Study On The Aminoacyltransferase BlsK And The Methyltransferase BlsL In The Biosynthesis Of Blasticidin S

Blasticidin S?BS?is a member of the peptide nucleoside antibiotic family.It was first discovered and isolated from Streptomyces griseochromogenes.It is the first biological agent replacing organic mercury pesticides for controlling rice blast fungus diseases.Its action mechanism is to kill the pathogens by inhibiting protein synthesis.Given that the biological activity of BS is higher than that of organic mercury and it is harmless to fish,it has been widely used in Asian markets such as Japan,and had achieved good control effects.Currently,BS is used as a transgenic engineering screening reagent for eukaryotic cells due to the high sensitivity of mammalian cells to BS.The difficulty of genetic manipulation of the BS native producer strain S.griseochromogenes has hindered the study of its biosynthetic pathway.To overcome this shortage,BS biosynthetic gene cluster was grafted into the S.lividans genome,and the deaminase gene-SLBSD was knocked out to obtain the BS heterologous expression strain WJ2.Fermentation analysis of WJ2 found that in addition to the final product BS,there are three other metabolites with similar core structural moiety to BS,namely demethylblasticidin S?DBS?,leucyldemethylblasticidin S?LDBS?and leucylblasticidin S?LBS?.What's the order of synthesis of these three intermediates with the final product and which proteins are involved in the mutual transformation among them?These questions remained unclear.This study focuses on above questions to finalise the biosynthetic pathway of BS.The blasticidin S biosynthetic gene cluster was reported in 2003,and its essential gene cluster was localized to a DNA stretch containing open reading frames from blsD,E,F,G,H,I,J,K to L and an oppositely oriented blsM.The initial step in BS biosynthesis is to hydrolyze CMP to produce cytosine by BlsM,and followed by transfer of free cytosine to the glucuronic acid to give cytosylglucuronic acid?CGA?by BlsD,however,the function of other coding proteins remains unknown.This study focused on the leucylation of the intermediate product DBS during BS synthesis,because the bacteriostatic activity of LDBS and LBS were significantly decreased after loading the leucyl group.The leucyl is hydrolyzed from the final product BS by PepN,the loading of the leucyl group is thus considered to be a unique self-resistance mechanism for BS-producers.blsK and blsL are the last two genes in terms of gene arrangement in the BS biosynthetic gene cluster.The sequence analysis revealed many homologous proteins to BlsK,but all of them were annotated as proteins with unknown function,indicating that they may constitue a new protein family.blsK mutant strain WXK3 no longer produces LDBS and LBS,but DBS and trace amounts of BS,suggesting that BlsK is responsible for the leucylation of DBS or BS.The purified BlsK does not catalyze the formation of LDBS when incubated with DBS,whereas cell-free extract?CFE?overexpressing BlsK does,suggesting the presence of cofactor?s?in CFE necessary for BlsK activity.We found that when RNase A is added to the CFE,the LDBS is no longer produced.When tRNAs were added to the in vitro reaction system of purified BlsK,LDBS was generated and LDBS production efficiency was further improved when leucyl tRNA synthetase?LRS?was added.When using the prepared Leu-tRNA^Leu as a substrate,BlsK can transfer the leucyl group to DBS to generate LDBS directly,indicating that BlsK is a leucyl-tRNA dependent aminoacyltransferase.When the leucine is excessive in the reaction system,BlsK can load a second leucine on the?amino group of LDBS leucine to form LLDBS.The above results indicate that BlsK is responsible for catalyzing the loading of leucyl group onto DBS to form LDBS in the biosynthetic pathway of BS.In the course of purification of BlsK,we noticed that the protein solution is brownish red and prone to precipitate if it is exposed to the air for a long time in the absence of DTT,it is speculated that BlsK is a[Fe-S]protein.UV-VIS spectral analysis showed that BlsK protein solution had the characteristic absorption peak of[Fe-S]cluster at about 420 nM.Electron Paramagnetic resonance?EPR?analysis showed that BlsK had a signal of g=2.01?S=1/2?,and the signal was disappeared when the reducing agent sodium dithionite?DT?was added.Inductively coupled plasma mass spectrometry?ICP-MS?examination determined that each protein contained 3.253 Fe atoms and 4.363 S atoms,these data together showed that BlsK coordinates one[3Fe-4S]cluster.To determine which cysteines are involved in the formation of the[3Fe-4S]cluster,we individually mutated the nine cysteine residues of the BlsK into serine and found that the C236S,C253S and C259S mutant proteins became colorless,indicating that the[3Fe-4S]cluster was destroyed.EPR analysis detected the disappearance of the characteristic signals of the mutant proteins[3Fe-4S]cluster,but there was a signal of Fe³⁺,indicating that they could chelate some of the iron ions,but could not form a complete[3Fe-4S]cluster.Biochemical activity assay showed that the ability of mutant C236S,C253S and C259S proteins to catalyze the formation of LDBS were disappeared,while circular dichroism analysis showed that their secondary structure was similar to that of wild-type protein,indicating that the disappearance of protein activity was due to the incompleteness of[3Fe-4S]cluster.When BlsK was overexpressed with M9 medium containing no ferrous ions,the proteins were all precipitated,indicating that iron ions are necessary for BlsK to fold correctly.In the presence of the reducing agent sodium dithionite?DT?,the[3Fe-4S]¹⁺is reduced to[3Fe-4S]⁰,BlsK catalyzes the production of a new unstable compound and its structure needs further determination.In the previous work,WJ2 no longer produced BS after deletion of blsL gene,and the accumulated intermediate product was DBS and a small amount of LDBS.This study found that purified BlsL could not convert DBS into BS,but LDBS can be converted into LBS efficiently.We determined the enzyme kinetic parameters of BlsL for these two substrates and found that the affinity of BlsL for LDBS?K_m?106.8?M?is only three times that of DBS?K_m?304.8?M?,but the catalytic efficiency of BlsL for LDBS(k_cat/K_m?1.475×10^-22 mM^-1S^-1)is about 6556 times that of DBS(k_cat/K_m?2.25×10^-66 mM^-1S^-1),demonstrating that LDBS is the real substrate for BlsL.Its leucyl group has a great influence on the catalytic activity of the enzyme,and the complex structure of the substrate and enzyme may explain the difference in activity.Therefore,the conversion relationship between these three intermediates and BS is:BlsK catalyzes the loading of leucyl group onto DBS to generate LDBS,BlsL catalyzes the loading of a methyl group to form LBS,and PepN hydrolyzes the leucyl group to form the final product BS.Because the efficiency of aminopeptidase PepN hydrolysis of LDBS to generate DBS is almost the same as that of hydrolysis of LBS to form BS,therefore,after the blsL gene is deleted,the strain accumulates a large amount of DBS instead of LDBS.In this paper,through systematic studies of BlsK and BlsL in BS biosynthesis,it was confirmed that BlsK is a protein responsible for loading leucine onto DBS to generate LDBS.BlsK is a Leucyl-tRNA dependent aminoacyltransferase containing a[3Fe-4S]cluster,the[3Fe-4S]cluster is not only related to protein activity,but also plays a role in maintaining proper folding of the protein.It was confirmed that BlsL is a SAM-dependent methyltransferase whose substrate is LDBS instead of DBS,indicating that BlsK conversion of DBS to LDBS not only reduces toxicity,but also provides a substrate for BlsL.In summary,our study revealed the sequence of the last metabolites DBS,LDBS and LBS in the BS synthesis pathway and the transformation relationship between them,which complements the biosynthetic pathway of BS.