Table 3.
Functionality of MjCobY variants.
| Protein Encoded |
Functional In vivo? |
Doubling Time (min) |
% in vitro activity (relative to CobYWT) |
Monomer: Dimer Ratio |
Specific Activity (pmol/min/µg protein) |
|---|---|---|---|---|---|
| WT | Yes | 63 ± 4 | 100 | 59 : 41 | 116 ± 3 |
| G8D | No | NA | 3 | 33 : 67 | 4 ± 0 |
| R13A | Yes | 187 ± 30 | 15 | 14 : 86 | 18 ± 1 |
| E18D | Yes | 61 ± 4 | 56 | 40 : 60 | 66 ± 2 |
| K19A | Yes | 39 ± 11 | 16 | 36 : 64 | 19 ± 1 |
| P20A | Yes | 61 ± 5 | 93 | 63 : 37 | 102 ± 4 |
| S50A | Yes | 64 ± 6 | 100 | ND | 112 ± 7 |
| T53S | Yes | 58 ± 3 | 84 | 76 : 24 | 97 ± 4 |
| P54A | Yes | 58 ± 4 | 62 | 50 : 50 | 73 ± 3 |
| T56A | Yes | 119 ± 12 | 102 | ND | 107 ± 3 |
| Y80F | Yes | 60 ± 4 | 65 | 74 : 26 | 69 ± 4 |
| D83G | Yes | 60 ± 5 | 100 | 82 : 18 | 110 ± 5 |
| P94A | Yes | 68 ± 5 | 43 | 52 : 48 | 71 ± 3 |
| S100A | Yes | 117 ± 7 | 100 | ND | 101 ± 3 |
| D101G | Yes | 64 ± 5 | 71 | 34 : 66 | 79 ± 5 |
| G153D | No | NA | 2 | 54 : 46 | 3 ± 0 |
| N177R | No | NA | 5 | ND | 6 ± 0 |
| N179D | Yes | 67 ± 11 | 95 | ND | 93 ± 2 |
| T180A | Yes | 98 ± 6 | 73 | ND | 106 ± 5 |
The ability of the above-mentioned variants to restore AdoCbl synthesis from CNCby in the S. enterica ΔcobU ΔycfN strain was determined as described under Experimental Procedures. Doubling times of cultures grown in NCE glycerol medium supplemented with CNCby (15 nM) were calculated graphically from semi-log plots of OD595 as a function of time and are shown as the arithmetic mean ± standard error of the mean (n=3), using the Prism® v4 program (GraphPad Software). Monomer:dimer ratios and specific activities were calculated as described elsewhere (23); NA, not applicable; ND, not determined.