Table 1.
Alanine mutations of selected amino acids of Agl3: used primers, primer sequence, target mutation and overall mutant enzyme activity
| Primer name | Primer sequence | Target mutation | Mutant enzyme activitya |
|---|---|---|---|
| Sqsyn1-Forward-H95A | 3′-GCCATAGTGGCTTTCGCTGAG-5′ | Histidine 95 | |
| Sqsyn2-Reverse-H95A | 3′-CTCAGCGAAAGCCACTATGGC-5′ | Histidine 95 | − |
| Sqsyn3-Forward-R101A | 3′-GCTGAGCAGGCTTCTGCTCCG-5′ | Arginine 101 | |
| Sqsyn4-Reverse-R101A | 3′-CGGAGCAGAAGCCTGCTCAGC-5′ | Arginine 101 | − |
| Sqsyn5-Forward-T144A | 3′-AAGATGGGTGCTATGGGTGAG-5′ | Threonine 144 | |
| Sqsyn6-Reverse-T144A | 3′-CTCACCCATAGCACCCATCTT-5′ | Threonine 144 | ++ |
| Sqsyn7-Forward-M145A | 3′-ATGGGTACCGCTGGTGAGTAT-5′ | Methionine 145 | |
| Sqsyn8-Reverse-M145A | 3′-ATACTCACCAGCGGTACCCAT-5′ | Methionine 145 | − |
| Sqsyn19-Forward-E147A | 3′-ACCATGGGTGCCTATGGAACACCT-5′ | Glutamic acid 147 | |
| Sqsyn20-Reverse-E147A | 3′-AGGTGTTCCATAGGCACCCATGGT-5′ | Glutamic acid 147 | (±) |
| Sqsyn21-Forward-Y148A | 3′-CATGGGTGAGGCTGGAACACCT-5′ | Tyrosine 148 | |
| Sqsyn22-Reverse-Y148A | 3′-AGGTGTTCCAGCCTCACCCATG-5′ | Tyrosine 148 | (±) |
| Sqsyn9-Forward-S180A | 3′-GCGGGTGCTGTTTATCAC-5′ | Serine 180 | |
| Sqsyn10-Reverse-S180A | 3′-GTGATAAACAGCACCCGC-5′ | Serine 180 | (+) |
| Sqsyn11-Forward-Y182A | 3′-GGTTCTGTTGCTCACTGGACT-5′ | Tyrosine 182 | |
| Sqsyn12-Reverse-Y182A | 3′-AGTCCAGTGAGCAACAGAACC-5′ | Tyrosine 182 | − |
| Sqsyn15-Forward-T185A | 3′-TATCACTGGGCTAAGGTTCAT-5′ | Threonine 185 | |
| Sqsyn16-Reverse-T185A | 3′-ATGAACCTTAGCCCAGTGATA-5′ | Threonine 185 | (+) |
| Sqsyn17-Forward-K186A | 3′-CACTGGACTGCTGTTCATGAT-5′ | Lysine 186 | |
| Sqsyn18-Reverse-K186A | 3′-ATCATGAACAGCAGTCCAGTG-5′ | Lysine 186 | – |
aQualitative determination: ++ strongly active, + active, (+) lesser active, (±) less active, – inactive protein