A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S

Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of β-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the β-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron–sulfur cluster that is necessary for activity. These findings provide an example of an iron–sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)–dependent amide bond formation in a natural product biosynthetic pathway.

Amide bond formation in natural product biosynthesis can be catalyzed by nonribosomal peptide synthetases (NRPSs) and ATP-grasp ligases including the ATP-dependent activation of amino acid substrates. The carboxyl group is activated by phosphorylation and adenylation, respectively, in ATP-grasp ligase and NRPS catalyzed reactions (1). Recently, a new family of enzymes has been found to catalyze amide bond formation using aminoacyl-transfer RNA (aa-tRNA) as an activated cosubstrate. aa-tRNA plays a profound role in cells connecting the messenger RNA (mRNA) and protein synthesis at the ribosome in primary metabolism (2). Interestingly, recent studies have revealed that aa-tRNA can also be involved in natural product biosynthesis (3). aa-tRNA–dependent enzymes involved in natural product biosynthesis mainly form three groups: amide bond-forming ligases homologous with FemX peptidyltransferases from cell wall biosynthesis (4), synthase enzymes including the cyclodipeptide synthase family (5, 6), and dehydratase enzymes in RiPP (ribosomally synthesized and posttranslationally modified peptide) synthesis (7).Blasticidin S (BS), a representative of the amino hexose pyrimidine nucleoside antibiotics, was first isolated from Streptomyces griseochromogenes (8). The structure of BS features a C2′, C3′-dehydrated pyrose ring that is connected with cytosine at C1′ and β-arginine at C4′ (9). BS shows a broad spectrum of biological activities and had been widely used as a fungicide to protect rice from blast diseases (10, 11). Presently, BS is commonly used as an efficient selection antibiotic for transformed mammalian cells that express appropriate resistance genes (12, 13).The structural features and commercial value of BS stimulated interest in its biosynthetic studies. Early feeding experiments and characterization of related metabolites determined that glucose, cytosine, L-arginine, and methionine are the basic precursors for BS biosynthesis (14). The biosynthesis gene cluster was reported in 2003 and enabled a proposal for the BS biosynthetic pathway (Fig. 1) (15). However, difficulty carrying out gene knockout experiments in the native producer, S. griseochromogenes, hindered the further characterization of each biosynthetic step. More recently, the BS biosynthetic cluster was engrafted into the chromosome of Streptomyces lividans to generate the genetically stable mutant strain WJ2, which is able to produce BS and thereby facilitating in vivo studies of the function of BS biosynthetic genes (13). Gene deletions in WJ2 determined the essential genes for BS biosynthesis including blsD-blsL that are transcribed in one direction and the divergently transcribed blsM. The first committed step of BS biosynthesis is the hydrolysis of cytidine monophosphate by BlsM to produce free cytosine, which is then condensed with UDP-glucuronic acid by BlsD to form cytosyl-glucuronic acid (CGA) (15, 16). The process from CGA to cytosinine, a putative intermediate for BS biosynthesis, remains to be determined. A radical SAM protein BlsE and an aminotransferase BlsH are possibly involved in this transformation (13, 15). The β-arginine moiety of BS is derived from L-arginine through a radical-mediated reaction catalyzed by BlsG, a 2,3-arginine aminomutase (15, 17). It remains unknown whether BlsI, a putative ligase, is involved in the formation of DBS (demethylblasticidin S) by coupling of the carboxyl group of β-arginine and the amido group at C4′ of cytosinine since mutant WJ2∆blsI only accumulates the very early intermediate CGA. It is worth noting that DBS cannot be directly methylated to BS by the N-methyltransferase BlsL, which was confirmed by in vitro biochemical characterization (18).Open in a separate windowFig. 1.The proposed biosynthetic pathway of BS. CMP: Cytidine 5-monophosphate; AdoMet: S-Adenosylmethionine.Addition of leucine to DBS at the β-amino group of the arginine side chain forms leucyldemethylblasticidin S (LDBS) which is then methylated by BlsL to form the penultimate product leucylblasticidin S (LBS) (18). Finally, PepN catalyzes the maturation of BS biosynthesis via the hydrolysis of the leucyl group of LBS (19). The cell toxicity of LDBS and LBS are much lower than DBS and BS. Therefore, it is proposed that the circuitous modifications of BS and DBS with a leucyl group function as the self-resistance mechanism during BS biosynthesis (16). Attempts to form LDBS from DBS in a S. griseochromogenes cell-free system were not successful. A plausible reason for the failure of LDBS formation was that even if LDBS could be formed, it would be readily hydrolyzed back to DBS by the conserved aminopeptidase PepN in the cell-free system (16, 19).The intermediacy of LBS and LDBS hints at the necessity of an extra ligase other than BlsI in BS biosynthesis (15). In this report, we disclose the mechanism of the cryptic leucylation involved in BS biosynthesis. By combining in vivo gene inactivation and in vitro biochemical assays, we demonstrate that BlsK catalyzes the tRNA-dependent transfer of a leucyl group to the DBS β-arginine moiety by directing leucyl-tRNA^Leu to the BS biosynthetic pathway. More interestingly, BlsK is determined to be an iron–sulfur enzyme that does not show similarity with any known iron–sulfur enzymes. BlsK contains a [3Fe-4S] cluster that is critical for its activity. An iron–sulfur protein was shown to be involved in the amide bond formation. These results pave the way to fully understand the self-resistance and biosynthesis of BS.