Modular polyketide synthases(PKSs)are a multidomain megasynthase class of biosynthetic enzymes that have great promise for the development of new compounds,from new pharmaceuticals to high value commodity and specialt...Modular polyketide synthases(PKSs)are a multidomain megasynthase class of biosynthetic enzymes that have great promise for the development of new compounds,from new pharmaceuticals to high value commodity and specialty chemicals.Their colinear biosynthetic logic has been viewed as a promising platform for synthetic biology for decades.Due to this colinearity,domain swapping has long been used as a strategy to introduce molecular diversity.However,domain swapping often fails because it perturbs critical protein-protein interactions within the PKS.With our increased level of structural elucidation of PKSs,using judicious targeted mutations of individual residues is a more precise way to introduce molecular diversity with less potential for global disruption of the protein architecture.Here we review examples of targeted point mutagenesis to one or a few residues harbored within the PKS that alter domain specificity or selectivity,affect protein stability and interdomain communication,and promote more complex catalytic reactivity.展开更多
B.Subtilis expression plasmids generally require a stringent Shine Dalgarno Sequence(SDS). Site directed mutagenesis was explored to change the Shine Dalgarno Sequence from AAAAATGGGG (mutant type) to AAAAAGGGGG (...B.Subtilis expression plasmids generally require a stringent Shine Dalgarno Sequence(SDS). Site directed mutagenesis was explored to change the Shine Dalgarno Sequence from AAAAATGGGG (mutant type) to AAAAAGGGGG (wild type) in recombinant plasmid PSM604. The single base substitution made the plasmid with wild SDS unstable in structure and segregation. The interaction of SDS with subtilisin leader sequence of PSM604 might be responsible for the instability of plasmid.展开更多
Homology comparison of human, murine and E coli adenosine deaminases revealed a single conserved cysteine residue. On the basis of many previous reports indicating that there is an essential Cys residue involved in ...Homology comparison of human, murine and E coli adenosine deaminases revealed a single conserved cysteine residue. On the basis of many previous reports indicating that there is an essential Cys residue involved in enzymatic activity, this conserved Cys was proposed to be the essential one involved in the catalytic activity of the enzyme. Using site directed mutagenesis and steady state kinetics, the role of the single conserved Cys residue was tested. However, results presented here indicate that this conserved Cys is not required for enzyme activity, strongly suggesting that cysteine should not be required for the enzymatic activity of adenosine deaminase. The preponderance of previously reported data indicating the “essentiality” of the Cys residue for enzymatic activity are discussed .展开更多
基金This work was supported by funding provided by the University of Tennessee,Knoxville.
文摘Modular polyketide synthases(PKSs)are a multidomain megasynthase class of biosynthetic enzymes that have great promise for the development of new compounds,from new pharmaceuticals to high value commodity and specialty chemicals.Their colinear biosynthetic logic has been viewed as a promising platform for synthetic biology for decades.Due to this colinearity,domain swapping has long been used as a strategy to introduce molecular diversity.However,domain swapping often fails because it perturbs critical protein-protein interactions within the PKS.With our increased level of structural elucidation of PKSs,using judicious targeted mutations of individual residues is a more precise way to introduce molecular diversity with less potential for global disruption of the protein architecture.Here we review examples of targeted point mutagenesis to one or a few residues harbored within the PKS that alter domain specificity or selectivity,affect protein stability and interdomain communication,and promote more complex catalytic reactivity.
文摘B.Subtilis expression plasmids generally require a stringent Shine Dalgarno Sequence(SDS). Site directed mutagenesis was explored to change the Shine Dalgarno Sequence from AAAAATGGGG (mutant type) to AAAAAGGGGG (wild type) in recombinant plasmid PSM604. The single base substitution made the plasmid with wild SDS unstable in structure and segregation. The interaction of SDS with subtilisin leader sequence of PSM604 might be responsible for the instability of plasmid.
文摘Homology comparison of human, murine and E coli adenosine deaminases revealed a single conserved cysteine residue. On the basis of many previous reports indicating that there is an essential Cys residue involved in enzymatic activity, this conserved Cys was proposed to be the essential one involved in the catalytic activity of the enzyme. Using site directed mutagenesis and steady state kinetics, the role of the single conserved Cys residue was tested. However, results presented here indicate that this conserved Cys is not required for enzyme activity, strongly suggesting that cysteine should not be required for the enzymatic activity of adenosine deaminase. The preponderance of previously reported data indicating the “essentiality” of the Cys residue for enzymatic activity are discussed .
