TABLE 2

Primers used in pva site-directed mutagenesis studies and effect of mutations on rSlPVA catalytic activity

Rhodococcus sp. strainMutationSynthetic oligonucleotide and sequenceaRemaining activityb (%)
Control pNPAATG100
pNPAATGS1CSβ1CPVAS1C1; 5′GCAGAACGCCGACATGGGGCTGCAACGCGGTCGCCTTCCGGGGG3′0
PVAS1C2; 5′CCCCCGGAAGGCGACCGCGTTGCAGCCCCATGTCGGCGTTCTGC3′
pNPAATGS1ASβ1APVAS1A1; 5′GCAGAACGCCGACATGGGGCGCCAACGCGGTCGCCTTCCGGGGG3′0
PVAS1C2; 5′CCCCCGGAAGGCGACCGCGTTGGCGCCCCATGTCGGCGTTCTGC3′
pNPAATGS1DSβ1DPVAS1D1; 5′GCAGAACGCCGACATGGGGCGATAACGCGGTCGCCTTCCGGGGG3′0
PVAS1D2; 5′CCCCCGGAAGGCGACCGCGTTATCGCCCCATGTCGGCGTTCTGC3′
pNPAATGS1KSβ1KPVAS1K-1; 5′GCAGAACGCCGACATGGGGCAAGAACGCGGTCGCCTTCCGGGGG3′0
PVAS1K-2; 5′CCCCCGGAAGGCGACCGCGTTCTTGCCCCATGTCGGCGTTCTGC3′
pNPAATGS1HSβ1HPVAS1H-1; 5′GCAGAACGCCGACATGGGGCCACAACGCGGTCGCCTTCCGGGGG3′0
PVAS1H-2; 5′CCCCCGGAAGGCGACCGCGTTGTGGCCCCATGTCGGCGTTCTGC3′
pNPAATGHQHβ23QPVAH23Q-1; 5′GGGCTGCTCCTCGGCAACCCGCAGTATCCGTGGGAGGGCGGCCGC3′0
PVAH23Q-2; 5′GCGGCCGCCGTCCCACGGATACTGCGGGTTGCCGAGGAGCAGCCC3′
pNPAATGHDHβ23DPVAH23D-1; 5′GGGCTGCTCCTCGGCAACCCGGACTATCCGTGGGAGGGCGGCCGC3′0
PVAH23D-2; 5′GCGGCCGCCGTCCCACGGATAGTCCGGGTTGCCGAGGAGCAGCCC3′
pNPAATGVAVβ70APVAV70A-1; 5′GTGGCCTGGAGCCACACGGCCGCGACCGGCGTCACGCTG3′0.25
PVAV70A-2; 5′CAGCGTGACGCCGGTCGCGGCCGTGTGGCTCCAGGCCAC3′
pNPAATGVDVβ70DPVAV70D-1; 5′CGTGGCCTGGAGCCACACGGACGCGACCGGCGTCACGCTG3′0.15
PVAV70D-2; 5′CAGCGTGACGCCGGTCGCGTCCGTGTGGCTCCAGGCCACG3′
pNPAATGNQNβ272QPVAN272Q-1; 5′CGCCCCGTACGTCGAGAACTCCCAGGACAGCGCCTGGCTGACC3′0.35
PVAN272Q-2; 5′GGTCAGCCAGGCGCTGTCCTGGGAGTTCTCGACGTACGGGGCG3′
pNPAATGNDNβ272DPVAN272D-1; 5′CGCCCCGTACGTCGAGAACTCCGACGACAGCGCCTGGCTGACC3′0.33
PVAN272D-2; 5′GGTCAGCCAGGCGCTGTCGTCGGAGTTCTCGACGTACGGGGCG3′
pNPAATGNVNβ272VPVAN272V-1; 5′CGCCCCGTACGTCGAGAACTCCGTCGACAGCGCCTGGCTGACC3′0
PVAN272V-2; 5′GGTCAGCCAGGCGCTGTCGACGGAGTTCTCGACGTACGGGGCG3′
  • a Mutated codons are underlined.

  • b Activity was determined under standard conditions using penicillin V as the substrate.