings. The in vitro activity of Neo16, the only deacetylase so far recognised in the neo gene cluster, showed that it is capable of acting in tandem with both Neo8 and Neo15 as previously proposed. However, the deacetylation of N-acetylglucosaminylribostamycin was still observed in a strain deleted of the neo16 gene and fed with suitable pathway precursors, providing evidence for the existence of a second enzyme in S. fradiae with this activity.     

Reversed-phase liquid chromatographic method for the analysis of aminoglycoside antibiotics using pre-column derivatization with phenylisocyanate

A pre-column derivatization liquid chromatographic method has been developed for the analysis of aminoglycoside antibiotics using phenylisocyanate as a derivatization reagent. Derivatives including kanamycin, neomycin and gentamicin were formed by reaction of the analytes with phenylisocyanate in the presence of triethylamine. Phenylisocyanato groups were attached to corresponding amino groups of aminoglycoside and their molecular mass was confirmed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The experimental conditions for derivatization and separation of aminoglycoside derivatives were optimized and validated. A simple liquid chromatographic method for the determination of aminoglycoside antibiotics was demonstrated.     

Active site mapping of 2-deoxy-scyllo-inosose synthase, the key starter enzyme for the biosynthesis of 2-deoxystreptamine. Mechanism-based inhibition and identification of lysine-141 as the entrapped nucleophile

A key enzyme in the biosynthesis of clinically important aminoglycoside antibiotics including neomycin, kanamycin, gentamicin, etc. is 2-deoxy-scyllo-inosose synthase (DOIS), which catalyzes the carbocycle formation from d-glucose-6-phosphate to 2-deoxy-scyllo-inosose (DOI). To clarify its precise reaction mechanism and crucial amino acid residues in the active site, we took advantage of a mechanism-based inhibitor carbaglucose-6-phosphate (pseudo-dl-glucose, C-6-P) with anticipation of its conversion to a reactive alpha,beta-unsaturated carbonyl intermediate. It turned out that C-6-P clearly showed time- and concentration-dependent inhibition against DOIS, and the molecular mass of the resulting modified-DOIS with C-6-P was 160 mass units larger than that of native DOIS. Thus, the expected alpha,beta-unsaturated intermediate appeared to trap a specific nucleophilic group in the active site through the Michael-type 1,4-addition. The covalently modified amino acid residue was determined to be Lys-141 by means of enzymatic digestion and subsequent LC/MS and LC/MS/MS of the digest. Also discussed are the role of Lys-141 in the substrate recognition and the reaction pathway and comparison with evolutionary related dehydroquinate synthase.     

Rational Design of Modular Polyketide Synthases: Morphing the Aureothin Pathway into a Luteoreticulin Assembly Line

The unusual nitro‐substituted polyketides aureothin, neoaureothin (spectinabilin), and luteoreticulin, which are produced by diverse Streptomyces species, point to a joint evolution. Through rational genetic recombination and domain exchanges we have successfully reprogrammed the modular (type I) aur polyketide synthase (PKS) into a synthase that generates luteoreticulin. This is the first rational transformation of a modular PKS to produce a complex polyketide that was initially isolated from a different bacterium. A unique aspect of this synthetic biology approach is that we exclusively used genes from a single biosynthesis gene cluster to design the artificial pathway, an avenue that likely emulates natural evolutionary processes. Furthermore, an unexpected, context‐dependent switch in the regiospecificity of a pyrone methyl transferase was observed. We also describe an unprecedented scenario where an AT domain iteratively loads an extender unit onto the cognate ACP and the downstream ACP. This aberrant function is a novel case of non‐colinear behavior of PKS domains.     

Development and validation of an improved reversed‐phase liquid chromatographic method combined with pulsed electrochemical detection for the analysis of netilmicin

Netilmicin is one of the aminoglycoside antibiotics that lacks a strong UV absorbing chromophore. However, the application of pulsed electrochemical detection has been used successfully for the direct analysis of aminoglycoside antibiotics. This study describes an improved LC method combined with pulsed electrochemical detection for the analysis of netilmicin. Using a Zorbax SB C‐18 column (250 mm×4.6 mm id, 5 μm), isocratic elution was carried out with a mobile phase containing sodium sulfate (20 g/L), sodium octanesulfonate (0.3 g/L), THF (20 mL/L), and 0.2 M phosphate buffer pH 3.0 (50.0 mL/L). The robustness of the method was examined by means of an experimental design. The method proved to be sensitive, repeatable, linear, and robust. The method has also been used to analyze some commercial netilmicin samples.     

Estimation of the molecular volumes of the reaction products between beta-lactam antibiotics and aminoglycoside antibiotics, and those of the degradation products of beta-lactam antibiotics by isotachophoresis

The correlative equations between the molecular volume and the qualitative indication (hR) for beta-lactam antibiotics, the reaction products between beta-lactam antibiotics and kanamycin, and the degradation products of beta-lactam antibiotics were hR = 0.32 + 0.080 VA2/3//Z/(N = 15, r = 0.972 for penicillins) and hR = 0.04 + 0.072 VA2/3//Z/(N = 12, r = 0.987 for cephems). Where VA is van der Waals volume (A3/molecule), hR is the relative step height in the isotachopherogram, and Z is the electric change, respectively. According to these equations, the molecular volumes of the reaction products between beta-lactam antibiotics and the other aminoglycoside antibiotics, and those of the degradation products of beta-lactam antibiotics can be estimated from the value of hR. Also according to the step height in the isotachopherogram, the reaction products or the degradation products may be estimated directly when the electric charge is known. It was confirmed that a molecule of aminoglycoside antibiotics reacted with a molecule of beta-lactam antibiotics. Therefore, the inactivation of aminoglycoside antibiotics is much greater than for beta-lactam antibiotics when the clinical doses of these antibiotic combinations are used.     

曲利本红与氨基糖苷类抗生素相互作用的共振瑞利散射光谱及其分析应用

在弱酸条件下，酸性双偶氮染料曲利本红（TR）或硫酸卡那霉（KANA）、硫酸 新霉素（NEO）、硫酸庆大霉素（GEN）和硫酸妥布霉素（TOB）等氨基糖苷类抗生 素的各自共振瑞利散射（RRS）十分微弱，但两者相互作用形成离子缔合物时能使 RRS急剧提高并产生新的RRS光谱，在400～535nm之间有一个强的散射带，最大散射 峰位于400nm处，在0.013～6.0μg·mL~(-1)范围内RRS强度与抗生素浓度成正比， 可用于氨基糖苷类抗生素的测定，对不同抗生素的检出限（3σ）在12.9～17.6ng ·mL~(-1)之间，其灵敏度的顺序是KANA＞NEO＞TOB＞GEN,方法有较好的选择性， 可用于市售抗生素注射液或滴耳液中药物含量和临床血药浓度的快速测定，中还用 量子化学方法对反应机理进行探讨，并讨论了的RRS光谱特性的影响因素和RRS增强 的原因。     

Conformational constraint as a means for understanding RNA-aminoglycoside specificity

The lack of high RNA target selectivity displayed by aminoglycoside antibiotics results from both their electrostatically driven binding mode and their conformational adaptability. The inherent flexibility around their glycosidic bonds allows them to easily assume a variety of conformations, permitting them to structurally adapt to diverse RNA targets. This structural promiscuity results in the formation of aminoglycoside complexes with diverse RNA targets in which the antibiotics assume distinct conformations. Such differences suggest that covalently linking individual rings in an aminoglycoside could reduce its available conformations, thereby altering target selectivity. To explore this possibility, conformationally constrained neomycin and paromomycin analogues designed to mimic the A-site bound aminoglycoside structure have been synthesized and their affinities to the TAR and A-site, two therapeutically relevant RNA targets, have been evaluated. As per design, this constraint has minimal deleterious effect on binding to the A-site. Surprisingly, however, preorganizing these neomycin-class antibiotics into a TAR-disfavored structure has no deleterious effect on binding to this HIV-1 RNA sequence. We rationalize these observations by suggesting that the A-site and HIV TAR possess inherently different selectivities toward aminoglycosides. The inherent plasticity of the TAR RNA, coupled to the remaining flexibility within the conformationally constrained analogues, makes this RNA site an accommodating target for such polycationic ligands. In contrast, the deeply encapsulating A-site is a more discriminating RNA target. These observations suggest that future design of novel target selective RNA-based therapeutics will have to consider the inherent "structural" selectivity of the RNA target and not only the selectivity patterns displayed by the low molecular weight ligands.     

Exploring the biosynthetic potential of bimodular aromatic polyketide synthases

Polycyclic aromatic polyketides such as actinorhodin and tetracenomycin are synthesized from acetate equivalents by type II polyketide synthases (PKS). Their carbon chain backbones are derived from malonyl-CoA building blocks through the action of a minimal PKS module consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and a malonyl-CoA/ACP transacylase. In contrast to these acetogenic polyketides, the backbones of a few aromatic polyketide natural products, such as the R1128 antibiotics, are primed by non-acetate building blocks. These polyketides are synthesized by bimodular PKSs comprising of a dedicated initiation module, which includes a ketosynthase, acyl transferase and ACP, as well as a minimal PKS module. Recently we showed that regioselectively modified polyketides could be synthesized through the genetic recombination of initiation modules and minimal PKS modules from different polyketide biosynthetic pathways (Tang et al. PLoS Biol. 2004, 2, 227-238). For example, the actinorhodin and tetracenomycin minimal PKSs could accept and elongate unnatural primer units from the R1128 initiation module. In this report we provide further examples of using heterologous bimodular PKSs for the engineered biosynthesis of new aromatic polyketides. In addition to providing insights into the biosynthetic mechanisms of aromatic PKSs, our findings also highlight considerable potential for crosstalk between amino acid catabolism and aromatic polyketide biosynthesis. For example, exogenously supplied unnatural amino acids are efficiently incorporated into bioactive anthraquinone antibiotics.     

Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin.

BACKGROUND: Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4'-phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides. PPTases have been classified according to their carrier protein specificity. In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity. However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene. RESULTS: A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp gene from S. verticillus. The svp gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters. The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution. Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S. verticillus or other Streptomyces species. As compared to Sfp, the only 'promiscuous' PPTase known previously, Svp displays a similar catalytic efficiency (k(cat)/K(m)) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP. CONCLUSIONS: PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products.