Disruption of Macrodomain Protein SCO6735 Increases Antibiotic Production in Streptomyces coelicolor

Abstract

ADP-ribosylation is a post-translational modification that can alter the physical and chemical properties of target proteins and that controls many important cellular processes. Macrodomains are evolutionarily conserved structural domains that bind ADP-ribose derivatives and are found in proteins with diverse cellular functions. Some proteins from the macrodomain family can hydrolyze ADP-ribosylated substrates and therefore reverse this post-translational modification. Bacteria and Streptomyces, in particular, are known to utilize protein ADP-ribosylation, yet very little is known about their enzymes that synthesize and remove this modification. We have determined the crystal structure and characterized, both biochemically and functionally, the macrodomain protein SCO6735 from Streptomyces coelicolor This protein is a member of an uncharacterized subfamily of macrodomain proteins. Its crystal structure revealed a highly conserved macrodomain fold. We showed that SCO6735 possesses the ability to hydrolyze PARP-dependent protein ADP-ribosylation. Furthermore, we showed that expression of this protein is induced upon DNA damage and that deletion of this protein in S. coelicolor increases antibiotic production. Our results provide the first insights into the molecular basis of its action and impact on Streptomyces metabolism.

Keywords: ADP-ribose; ADP-ribosylation; DNA damage; PARP; Streptomyces; antibiotics; enzyme mechanism; microbiology; nicotinamide adenine dinucleotide (NAD); post-translational modification (PTM).

FIGURE 1.

Maximum likelihood phylogenetic tree illustrating the relationship between selected representative macrodomains. Human proteins (Hsap) and bacterial PARG from Thermomonospora curvata (Tcur) are shown in bold type. S. coelicolor proteins are circled. Remaining macrodomain proteins were selected from the following bacterial species: B. thetaiotaomicron (Bthe), Deinococcus frigens (Dfri), Deinococcus pimensis (Dpim), D. radiodurans (Drad), E. coli (Ecol), Herpetosiphon aurantiacus (Haur), Listeria monocytogenes (Lmon), Myxococcus fulvus (Mful), and M. xanthus (Mxan). Sequence accession numbers are given in parentheses. Bootstrap values ML (>60%) are given above the lines, and MCMC values are given (>0.6) below the lines. The scale bar indicates the genetic distance of the branch lengths.

FIGURE 2.

Structure-based alignment of selected macrodomain protein sequences. Designated secondary structure elements refer to SCO6735. Residues responsible for binding and hydrolysis of ADP-ribose in human TARG1 (Lys⁸⁴ and Asp¹²⁵) are marked with asterisks.

FIGURE 3.

Activity of SCO6735 protein. A, activity of SCO6735 on ³²P-automodified SCO5461ΔN34 substrate. The arrow indicates the position of SCO5461ΔN34 protein. B, activity of SCO6735 on automodified PARP1 E988Q protein. Recombinant PARP1 E988Q (0.5 μm) was automodified in presence of [³²P]NAD⁺ and treated or not with 1 μm of SCO6735 or with 1 μm of SCO6735 G128E mutant. Right panel, Coomassie staining of dried gel exposed in left panel. C, quantification of residual ³²P-labeled ADP-ribose on PARP1 E988Q after treatment with buffer, SCO6735 or SCO6735 G128E mutant. The ratio between the intensity of each autoradiography band and correspondent intensity in Coomassie staining data are the means ± S.D. of the values obtained in two independent experiments. D, TLC analyzing the by-products of incubation of GST-PARP10cd immobilized on beads with buffer or SCO6735. ADP-ribose marker was obtained treating recombinant wild type PARP1 with recombinant human PARG enzyme.

FIGURE 4.

Three-dimensional structure of SCO6735 (magenta) superimposed on a human TARG1/C6orf130 (PDB code 4J5R, green) and bacterial, B. thetaiotaomicron (PDB code 2FG1, blue) proteins. Human protein is in complex with PARG inhibitor ADP-HPD. ADP-HPD, as well as amino acids important for ligand binding (Lys⁸⁴) and catalytic activity (Asp¹²⁵) and corresponding amino acids in bacterial structures (Gln⁸⁵ and Ala¹³⁰ in SCO6735 and Gln⁸² and Ala¹²⁸ in BT1257) are shown in stick representation. Amino acids that could not be well defined in the electron density maps are omitted and shown by dashed line.

FIGURE 5.

Alignment of the RecA-NDp promoter sequences of Mycobacterium tuberculosis (Mtub) and S. coelicolor (Scoe) recA genes with SCO6735 and its orthologues in other Streptomyces species. Sliv, S. lividans; Sgri, S. griseus; Save, Streptomyces avermitilis; Sdav, Streptomyces davawensis; Sful, Streptomyces fulvissimus. Putative promoter sequences are boxed, and the (potential) transcription start sites are underlined. The numbers indicate the distance to the starts of translation with respect to the shown sequences. GenBank^TM accession numbers for the sequences are given in parentheses.

FIGURE 6.

Expression of SCO6735 gene is up-regulated upon DNA damage. cDNA from untreated (UT) and UV-irradiated (200 J m⁻²) mycelium (UV) of S. coelicolor wild type strain was used as a template for PCR (A) and qRT-PCR analyses (B). S. coelicolor genomic DNA (gDNA) was used as a control template in PCR analysis. The data represent the mean values from three independent experiments. The error bars represent standard error of the mean.

FIGURE 7.

UV and MMS sensitivity of the S. coelicolor Δ6735 mutant compared with wild type. The data represent the mean values from three independent experiments. The error bars represent standard error of the mean.

FIGURE 8.

Blue phenotype of S. coelicolor Δ6735 mutant in liquid (A) and on solid (B) minimal medium compared with wild type and complementation strain (CΔ6735) phenotypes.

FIGURE 9.

The content of intracellular and extracellular actinorhodin in S. coelicolor WT, Δ6735 mutant, and complementation strain (CΔ6735) over 5 days of growth in minimal medium. The data represent the mean values from three independent experiments. The error bars represent standard error of the mean.

FIGURE 10.

Quantitative real time PCR analysis of genes involved in the biosynthesis of actinorhodin, SCO5085, and SCO5087 in S. coelicolor Δ6735 mutant, wild type and complementation strain. Samples for RNA isolation were taken during the 5 days of growth in minimal medium. The data represent the mean values from three independent experiments. The error bars represent standard error of the mean.