Table 1 Summary of features of the Thermus sp.enzyme family
REase | TspDTI | Tth111II | TthHB27I | TsoI | TspGWI | TaqII |
|---|---|---|---|---|---|---|
Bacterial host | Thermus sp. DT | Thermus thermophilus 111 | Thermus thermophilus HB27 | Thermus scotoductus | Thermus sp. GW | Thermus aquaticus YTI |
Recognition and cleavage site | ATGAA(11/9) | CAARCA(11/9) | CAARCA(11/9) | TARCCA(11/9) | ACGGA(11/9) | GACCGA(11/9) |
Recognition site length (bp) | 5 | 6, (degenerated) | 6 (degenerated) | 6 (degenerated) | 5 | 6 |
ORF GC% | 57.99 | 61.67 | 61.59 | 56.54 | 69.19 | 66.27 |
Polypeptide length (aa) | 1112 | 1106 | 1121 | 1116 | 1097 | 1106 |
Molecular weight (kDa) | 126.885 | 125.956 | 127.672 | 126.474 | 120.202 | 125.675 |
Native molecular size (kDa)/protein organization | 110-130 monomer | 110-130 monomer | ND | ND | 110-130 monomer | 110-130 monomer |
Optimum reaction temperature | 65°C | 65-70°C | ND | 55°C | 65°C | 65°C |
Single site cleavage | Yes | ND | ND | ND | Two sites preferred | Yes |
Isoelectric point (calculated) | 6.68 | 6.60 | 6.80 | 8.11 | 6.58 | 5.40 |
Specific DNA + methylation | Â | ND | ND | + | + | + |
Cleavage stimulation with SAM | Yes | ND | ND | Yes | Slight inhibition | Yes |
Cleavage stimulation with SIN (SAM analogue) | Yes | ND | ND | ND | Yes, specificity change | Yes, specificity change |
Methylation stimulation with Ca2+ | No | ND | ND | ND | Yes | Yes |
Cleavage stimulation with divalent cations | Ca2+ | ND | ND | ND | Mn2+, Fe2+, Co2+ not stimulated by Ca2+ | ND |
Domain organization* | R-Mh-Mc-S | R-Mh-Mc-S | R-Mh-Mc-S | R-Mh-Mc-S | R-Mh-Mc-S | R-Mh-Mc-S |
Cleavage catalytic motif (B/E)** | atypical D-EXE (B) | atypical D-EXE (B) | atypical D-EXE (B) | atypical D-EXE (B) | PDX13EX1K (B + E) | PDX13EX1K (B + E) |
SAM binding motif: (B/E)** | DPACGSG (B) | DPACGSG (B) | DPACGSG (B) | PPACGSG (B) | DPAVGTG (B + E) | DPAMGTG (B + E) |
Methylation catalytic motif (B/E)** | NPPW (B) | NPPW (B) | NPPW (B) | NPPW (B) | NPPY (B + E) | NPPY (B + E) |
Subfamily homology* | Subfamily TspDTI | Subfamily TspDTI | Subfamily TspDTI | Subfamily TspDTI | Subfamily TspGWI | Subfamily TspGWI |
Reference | [20] | GenBank: AE017221.1 | This work, manuscript in preparation | GenBank: AY057443, 21 |