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 Acyl Carrier Protein-Dependent Glycosyltransferase in Mitomycin C Biosynthesis.

Mitomycins make up a group of antitumor natural products that are biosynthesized from aminohydroxybenzoic acid (AHBA) and N-acetylglucosamine (GlcNAc). While the biosynthetic gene cluster was reported two decades ago, the mechanism by which the two building blocks, AHBA and GlcNAc, are coupled during biosynthesis remained uncharacterized. Here we report evidence that AHBA is first loaded onto an MmcB acyl carrier protein (ACP) by a MitE acyl ACP synthetase, followed by a transfer of GlcNAc from UDP-GlcNAc by MitB. The results suggest that the early steps of mitomycin biosynthesis proceed via intermediates linked to MmcB.

Biosynthesis of 3,5-AHBA-derived natural products.

Covering: 1957 to 2011. 3-Amino-5-hydroxy benzoic acid (3,5-AHBA) is a precursor for a large group of natural products, including the family of naphthalenic and benzenic ansamycins, the unique saliniketals, and the family of mitomycins. This review covers the biosynthesis of AHBA-derived natural products from a molecular genetics, chemical, and biochemical perspectives, and 174 references are cited.

The biosynthesis of 3-amino-5-hydroxybenzoic acid (AHBA), the precursor of mC7N units in ansamycin and mitomycin antibiotics: a review.

The aminoshikimate pathway of formation of 3-amino-5-hydroxybenzoic acid (AHBA), the precursor of ansamycin and other antibiotics is reviewed. In this biosynthesis, genes for kanosamine formation have been recruited from other genomes, to provide a nitrogenous precursor. Kanosamine is then phosphorylated and converted by common cellular enzymes into 1-deoxy-1-imino-erythrose 4-phosphate, the substrate for the formation of aminoDAHP. This is converted via 5-deoxy-5-aminodehydroquinic acid and 5-deoxy-5-aminodehydroshikimic acid into AHBA. Remarkably, the pyridoxal phosphate enzyme AHBA synthase seems to have two catalytic functions: As a homodimer, it catalyzes the last reaction in the pathway, the aromatization of 5-deoxy-5-aminodehydroshikimic acid, and at the beginning of the pathway in a complex with the oxidoreductase RifL it catalyzes the transamination of UDP-3-keto-D-glucose. The AHBA synthase gene also serves as a useful tool in the genetic screening for new ansamycins and other AHBA-derived natural products.

Synthesis of potential early-stage intermediates in the biosynthesis of FR900482 and mitomycin C.

Beyond the identification of 3-amino-5-hydroxybenzoic acid (AHBA) and D-glucosamine as biosynthetic precursors to mitomycin C (5) and FR900482 (6), little is known about the pathway Nature uses to prepare these antitumor antibiotics. To gain some insight into their biosynthesis, amino acids 1 and 2 as well as C-2 N-acetylated derivatives 3 and 4 were prepared. Preparation of these putative biosynthetic intermediates and N-acetylcysteamine thioester analogues 28 and 29 should enable confirmation of their involvement in FR900482 and mitomycin C biosynthesis.

Analysis of a parallel branch in the mitomycin biosynthetic pathway involving the mitN-encoded aziridine N-methyltransferase.

Mitomycin C is a natural product with potent alkylating activity, and it is an important anticancer drug and antibiotic. mitN, one of three genes with high similarity to methyltransferases, is located within the mitomycin biosynthetic gene cluster. An inframe deletion in mitN of the mitomycin biosynthetic pathway was generated in Streptomyces lavendulae to produce the DHS5373 mutant strain. Investigation of DHS5373 revealed continued production of mitomycin A and mitomycin C in addition to the accumulation of a new mitomycin analog, 9-epi-mitomycin C. The mitN gene was overexpressed in Escherichia coli, and the histidine-tagged protein (MitN) was purified to homogeneity. Reaction of 9-epi-mitomycin C with MitN in the presence of S-adenosylmethionine yielded mitomycin E showing that the enzyme functions as an aziridine N-methyltransferase. Likewise, MitN was also shown to convert mitomycin A to mitomycin F under the same reaction conditions. We conclude that MitN plays an important role in a parallel biosynthetic pathway leading to the subclass of mitomycins with 9alpha-stereochemistry but is not involved directly in the biosynthesis of mitomycins A and C.

The mitomycin C (MMC)-binding protein from MMC-producing microorganisms protects from the lethal effect of bleomycin: crystallographic analysis to elucidate the binding mode of the antibiotic to the protein.

Antibiotic-producing microorganisms must be protected from the lethal effect of their own antibiotic. We have previously determined the X-ray crystal structure of the bleomycin (Bm)-binding protein, designated BLMA, as a self-resistance determinant from Bm-producing Streptomyces verticillus, which suggests that the binding of the first Bm to one of two pockets formed in the BLMA homodimer induces the cooperative binding of the second Bm to the other pocket. In the present study, we noticed that the X-ray crystallographic structure of a self-resistance determinant from a mitomycin C-producing microorganism, designated MRDP, reveals similarity to the folding pattern on the BLMA, although no sequence homology exists. To clarify the hypothesis that MRDP may function as a resistance determinant to Bm, we characterized and determined the crystal structure of MRDP complexed with the Cu(II)-bound form of BmA(2) grouped into the Bm family of antibiotics. The biochemical and structural studies for Bm binding provide evidence that the first Bm binds anti-cooperatively to a pocket of MRDP with binding affinity of the nanomolar order, whereas the second Bm binds to the other pocket, which has binding affinity of the micromolar order. The invisibility of the second Bm in the structure agrees with the observation that Escherichia coli-expressing MRDP displays lower resistance to Bm than that expressing BLMA. The structure of MRDP, which is complexed with the Cu(II)-bound BmA(2), revealed that the gamma-aminopropyldimethylsulphonium moiety of the antibiotic is sandwiched between the peripheral residues of the binding pocket and that its positively charged sulphonium head is accommodated completely in the negatively charged region of the MRDP pocket. Furthermore, the Cu(II)-bound BmA(2) has a very compact structure, in which the bithiazole ring of BmA(2) is folded back to the metal-binding domain.

The genetic switch regulating activity of early promoters of the temperate lactococcal bacteriophage TP901-1.

A functional analysis of open reading frame 4 (ORF4) and ORF5 from the temperate lactococcal phage TP901-1 was performed by mutant and deletion analysis combined with transcriptional studies of the early phage promoters p(R) and p(L). ORF4 (180 amino acids) was identified as a phage repressor necessary for repression of both promoters. Furthermore, the presence of ORF4 confers immunity of the host strain to TP901-1. ORF5 (72 amino acids) was found to be able to inhibit repression of the lytic promoter p(L) by ORF4. Upon transformation with a plasmid containing both ORF4 and ORF5 and their cognate promoters, clonal variation is observed: in each transformant, either p(L) is open and p(R) is closed or vice versa. The repression is still dependent on ORF4, and the presence of ORF5 is needed for the clonal variation. Induction of a repressed p(L) fusion containing orf4 and orf5 was obtained by addition of mitomycin C, and the induction was also shown to be dependent on the presence of the RecA protein, even though ORF4 does not contain a recognizable autocleavage site. Our results suggest that the relative amounts of the two proteins ORF4 and ORF5 determine the decision between lytic or lysogenic life cycle after phage infection and that a protein complex consisting of ORF4 and ORF5 may constitute a new type of genetic switch in bacteriophages.

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.

Genetic localization and molecular characterization of two key genes (mitAB) required for biosynthesis of the antitumor antibiotic mitomycin C.

Mitomycin C (MC) is an antitumor antibiotic derived biosynthetically from 3-amino-5-hydroxybenzoic acid (AHBA), D-glucosamine, and carbamoyl phosphate. A gene (mitA) involved in synthesis of AHBA has been identified and found to be linked to the MC resistance locus, mrd, in Streptomyces lavendulae. Nucleotide sequence analysis showed that mitA encodes a 388-amino-acid protein that has 71% identity (80% similarity) with the rifamycin AHBA synthase from Amycolatopsis mediterranei, as well as with two additional AHBA synthases from related ansamycin antibiotic-producing microorganisms. Gene disruption and site-directed mutagenesis of the S. lavendulae chromosomal copy of mitA completely blocked the production of MC. The function of mitA was confirmed by complementation of an S. lavendulae strain containing a K191A mutation in MitA with AHBA. A second gene (mitB) encoding a 272-amino-acid protein (related to a group of glycosyltransferases) was identified immediately downstream of mitA that upon disruption resulted in abrogation of MC synthesis. This work has localized a cluster of key genes that mediate assembly of the unique mitosane class of natural products.

[Cloning of mitomycin C resistance gene from Streptoverticillium caespitosum ATCC27422 and studying its function].

Streptoverticillium caespitosum is the major producer of anti-cancer drug mitomycin. It demonstrated that there exists a strong restriction system for the transformation of foreign DNA in this strain. Using the blocked mutant S-6 which is sensitive to mitomycin C (MC) and does not produce mitomycin as a recepient and plasmid pIJ699 as vector, the gene library of the strain ATCC27422 was constructed. A clone of mitomycin C resistance gene(mcr) was obtained by screening from this library. The mitomycin C resistance gene cloned on the 6.2 kb fragment of plasmid pLX5 could be expressed in the Strptonmyces lividans TK54. It was the first time to introduce successfully the plasmid pLX5 into the wild type strain ATCC27422 by electroporation technique. And the transformant's resistance to mitomycin C has risen greatly: the MIC has gone up from 200 micrograms/ml to more than 1000 micrograms/ml. The result of fermentation showed that the yield of mitomycin of ATCC27422 harboring plasmid pLX5 was more than the wild strain ATCC27422's. It suggested that there is some relationship between the level of the resistance to mitomycin C and its biosynthesis.