An archaeal protein evolutionarily conserved in prokaryotes is a zinc-dependent metalloprotease

Abstract

A putative protease gene (tldD) was previously identified from studying tolerance of letD encoding the CcdB toxin of a toxin–antidote system of the F plasmid in Escherichia coli. While this gene is evolutionarily conserved in archaea and bacteria, the proteolytic activity of encoded proteins remained to be demonstrated experimentally. Here we studied Sso0660, an archaeal TldD homologue encoded in Sulfolobus solfataricus by overexpression of the recombinant protein and characterization of the purified enzyme. We found that the enzyme is active in degrading azocasein and FITC–BSA substrates. Protease inhibitor studies showed that EDTA and o-phenanthroline, two well-known metalloprotease inhibitors, either abolished completely or strongly inhibited the enzyme activity, and flame spectrometric analysis showed that a zinc ion is a cofactor of the protease. Furthermore, the protein forms disulfide bond via the Cys416 residue, yielding protein dimer that is the active form of the enzyme. These results establish for the first time that tidD genes encode zinc-containing proteases, classifying them as a family in the metalloprotease class.

Crystal Structure of a Putative Modulator of Gyrase (TldE) from Thermococcus kodakarensis

“…The hydrogen bond/salt bridge network contributed to the stability of all dimers. Multiple alignments based on secondary structure of eight representative sequences, TldE from T. kodakarensis (TkTldE, 6J6A), TldE and TldD from E. coli (EcTldE/TldD, 5NJ9), TldE from T. maritime (TmTIDE, 1VL4), TldE from Shigella flexneri (SsTldE, 3TV9), PmbA from Pseudomonas aeruginosa (PaPmbA, 3QTD), PmbA from Bacteroides thetaiotaomicron (BtPmbA, 1VPB), TldD from T. kodakarensis (TkTldD, tk0502), and TldD from S. solfataricu (SsTldD, SsO0660), revealed several conserved motifs including HExxxH, a metalloprotease-like motif, RMxNTxxxPG, and GxC in TldDs but TldEs [14] (Figure 3). The conserved residues in HExxxH and GxC motify coordinated a metal ion and was response for catalytically activity [14].…”

“…Multiple alignments based on secondary structure of eight representative sequences, TldE from T. kodakarensis (TkTldE, 6J6A), TldE and TldD from E. coli (EcTldE/TldD, 5NJ9), TldE from T. maritime (TmTIDE, 1VL4), TldE from Shigella flexneri (SsTldE, 3TV9), PmbA from Pseudomonas aeruginosa (PaPmbA, 3QTD), PmbA from Bacteroides thetaiotaomicron (BtPmbA, 1VPB), TldD from T. kodakarensis (TkTldD, tk0502), and TldD from S. solfataricu (SsTldD, SsO0660), revealed several conserved motifs including HExxxH, a metalloprotease-like motif, RMxNTxxxPG, and GxC in TldDs but TldEs [14] (Figure 3). The conserved residues in HExxxH and GxC motify coordinated a metal ion and was response for catalytically activity [14]. The C α root mean square deviations between TkTldE and the other structural homologues EcTldE, EcTldD, 1VL4, 3TV9, 1VPB, and 3QTD were 2.6, 3.6, 2.1, 2.6, 2.5, and 2.4 Å, respectively, as determined using the DALI server (see Supplementary Materials Table S1) [25].…”

“…The function of EcTldD/TldE as a cleaving modified MccB17 precursor in vitro was reported [14]. The structural comparison between TkTldE and EcTldE/TldD indicated that the overall structures were similar, but there were additional folds termed the "clamp" in EcTldD, which made its β-strands longer than those of EcTldE; this clamp was also missing in TkTldE and other TldEs (Figure 4) [14]. Cys454 coordinating a Zn 2+ ion with two histidine residues from HExxxH motif was located in the clamp motif of EcTldD ( Figure 4C) [15].…”

“…It has been reported that in pmbA (TldE) or its homologous TldD mutant, pro-MccB17 cannot be released from cells, and so inhibited DNA replication [1,3]. Although the TldD and TldE proteins had high sequence similarity, only TldD contained a conserved metalloprotease-like HExxxH sequence motif that might contribute to catalysis [14]. Ghilarov reported that a heterodimeric TldD/TldE complex was needed for the cleavage of the N-terminal of the modified MccB17 precursor peptide to yield the mature antibiotic [15].…”

“…The structure of a putative modulator of DNA gyrase (TldE) from Thermotoga maritima was reported, but shed little light on its functions as a modulator of DNA gyrase [18]. Sso0660 from Sulfolobus solfataricus, a TldD homologue, could function alone as a metalloprotease, as it could proteolytically degrade azocasein and FITC-BSA substrates with a zinc ion as its cofactor [14]. The function of these homologous TldD/TldE proteins in Thermococcus kodakarensis has not been reported.…”