Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
A novel serralysin metalloprotease from Deinococcus radiodurans
Introduction
Metalloendopeptidases are ubiquitously distributed across all kingdoms of living organisms. They can extensively degrade proteins or selectively hydrolyze specific peptide bonds and are involved in metabolic regulation [1]. A majority of these enzymes are zinc-dependent, named zincins and possess a short consensus sequence, HEXXH, with the two histidines acting as ligands of the catalytic zinc and the glutamate as the general base. However, the pentapeptide sequence HEXXH also occurs in many other proteins that are not peptidases. A longer and more reliable consensus sequence, HEXXHXXGXXH/D, contains an additional strictly conserved glycine and a third zinc-binding histidine or aspartate [2], [3]. The zincins containing a conserved methionine which forms a “Met-turn” are named metzincins [4], [5], [6].
The seralysin family is a subfamily of metzincins that shares common features like a glycine rich C-terminal Ca+2 binding motif which overlaps with glycine rich secretion signal [7], and absence of conserved cysteine residue responsible for “cysteine switch” mechanism of proenzyme activation [8]. The proteases so far assigned to the serralysin family are reported from Gram-negative bacteria such as Serratia marcescens, Pseudomonas aeruginosa, Erwinia chrysanthemi, Proteus mirabilis and Caulobacter crecentus [3], [9], [10] and are secreted using glycine rich Gram-negative type secretion signals. Serralysins are bacterial extracellular metalloendopeptidases that show complete conservation of Zn+2 binding domain, Met-turn and Ca+2 binding repeats. The protease serralysin from Serratia marcescens, discovered first, is a type example of the family. Although primary function of serralysins is nutrient uptake, they are also involved in pathogenesis [11]. So far no functional serralysins have been reported from Gram positive organisms.
Deinococcus radiodurans, a red pigmented, Gram-positive, non-pathogenic, nonsporulaing, nonmotile bacterium [12], [13] is well known for its extreme radioresistance. The genome of this organism has been completely sequenced and contains over 1000 ORFs unique to Deinococcus radiodurans [14]. Apart from its extreme radioresistance and phenomenal DNA repair capabilities, Deinococcus radiodurans presents a novel combination of genes and proteins from prokaryotes and eukaryotes including plants and humans [15]. Some of these proteins with novel domain combinations are speculated to be important for its radioresistance. The organism exhibits a proteolytic life style [16]. This paper reports the identification and characterization of a novel serralysin-like secreted metalloprotease encoded by a hypothetical ORF DR2310 in Deinococcus radiodurans.
Section snippets
Bacterial strains and growth conditions
The bacterial strains used in this study are listed in Table 1. D. radiodurans was grown aerobically in TGY (1% Bacto Tryptone, 0.1% glucose, and 0.5% yeast extract) medium at 32 °C and supplemented with 8 μg/ml kanamycin when required. Alternatively, D. radiodurans was grown aerobically in synthetic minimal medium [17] at 32°C and supplemented with either 0.5% cassamino acids or 0.1% gelatin as a sole source of amino acids. E. coli was grown in Luria–Bertani medium at 37 °C and supplemented
Bioinformatic analysis and identification of DR2310 protein
The Conserved Domain Database (CDD) program was used to screen bacterial genome databases searching for regions of sequence similarity to DR2310 protein [23]. The program detected two highly conserved sequences. (1) ZnMc: Zn+2-binding metalloprotease (HEXXH) domain spanning from 581–585 amino acid residues and (2) “Met turn” of metzincins (SVMSY, 620–625) C-terminal to the Zn+2 binding domain. These conserved sequences showed highest sequence similarities with the metalloproteases of organisms
Discussion
This study, examined a hypothetical protein DR2310 from the radioresistant microbe Deinococcus radiodurans. The in-silico detection of completely conserved Zn+2 binding domain and Met-turn suggested it to be a potential candidate of serralysin family of metalloproteases. The mutational analysis confirmed that ORF DR2310 encodes an 85 kDa constitutively expressed protease that is secreted into the culture medium, a property similar to serralysins. Biochemical data also confirmed that DR2310 is a
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2019, Biochimica et Biophysica Acta - Proteins and ProteomicsCitation Excerpt :Following irradiation, the cells were recovered in fresh TGY (OD600 = 0.5) under optimal growth conditions. The DR_0907 deletion mutant was constructed as described previously [18]. In brief, the upstream (PprM-up, 0.5 kb) and downstream (PprM-dn, 0.5 kb) sequences of the DR_0907 gene were PCR amplified using primer pairs PprM-up-F/R and PprM-dn-F/R, respectively (Table 1).
Putative DNA modification methylase DR_C0020 of Deinococcus radiodurans is an atypical SAM dependent C-5 cytosine DNA methylase
2017, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :Sequence specificity of this enzyme clearly suggests that it is an unusual bacterial cytosine methyltransferase. To assess the contribution of cytosine methylation to cellular physiology, the dcm gene was knocked out by replacing the dcm gene with a kanamycin resistance cassette [19] as shown in Fig. 4A. Complete deletion of the dcm gene in a Δdcm mutant, as compared to wild type D. radiodurans, was confirmed by PCR (Fig. 4B) and the absence of a corresponding Dcm protein in the cell extracts was ascertained by western blotting using anti-Dcm antibodies (Fig. 4C). The Δdcm mutant showed a complete loss of C-5 cytosine methyl transferase activity from the cellular extracts (Fig. 4D, Table 2) and of m5C content in the genome of the Δdcm mutant (Fig. 4E, Table 3).
DNA adenine hypomethylation leads to metabolic rewiring in Deinococcus radiodurans
2015, Journal of ProteomicsCitation Excerpt :Alternatively, the enzyme activity was estimated by a non-radioactive colorimetric methyltransferase assay (SAM510: SAM Methyltransferase Assay kit, G-Biosciences, USA), as per the manufacturer's protocol. The DR_0643 knockout mutant was generated using strategies described earlier [21]. In brief, the immediate upstream (780 bp, DR_0643-up) or downstream (700 bp, DR_0643-dn) region of the DR_0643 gene was amplified using primer pairs DR_0643-up-F/DR_0643-up-R or DR_0643-dn-F/DR_0643-dn-R (Supplementary Table 1), respectively, and cloned into the BamHI/XhoI sites of pBlueScript vector to obtain plasmid p∆dam1.