2.3.1.28: chloramphenicol O-acetyltransferase

mutant enzyme is less stable than wild-type enzyme

A203I

mutant enzyme is more thermostable than wild-type

I191V

mutant enzyme is less stable than wild-type enzyme

Y33F/A203V

mutant enzyme is more thermostable than wild-type

C214A

95% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 31% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

C214D

50% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 85% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

C214E

75% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 84% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

C214F/G219S

95% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 81% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

C214G

80% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 44% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

C214L

100% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 33% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

C214P

95% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 88% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

C214Q

95% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 73% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

C214R

55% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 84% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

C214S

95% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 32% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

C214T

90% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 59% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

C214V

95% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 45% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

C214W

50% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 70% of activity after 30 min at 65 C compared to 15% for the wild-type enzyme

C214Y

90% of the in vivo produced mutant polypeptide is soluble compared to 90% for the wild-type enzyme. Mutant enzyme loses 81% of activity after 30 min at 65°C compared to 15% for the wild-type enzyme

CATIII (F24A/Y25F/L29A)

Km-value for acetyl-CoA is 0.095 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.023 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 30% of the wild-type enzyme CAT III

CATIII(K14E/H195A/K217A)

no activity

CATIII(Q92C/N146F/Y169F/I172V)

Km-value for acetyl-CoA is 0.165 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.02 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 60% of the wild-type enzyme CAT III

K14/K217E

Km-value for acetyl-CoA is 0.166 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.017 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 87% of the wild-type enzyme CAT III

L145F

folding of chloramphenicol acetyltransferase is hampered by deletion of the carboxy-terminal tail including the last residue of the carboxy-terminal alpha-helix. Such truncated CAT polypeptides quantitatively aggregate into cytoplasmic inclusion bodies, which results in absence of chloramphenicol-resistant phenotype for the producing host. Introduction of Phe at amino acid position 145 improves the ability of the protein to fold into a soluble, enzymatically active conformation

486730

L158I

fluorinated mutant expressed in trifluoroleucine shows enhanced thermostability compared to CAT T (CAT expressed in trifluoroleucine), suggesting that trifluoroleucine at position 158 contributes to a portion of the observed loss in thermostability upon global fluorination. Relative activity: 89% (non-fluorinated mutant), 51.7% (fluorinated mutant)

685590

L208I

fluorinated mutant expressed in trifluoroleucine shows loss in thermostability

685590

L821I

fluorinated mutant expressed in trifluoroleucine shows loss in thermostability

685590

[CATI (H195A)]2[CATIII(K14E/K217E)]

hybrid trimer, Km-value for acetyl-CoA is 0.072 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.018 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 14% of the wild-type enzyme CAT III

[CATIII]2[CATIII(K14E/H195A/K217A)]

Km-value for acetyl-CoA is 0.143 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.016 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 80% of the wild-type enzyme CAT III

[CATIII][CATIII(K14E/H195A/K217A)]2

Km-value for acetyl-CoA is 0.198 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.02 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 82% of the wild-type enzyme CAT III

[CATI][CATIII(K14E/H195A/K217E)]2

hybrid trimer, Km-value for acetyl-CoA is 0.107 mM compared to 0.093 mM for wild-type CATIII, Km-value for chloramphenicol is 0.02 mM compared to 0.012 mM for the wild-type CATIII, turnover number is 50% of the wild-type enzyme CAT III