Streptomyces Lavendulae Research Articles

Screening and Identification of Streptomyces lavendulae against Walnut Rot Disease Cytospora chrysosperma

Streptomyces is a biocontrol agent capable of protecting walnut from stem rot disease caused by the fungal pathogen Cytospora chrysosperma. Antagonistic strains were selected from walnut rhizosphere soil for biocontrol of the disease. The strains were screened using C. chrysosperma as the target pathogen. Using dilution coating plate separation method, plate confrontation method, growth rate method and in vitro branch protection experiment, screening the antagonistic actinobacteria with inhibitory effect and control effect on walnut tree rot disease. Strains were identified based on their morphological, physiological, and biochemical characteristics, 16S rDNA sequencing, and phylogenetic analysis. The strain F-02 was identified as Streptomyces Lavendulae. Plate confrontation method tests showed that the inhibition rates of the Streptomyces strain F-02 on the pathogens were approximately 80.30%. Further study showed that the filtrate of strain F-02 couldinhibit the C.chrysosperma, and the widths of their inhibition zone reached 1.24±0.15 mm, the prevention effect of 71.03%. For the first time, S.lavendulae was used for the biological control of walnut tree rot. It provides strain resources for the development of walnut rot agents. It has potential applications for biological control of walnut tree rot disease.

Streptomyces peucetius M1 and Streptomyces lavendulae M3 Soil Isolates as a Promising Source for Antimicrobials Discovery

The ever-increasing fatality due to the surge of drug resistance has demanded the development of therapeutic metabolites with novel modes of mechanism. Despite the discovery of new antimicrobials, antibiotic resistance is increasing at an alarming rate. The emergence of novel molecules is necessary for targeting the Multidrug (MDR) and Pan-Drug resistant (PDR) pathogens which are the principal cause of life-threatening infections. Streptomyces strains commonly found in the soil produce secondary metabolites which have outstanding potential for being an antifungal and antibacterial agent. This study aims to isolate, identify, and characterize Streptomyces strains isolated from soil and extract their crude metabolites to determine their hidden therapeutic property. Out of four isolates identified, Streptomyces peucetius and Streptomyces lavendulae have shown significant antimicrobial activity against test organisms. To further determine the activity of their extracts GC-MS analysis, and MIC, as well as radical scavenging activity, were determined. GC-MS analysis revealed the presence of compounds 2,3-butanediol, 1-methyl-2-pyrrolidone-4-carboxamide, and L-Lactic acid which are frequently associated with potent antimicrobial nature. The most vulnerable pathogen to M1 extract was Candida albicans with a MIC value of 7.8125 μg/mL while the susceptible bacteria to M3 extract included Staphylococcus aureus with a MIC value of 3.90 μg/mL. The data obtained for antioxidant activity in the case of metabolic extracts M1 and M3 had an IC50 value of 43.17 µg/mL and 22.65 µg/mL respectively. The above findings hint at the unrealized potential of Streptomyces strains.

Isolation and molecular identification of lignin peroxidase-producing bacterial isolates from Jeddah City

Background The identification of naturally occurring bacteria with lignin-oxidizing enzymes would be significant. Several species of filamentous bacteria belonging to the genus Streptomyces (Actinomycetes) have been identified as degraders of lignin. Such species play the most important role in biodegradation of lignin. Objective This study aimed to isolate and discover promising isolates and ideal conditions for lignin peroxidase (LiP) production as well as 16S-rRNA identification of the ligninolytic bacterial strains. Materials and methods Lignin was isolated and purified from black wood liquor. The ligninolytic bacterial colonies were isolated from three types of soil farms (F1, F2, and F3) from Jeddah, KSA. Fermentation medium (FM) was used for screening of lignin-degrading bacteria after dilution of the soil sample using lignin (0.1% v/v). The FM medium was supplemented with 50 mg/l of Azure B and toluidine dyes and 100 mg/l of tannic acid. FM was used without any supplements and agar for isolation of lignin-degrading bacteria after dilution of the soil samples. Different concentrations of lignin (0.1–0.9%) were applied to optimize LiP production by the selected strains under different temperatures (30, 35, 40, and 45°C); different pH values (7, 7.5, 8.0, and 8.5); eight different carbon sources (0.1%, w/v), such as glucose, fructose, xylose, lactose, sucrose, carboxymethyl cellulose, and xylan; and four organic sources (0.1%, w/v), such as peptone, meat extract, sodium nitrate, and potassium nitrate. The enzyme productivity was evaluated in the culture supernatant. The bacterial strain genomic DNA was extracted from pure culture isolated from soil and subjected to amplification and sequencing of 16 S ribosomal RNA gene. Results and discussion Nine ligninolytic bacterial colonies that excrete peroxidases based on the use of lignin (as sole carbon source) were isolated from three types of soil farms (F1, F2, and F3) from Jeddah, KSA, and the promising isolates and the optimum conditions for LiP production using FM under three incubation periods were evaluated. Two most active isolates for production of LiP belonging to Actinomycetes and Bacilli designated (R-St-1 and R-B-1) were identified using 16S-rRNA. Results showed that the highest LiP producer was Streptomyces R-St-1 isolate (3.8 U/ml) followed by Bacilli R-B-1 isolate (2.4 U/ml) after 3 days of fermentation. Different concentrations of lignin (0.1–0.9%) were tested for their effect on LiP production by Streptomyces R-St-1 . As lignin concentration increased, LiP production increased, and the maximum productivity of 4.9 U.mL−1 was observed at 0.5% lignin after which the LiP production was decreased. At the ideal temperature recorded of 35°C and at the optimum pH of 7.5, the production of LiP rose significantly (4.6 U.mL-1 and 4.0 U.mL-1). Various carbon sources were examined for LiP production, and glucose was shown to be the best option for producing a high yield of LiP by Streptomyces R-St-1 , followed by lactose (4.6 and 4.0 U/ml, respectively). However, neither organic nor inorganic nitrogen sources were shown to be suited for high LiP output. As lignin concentration increased, LiP production increased, and the maximum productivity of 4.4 U/ml from Bacilli R-B-1 isolate was observed at 0.7% lignin, after which LiP production was decreased. The optimum temperature was 40°C, where LiP production showed a significant increase (4.5 U/ml), whereas the optimum pH was 8.0, and sucrose was found to be more suitable for high yield of LiP production followed by glucose (4.3 and 3.3 U/ml, respectively). The identified bacterial DNA sequences were conserved in the GenBank under two accession numbers OL697233.1 (Streptomyces lavendulae R-St-1) and Priestia aryabhattai R-B-1 (OL697234.1) (formerly known as Bacillus aryabhattai).

Upgrading the Potential of Biopesticide from Actinobacteria for Controlling Onion Bacterial Rots by Mixing two Identified Streptomyces species in Egypt

Bacterial rots of onion are considered from the most dangerous stored and heaped onion crop diseases that cause huge loss in bulbs. Many bacterial species can induce onion bulb rots, with predominance of Pectobacterium carotovorum and Burkholderia cepacia that were used in this study. This research was conducted as an approach for improving the potential of biopesticide from actinobacteria in controlling onion bacterial rots by mixing Streptomyces coelicolor and Streptomyces lavendulae in one treatment against both P. carotovorum and B. cepacia each separately. The highest decrease in disease incidence was recorded in the mixed treatment of S. coelicolor and S. lavendulae that was recorded as 88.8% and 86.3% in case of P. carotovorum and B. cepacia, respectively, followed by the single treatment of S. lavendulae then the single treatment of S. coelicolor. Application of S. coelicolor and S. lavendulae, singly or in mixture had significantly increased the total phenols in onion bulbs but the highest increase was recorded in the mixed application treatment that recorded as 356.1% and 353.8% in case of P. carotovorum and B. cepacia, respectively. Also, the antioxidant enzymes (POX and PPO) activity was observed to be highest in case of the mixed treatment against both P. carotovorum and B. cepacia. On the otherhand lipid peroxidation was significantly decreased in all treatments but the highest decrease percentage was detected in the mixed treatment which was 53.3% and 57.7% in case of P. carotovorum and B. cepacia, respectively. Based on their efficiency and eco-safety, S. coelicolor and S. lavendulae have been recommended to be mixed in one treatment to increase the potential of biopesticide used for controlling onion bacterial rots.

Identification of Rhizospheric Actinomycete Streptomyces lavendulae SPS-33 and the Inhibitory Effect of its Volatile Organic Compounds against Ceratocystis Fimbriata in Postharvest Sweet Potato (Ipomoea Batatas (L.) Lam.).

Black spot disease, which is caused by the pathogenic fungal Ceratocystis fimbriata, seriously affects the production of sweet potato and its quality during postharvest storage. In this study, the preliminary identification of the rhizosphere actinomycete strain SPS-33, and its antifungal activity of volatiles in vitro and in vivo was investigated. Based on morphological identification and phylogenetic analysis of the 16S rRNA gene sequence, strain SPS-33 was identified as Streptomyces lavendulae. Volatile organic compounds (VOCs) emitted by SPS-33 inhibited mycelial growth and sporulation of C. fimbriata in vitro and also induced a series of observable hyphae morphological changes. In an in vivo pathogenicity assay, exposure to SPS-33 significantly decreased the lesion diameter and water loss rate in sweet potato tuberous roots (TRs) inoculated with C. fimbriata. It increased the antioxidant enzymes’ activities of peroxidase, catalase, and superoxide dismutase as well as decreased malondialdehyde and increased total soluble sugar. In the VOC profile of SPS-33 detected by a headspace solid-phase micro extraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS), heptadecane, tetradecane, and 3-methyl-1-butanol were the most abundant compounds. 2-Methyl-1-butanol, 3-methyl-1-butanol, pyridine, and phenylethyl alcohol showed strong antifungal effects against C. fimbriata. These findings suggest that VOCs from S. lavendulae SPS-33 have the potential for pathogen C. fimbriata control in sweet potato postharvest storage by fumigant action.

Fosmidomycin biosynthesis diverges from related phosphonate natural products.

Fosmidomycin and related molecules comprise a family of phosphonate natural products with potent antibacterial, antimalarial and herbicidal activities. To understand the biosynthesis of these compounds, we characterized the fosmidomycin producer, Streptomyces lavendulae, using biochemical and genetic approaches. Surprisingly, we were unable to elicit production of fosmidomycin, instead observing the unsaturated derivative dehydrofosmidomycin, which we showed potently inhibits 1-deoxy-D-xylulose 5-phosphate reductoisomerase and has bioactivity against a number of bacteria. The genes required for dehydrofosmidomycin biosynthesis were established by heterologous expression experiments. Bioinformatics analyses, characterization of intermediates, and in vitro biochemistry show that the biosynthetic pathway involves conversion of a two-carbon phosphonate precursor into the unsaturated three-carbon product via a highly unusual rearrangement reaction, catalyzed by the 2-oxoglutarate dependent dioxygenase DfmD. The required genes and biosynthetic pathway for dehydrofosmidomycin differ substantially from that of the related natural product FR-900098, suggesting that the ability to produce these bioactive molecules arose via convergent evolution.

Isolation of depsipeptides and optimization for enhanced production of valinomycin from the North-Western Himalayan cold desert strain Streptomyces lavendulae

Exploration of microbial dynamics of Streptomyces lavendulae ACR-DA1, a psychrotrophic isolate from the North-Western Himalayan cold desert, was carried out using matrix-assisted laser desorbtion ionisation-time of flight mass spectrometer. Valinomycin was found as a major produce and cyclic depsipeptide montanastatin as a minor produce. The yield of the valinomycin was found to be 0.3 mg l-1 in submerged growth condition at the batch scale. Miniaturization of optimization experiments was adept to maximize the production using the expeditious and efficient technique of intact cell mass spectrometry. The present study showed that using optimized conditions and growing the culture in synthetic mineral base starch medium at 10 °C enhanced the production to 19.4 mg l-1. Our results demonstrated 64-fold increase in yield from the wild-type S. lavendulae ACR-DA1 strain using a simple and economical downstream process.

Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae

BackgroundBeyond pathway engineering, the metabolic state of the production host is critical in maintaining the efficiency of cellular production. The biotechnologically important yeast Saccharomyces cerevisiae adjusts its energy metabolism based on the availability of oxygen and carbon sources. This transition between respiratory and non-respiratory metabolic state is accompanied by substantial modifications of central carbon metabolism, which impact the efficiency of metabolic pathways and the corresponding final product titers. Non-ribosomal peptide synthetases (NRPS) are an important class of biocatalysts that provide access to a wide array of secondary metabolites. Indigoidine, a blue pigment, is a representative NRP that is valuable by itself as a renewably produced pigment.ResultsSaccharomyces cerevisiae was engineered to express a bacterial NRPS that converts glutamine to indigoidine. We characterize carbon source use and production dynamics, and demonstrate that indigoidine is solely produced during respiratory cell growth. Production of indigoidine is abolished during non-respiratory growth even under aerobic conditions. By promoting respiratory conditions via controlled feeding, we scaled the production to a 2 L bioreactor scale, reaching a maximum titer of 980 mg/L.ConclusionsThis study represents the first use of the Streptomyces lavendulae NRPS (BpsA) in a fungal host and its scale-up. The final product indigoidine is linked to the activity of the TCA cycle and serves as a reporter for the respiratory state of S. cerevisiae. Our approach can be broadly applied to investigate diversion of flux from central carbon metabolism for NRPS and other heterologous pathway engineering, or to follow a population switch between respiratory and non-respiratory modes.

Insecticidal Activities of Some Actinomycete Strains Isolated from the Egyptian Sinai Soils

Seventy three pure actinomycete colonies were isolated from 48rhizospheric soil samples revealing different locations in Sinai. These isolates were subjected for measurement of their insecticidal activities against the greater wax moth Galleria mellonella L. of them seven isolates (S6, S13, S16, S23, S27, S35 and S36) were found as the most potent and were chosen for detailed toxicological studies. Their LC50 values were 25.23, 36.80, 55.96, 52.02, 54.19, 54.52 and 32.88 mg/ml, respectively. The most potent isolate (S6) was isolated from the rhizosphere of Tamarix nilatie plants grown in a sandy soil at El-Tor area and taxonomically was identified as Streptomyces lavendulae.

Discovering potential Streptomyces hormone producers by using disruptants of essential biosynthetic genes as indicator strains.

Autoregulators are low-molecular-weight signaling compounds that control the production of many secondary metabolites in actinomycetes and have been referred to as 'Streptomyces hormones'. Here, potential producers of Streptomyces hormones were investigated in 40 Streptomyces and 11 endophytic actinomycetes. Production of γ-butyrolactone-type (IM-2, VB) and butenolide-type (avenolide) Streptomyces hormones was screened using Streptomyces lavendulae FRI-5 (ΔfarX), Streptomyces virginiae (ΔbarX) and Streptomyces avermitilis (Δaco), respectively. In these strains, essential biosynthetic genes for Streptomyces hormones were disrupted, enabling them to respond solely to the externally added hormones. The results showed that 20% of each of the investigated strains produced IM-2 and VB, confirming that γ-butyrolactone-type Streptomyces hormones are the most common in actinomycetes. Unlike the γ-butyrolactone type, butenolide-type Streptomyces hormones have been discovered in recent years, but their distribution has been unclear. Our finding that 24% of actinomycetes (12 of 51 strains) showed avenolide activity revealed for the first time that the butenolide-type Streptomyces hormone is also common in actinomycetes.

Identification and characterization of a new agar-degrading strain with the novel properties of saccharides inhibition and nitrogen fixation.

In this study, a new agar-degrading strain was isolated from soil with agar as a sole carbon source and energy. Based on its morphological, physiological, biochemical characterization and 16S rDNA sequence, the strain was identified as Streptomyces lavendulae UN-8. The extracellular agarase activity reached 0.03 U/ml after fermentation in shake flask (250 ml), which was close to other reported non-marine microorganisms. Furthermore, it is interesting that the growth of UN-8 would be inhibited by glucose (40 g/L) and maltose (40 g/L) with the inhibitory rate of 100% and 70%, respectively. Besides, UN-8 could be grown on the solid medium without any nitrogen sources, then the possible nitrogen fixation gene nifU was cloned from its genomic DNA. The deduced amino acid sequence of nifU has high similarity (98%) with nitrogen fixation protein NifU from Streptomyces sp. NRRL S-104 (KJY22454.1) and Streptomyces sp. NRRL F-4428 (KJK52526.1) based on NCBI blast. It is suggested that the nifU gene of UN-8 also encoded nitrogen fixation protein NifU. These results provided some new information for the further understanding of agar-degrading strain.

Study on liquid paraffin decomposer microorganisms isolated from soils of Julfa-Iran

Paraffin compounds are derived from petroleum. Natural environment pollution with petroleum or petroleum products is still an environmental problem. The objective of the present study was to isolate the environmental liquid paraffin decomposer strains isolated from soil in Julfa, Iran. Initially, we collected soil samples from different areas and by preparation of serial dilution for 10-1 to 10-6 of them and culture in the Starch Casein Agar medium, 10 pure different colonies were isolated, from that, six strains in duplicate experiment showed paraffin decomposition ability about more than 70%. In the strains, resulted product was analyzed by gas chromatography (GC) mass test, as approval method of the paraffin decomposition. Then, for molecular recognition of isolates by specific primers of 16S rRNA, PCR were done and the resulted product of PCR was sequenced and considered strains were isolated. In this study, by sequencing of PCR products, the isolated strains were identified as Streptomyces lavendulae, Bacillus sp.and Streptomyces paracox. Key words: Liquid paraffin, degradation, microorganism, soil, petroleum, chromatography, Iran

Development of fed-batch strategies for the production of streptavidin by Streptomyces avidinii based on power input and oxygen supply studies

Streptavidin is a tetrameric protein with an extremely high affinity to biotin and different biotin-like peptide-tags. This characteristic causes its widespread use in biotechnology. Streptavidin is produced by the fermentation of wild type Streptomyces avidinii or by recombinant Streptomyces lavendulae, Escherichia coli, and Bacillus subtilis strains. However, little is known about the influence of power input and oxygen supply as well as feeding strategies on the production of streptavidin by S. avidinii. This paper provides a systematic analysis of the effect of rotary frequency of the stirrer, leading to a plateau-like streptavidin formation behaviour between 400 and 700min−1. This plateau was characterized by specific power inputs between 79 and 107WL−1 and corresponding maximal product concentrations of 6.90μM in 6 days. Lower as well as higher rotary frequencies were not beneficial. Subsequently, a linear fed-batch procedure could be established reproducibly yielding 39.20μM streptavidin in 14 days, characterized by a constant productivity of 114nMh−1. Fed-batch procedures based on dissolved oxygen were less efficient. The linear feeding strategy presented in this paper led to the highest streptavidin concentration ever reported and exceeded the maximal product level given in the literature drastically by a factor of 8.5.

A novel reporter system for bacterial and mammalian cells based on the non-ribosomal peptide indigoidine

The biosynthesis of non-ribosomal peptides, many of which have pharmaceutical activities, is an evolutionary privilege of microorganisms. Capitalizing on the universal set of the Streptomyces lavendulae non-ribosomal peptide synthase BpsA and the Streptomyces verticillus 4′-phosphopantetheinyl transferase Svp, we have engineered Escherichia coli as well as mammalian cells, including human stem cells, to produce the blue 3,3’-bipyridyl pigment keto-indigoidine and the reduced colorless but fluorescent leuco-isoform. Detailed characterization of a tailored substrate-free chromogenic assay and FACS analysis showed that indigoidine’s blue color and fluorescence could be reliably profiled in bacteria and mammalian cells using standard multiwell-compatible detection equipment. Besides serving as an inexpensive, reliable, versatile and easy-to-assay cross-kingdom reporter system, the potential of having mammalian cells produce non-ribosomal peptides, preferably ones with biopharmaceutical activities, may provide novel treatment opportunities in future gene- and cell-based therapies.

Hydroxyquinone O-Methylation in Mitomycin Biosynthesis

Mitomycins are bioreductively activated DNA-alkylating agents. One member of this family, mitomycin C, is in clinical use as part of combination therapy for certain solid tumors. The cytotoxicity displayed by mitomycins is dependent on their electrochemical potential which, in turn, is governed in part by the substituents of the quinone moiety. In this paper we describe studies on the biogenesis of the quinone methoxy group present in mitomycins A and B. An engineered Streptomyces lavendulae strain in which the mmcR methyltransferase gene had been deleted failed to produce the three mitomycins (A, B, and C) that are typically isolated from the wild type organism. Analysis of the culture extracts from the mmcR-deletion mutant strain revealed that two new metabolites, 7-demethylmitomycin A and 7-demethylmitomycin B, had accumulated instead. Production of mitomycins A and C or mitomycin B was selectively restored upon supplementing the culture medium of a S. lavendulae strain unable to produce the key precursor 3-amino-5-hydroxybenzoate with either 7-demethylmitomycin A or 7-demethylmitomycin B, respectively. MmcR methyltransferase obtained by cloning and overexpression of the corresponding mmcR gene was shown to catalyze the 7-O-methylation of both C9beta- and C9alpha-configured 7-hydroxymitomycins in vitro. This study provides direct evidence for the catalytic role of MmcR in formation of the 7-OMe group that is characteristic of mitomycins A and B and demonstrates the prerequisite of 7-O-methylation for the production of the clinical agent mitomycin C.

Novel family of cholesterol esterases produced by actinomycetes bacteria

Although cholesterol esterase (CHE; EC 3.1.1.13) is widespread in nature, CHEs from Streptomyces lavendulae and Streptomyces sp. X9 are the only known CHEs produced by actinomycetes. We purified CHEs from S. avermitilis JCM5070, and S. griseus IFO13350 and identified four new CHEs from actinomycetes. The enzymic properties of the CHEs from Streptomyces sp. X9, S. avermitilis, and S. griseus including substrate specificity, sensitivity to inhibitors and optimal conditions for catalysis were similar. We identified genes for the CHEs from Streptomyces sp. X9 and S. avermitilis and the encoded predicted sequences comprised 217 and 214 amino acid residues, respectively, with 64% similarity. The CHEs from Streptomyces sp. X9 and S. avermitilis were also 54 and 57% similar, respectively, to S. lavendulae CHE, indicating that these CHEs are orthologs. Phylogenetic analysis showed that they are distantly related to the conventional lipase/esterase type CHEs from mammals, yeasts and other bacteria. The actinomycetes CHEs did not have the Gly–Xaa–Ser–Xaa–Gly sequence that is conserved in the lipase/esterase family. A database search showed that orthologs of this type of CHE were restricted to actinomycetes. These findings imply that the actinomycetes CHEs constitute a novel family of cholesterol esterases.

Fluostatins C∼E, Novel Members of the Fluostatin Family Produced by Streptomyces Strain Acta 1383

Three new members of the fluostatin family, fluostatins C-E, were discovered in a culture filtrate extract of strain Acta 1383 during an HPLC screening program. The producing strain belongs to the genus Streptomyces and is closely related to type strains classified in the Streptomyces lavendulae 16S rRNA subclade. Fluostatins are named by their characteristic fluorenone chromophore. Fluostatin C shows moderate activity against selected human tumor cell lines.

Structural Evidence That Alanine Racemase from a d-Cycloserine-producing Microorganism Exhibits Resistance to Its Own Product

Alanine racemase (ALR), an enzyme that catalyzes the interconversion of Ala enantiomers, is essential for the synthesis of the bacterial cell wall. We have shown that it is harder to inhibit the catalytic activity of ALR from D-cycloserine (DCS)-producing Streptomyces lavendulae than that from Escherichia coli by DCS. To obtain structural evidence for the fact that Streptomyces ALR displays resistance to DCS, we determined the precise nature of the x-ray crystal structures of the cycloserine-free and cycloserine enantiomer-bound forms of Streptomyces ALR at high resolutions. Streptomyces ALR takes a dimer structure, which is formed by interactions between the N-terminal domain of one monomer with the C-terminal domain of its partner. Each of the two active sites of ALR, which is generated as a result of the formation of the dimer structure, is composed of pyridoxal 5'-phosphate (PLP), the PLP-binding residue Lys(38), and the amino acids in the immediate environment of the pyridoxal cofactor. The current model suggests that each active site of Streptomyces ALR maintains a larger space and takes a more rigid conformation than that of Bacillus stearothermophilus ALR determined previously. Furthermore, we show that Streptomyces ALR results in a slow conversion to a final form of a pyridoxal derivative arising from either isomer of cycloserine, which inhibits the catalytic activity noncompetitively. In fact, the slow conversion is confirmed by the fact that each enzyme bound cycloserine derivative, which is bound to PLP, takes an asymmetric structure.

Molecular Basis of Mitomycin C Resistance in Streptomyces: Structure and Function of the MRD Protein

Mitomycin C (MC) is a potent anticancer agent. Streptomyces lavendulae, which produces MC, protects itself from the lethal effects of the drug by expressing several resistance proteins. One of them (MRD) binds MC and functions as a drug exporter. We report the crystal structure of MRD and its complex with an MC metabolite, 1,2- cis-1-hydroxy-2,7-diaminomitosene, at 1.5 Å resolution. The drug is sandwiched by π-stacking interactions of His-38 and Trp-108. MRD is a dimer. The βαβββ fold of the MRD molecule is reminiscent of methylmalonyl-CoA epimerase, bleomycin resistance proteins, glyoxalase I, and extradiol dioxygenases. The location of the binding site is identical to the ones in evolutionarily related enzymes, suggesting that the protein may have been recruited from a different metabolic pathway.

Characterization of a quinone reductase activity for the mitomycin C binding protein (MRD): Functional switching from a drug-activating enzyme to a drug-binding protein

Self-protection in the mitomycin C (MC)-producing microorganism Streptomyces lavendulae includes MRD, a protein that binds MC in the presence of NADH and functions as a component of a unique drug binding-export system. Characterization of MRD revealed that it reductively transforms MC into 1,2-cis-1-hydroxy-2,7-diaminomitosene, a compound that is produced in the reductive MC activation cascade. However, the reductive reaction catalyzed by native MRD is slow, and both MC and the reduced product are bound to MRD for a relatively prolonged period. Gene shuffling experiments generated a mutant protein (MRD(E55G)) that conferred a 2-fold increase in MC resistance when expressed in Escherichia coli. Purified MRD(E55G) reduces MC twice as fast as native MRD, generating three compounds that are identical to those produced in the reductive activation of MC. Detailed amino acid sequence analysis revealed that the region around E55 in MRD strongly resembles the second active site of prokaryotic catalase-peroxidases. However, native MRD has an aspartic acid (D52) and a glutamic acid (E55) residue at the positions corresponding to the catalytic histidine and a nearby glycine residue in the catalase-peroxidases. Mutational analysis demonstrated that MRD(D52H) and MRD(D52H/E55G) conferred only marginal resistance to MC in E. coli. These findings suggest that MRD has descended from a previously unidentified quinone reductase, and mutations at the active site of MRD have greatly attenuated its catalytic activity while preserving substrate-binding capability. This presumed evolutionary process might have switched MRD from a potential drug-activating enzyme into the drug-binding component of the MC export system.