The Prodrug Activator EtaA from Mycobacterium tuberculosis Is a Baeyer-Villiger Monooxygenase

Abstract

EtaA is a newly identified FAD-containing monooxygenase that is responsible for activation of several thioamide prodrugs in Mycobacterium tuberculosis. It was found that purified EtaA displays a remarkably low activity with the antitubercular prodrug ethionamide. Hinted by the presence of a Baeyer-Villiger monooxygenase sequence motif in the EtaA sequence, we have been able to identify a large number of novel EtaA substrates. It was discovered that the enzyme converts a wide range of ketones to the corresponding esters or lactones via a Baeyer-Villiger reaction, indicating that EtaA represents a Baeyer-Villiger monooxygenase. With the exception of aromatic ketones (phenylacetone and benzylacetone), long-chain ketones (e.g. 2-hexanone and 2-dodecanone) also EtaA is also able to of of of the identified is with of identified is of the of that a in the is the of EtaA activity by of by and newly identified prodrug tuberculosis. EtaA is a newly identified FAD-containing monooxygenase that is responsible for activation of several thioamide prodrugs in Mycobacterium tuberculosis. It was found that purified EtaA displays a remarkably low activity with the antitubercular prodrug ethionamide. Hinted by the presence of a Baeyer-Villiger monooxygenase sequence motif in the EtaA sequence, we have been able to identify a large number of novel EtaA substrates. It was discovered that the enzyme converts a wide range of ketones to the corresponding esters or lactones via a Baeyer-Villiger reaction, indicating that EtaA represents a Baeyer-Villiger monooxygenase. With the exception of aromatic ketones (phenylacetone and benzylacetone), long-chain ketones (e.g. 2-hexanone and 2-dodecanone) also EtaA is also able to of of of the identified is with of identified is of the of that a in the is the of EtaA activity by of by and newly identified prodrug tuberculosis. is the of a of a with and number of that of to or of in the and for the of antitubercular several thioamide have been the for antitubercular and that also to the of of thioamide the by a Mycobacterium was identified that is responsible for the antitubercular of was was found to by the corresponding the a by with a of EtaA was by of the prodrug in Mycobacterium in the prodrug with in of of to and is that the antitubercular of also to activation by EtaA by that EtaA displays prodrug is also to a enzyme is to in Mycobacterium is in with the to been for a been in the the of been identified to in is also the for antitubercular the the prodrugs and by is by a been that EtaA is a monooxygenase of in the of the corresponding the to in a activation was also found to by EtaA antitubercular indicating that the is It been that the of to a the by a in the of been that the of activity by the of or a of of the been found that the for antitubercular for thioamide and and thioamide the of sequence the EtaA sequence with several the presence of a newly identified sequence that EtaA is a Baeyer-Villiger monooxygenase a in the sequence that the sequence with activity is monooxygenase. been and in and to the and of the identified prodrug and and and of the of purified the and and the monooxygenase was and of was by and of and the is and is a was in the have been the been by a the was with and and the of the in the of EtaA and of EtaA was by a in the is and is in with or and in with and a of EtaA was purified the a by and in and the was to with a of of the was with the and for the was by the with of and of the enzyme was the with the the enzyme was to a the enzyme was by with a and EtaA was purified in and the the was a enzyme the the enzyme was a with enzyme was a of by and to a and by and was a with or of the and a of a with the and activity with of the ketones was by the of of was by the of the and the for was the of and of the of EtaA was by the of and in the presence of and in was in a by with was by and a the of and enzyme was of the was of and EtaA for was by of was and and by was a and a with and the a to in by a to in and of was by a and of a and the was purified in the presence of of of purified enzyme a in corresponding to a of is to the of the of the the in low with the of that the enzyme is of EtaA by been identified in the EtaA sequence that a EtaA sequence with a number of that to or was found that EtaA to by in that the enzyme a of large to with purified EtaA and the activity of enzyme is EtaA been for of the was purified a indicating that a been identified of the purified enzyme a and in and for the purified was was found to the for of the indicating that is the of the enzyme by in a of the of the the and that the purified enzyme of the for EtaA the of EtaA of EtaA was by in to and of EtaA was by of EtaA with and that the enzyme is in the of of the in the enzyme was with of It was found that the enzyme was by the the with with the by with that the enzyme is that the enzyme is able to and with that the enzyme is and is in with the that of the for with that the enzyme a of low activity also been with and a of the thioamide the of of by the of the was the of the of EtaA for that is a remarkably for the prodrug a low of and a of a low of to of for FAD-containing range to is to that prodrug activation the in enzyme was found to in a of is to found for sequence EtaA and corresponding been identified by in a It is that with the exception of also the of a the FAD-containing the a of to the of the for is a was to the by EtaA we the of a that is by It was found that EtaA of the of to the the that the is in the of EtaA by with the of the a of EtaA in Baeyer-Villiger by the presence of a sequence motif in the EtaA sequence we a range of ketones that have been to by It was found that several and by with ketones of the or by the of the of a of a Baeyer-Villiger to was found that a range of ketones was by EtaA several long-chain 2-hexanone to ketones also via a Baeyer-Villiger the corresponding With the exception of the long-chain and of of the ketones in the of the the of and of the also the of Baeyer-Villiger the is by of the of the identified the of the in the range for ethionamide. the to a that is the found of the of the of in a found for of the that the of is by a the the of the enzyme the was the for the presence of the enzyme with of the of EtaA of and was of the was found that the enzyme is in the indicating that a in the is the of the a is the to the the of the the is to the of the by and the of EtaA was of indicating that the a the of with of the the of the and EtaA the for EtaA is the for and to for and is also in the range to of the by of EtaA and the activity of EtaA with of the is several for or the enzyme to the of several activity was of of the in sequence have been found the and EtaA was found of the activity by of was found that of EtaA activity was a in the also was the of that the of the of in the of the is in the and the and is of the is of is in with the in and a is found for of EtaA in a of EtaA by activity of EtaA was in the presence of of and in the of the of a was in the It was found that the a of for to was the of of that EtaA is in by or with is to in by been by also been with the of a was also was that the is for the is that is of EtaA for and was also and found to in the and a activity of the activity was with a low that to the the the of the of to the the of EtaA in the presence of was It was found that of in a of of by EtaA sequence with a monooxygenase. the sequence also to the activity of monooxygenase with was It was found that the enzyme converts the corresponding to EtaA monooxygenase was also found to the of the in the of the for and for that monooxygenase is in ethionamide. the corresponding is in the range found for to monooxygenase is also able to the in activity with indicating that the of the activity of monooxygenase with that monooxygenase represents a enzyme to the of have been to been that in the of the activation by a prodrug represents a FAD-containing monooxygenase that the monooxygenase is of a newly identified of we have been able to that EtaA is able to Baeyer-Villiger to and the of sequence with that in EtaA is a have that the low of the enzyme of a low of EtaA by It was found that by that the activity of EtaA in is by with a EtaA ketones the of the to identified in the of ketones to ketones have that of a to the by the is of been found to and by a of the sequence of and Mycobacterium and the is in the identified in the sequence identified in EtaA is the that in the in of of the in of the that in EtaA to of for the been that the of EtaA of EtaA to by or of the in the of in the of is of and the sequence motif that a large number of the been by with been that a large of for of in is It been that activity of a large number of in the to in the that EtaA a in the of of EtaA is in with a of the have been to in a the is in EtaA also a in a that is for of the It been that the of to the a Baeyer-Villiger responsible for the of esters been also been that a in the of is by a of EtaA to and with a in the of EtaA and in in of It been found that a of a number sequence the for of With the exception of a of in have also been found in a and have been identified in or that of of to prodrug in the of a wide range of of (e.g. that the EtaA prodrug a and corresponding for thioamide for for the activity of EtaA or of is the of a of a with and number of that of to or of in the and for the of antitubercular several thioamide have been the for antitubercular and that also to the of of thioamide the by a Mycobacterium was identified that is responsible for the antitubercular of was was found to by the corresponding the a by with a of EtaA was by of the prodrug in Mycobacterium in the prodrug with in of of to and is that the antitubercular of also to activation by EtaA by that EtaA displays prodrug is also to a enzyme is to in Mycobacterium is in with the to been for a been in the the of been identified to in is also the for antitubercular the the prodrugs and by is by a been that EtaA is a monooxygenase of in the of the corresponding the to in a activation was also found to by EtaA antitubercular indicating that the is It been that the of to a the by a in the of been that the of activity by the of or a of of the been found that the for antitubercular for thioamide and and thioamide the of sequence the EtaA sequence with several the presence of a newly identified sequence that EtaA is a Baeyer-Villiger monooxygenase a in the sequence that the sequence with activity is monooxygenase. been and in and to the and of the identified prodrug Baeyer-Villiger monooxygenase monooxygenase Baeyer-Villiger monooxygenase monooxygenase and and and of the of purified the and and the monooxygenase was and of was by and of and the is and is a was in the have been the been by a the was with and and the of the in the of EtaA and of EtaA was by a in the is and is in with or and in with and a of EtaA was purified the a by and in and the was to with a of of the was with the and for the was by the with of and of the enzyme was the with the the enzyme was to a the enzyme was by with a and EtaA was purified in and the the was a enzyme the the enzyme was a with enzyme was a of by and to a and by and was a with or of the and a of a with the and activity with of the ketones was by the of of was by the of the and the for was the of and of the of EtaA was by the of and in the presence of and in was in a by with was by and a the of and enzyme was of the was of and EtaA for was by of was and and by was a and a with and the a to in by a to in and of was by a and and and of the of purified the and and the monooxygenase was and of was by and of and the is and is a was in the have been the been by a the was with and and the of the in the of EtaA and of EtaA was by a in the is and is in with or and in with and a of EtaA was purified the a by and in and the was to with a of of the was with the and for the was by the with of and of the enzyme was the with the the enzyme was to a the enzyme was by with a and EtaA was purified in and the the was a enzyme the the enzyme was a with enzyme was a of by and to a and by and was a with or of the and a of a with the and activity with of the ketones was by the of of was by the of the and the for was the of and of the of EtaA was by the of and in the presence of and in was in a by with was by and a the of and enzyme was of the was of and EtaA for was by of was and and by was a and a with and the a to in by a to in and of was by a and of a and the was purified in the presence of of of purified enzyme a in corresponding to a of is to the of the of the the in low with the of that the enzyme is of EtaA by been identified in the EtaA sequence that a EtaA sequence with a number of that to or was found that EtaA to by in that the enzyme a of large to with purified EtaA and the activity of enzyme is EtaA been for of the was purified a indicating that a been identified of the purified enzyme a and in and for the purified was was found to the for of the indicating that is the of the enzyme by in a of the of the the and that the purified enzyme of the for EtaA the of EtaA of of the in the enzyme was with of It was found that the enzyme was by the the with with the by with that the enzyme is that the enzyme is able to and with that the enzyme is and is in with the that of the for with that the enzyme a of low activity also been with and a of the thioamide the of of by the of the was the of the of EtaA for that is a remarkably for the prodrug a low of and a of a low of to of for FAD-containing range to is to that prodrug activation the in enzyme was found to in a of is to found for sequence EtaA and corresponding been identified by in a It is that with the exception of also the of a the FAD-containing the a of to the of the for is a was to the by EtaA we the of a that is by It was found that EtaA of the of to the the that the is in the of EtaA by with the of the a of EtaA in Baeyer-Villiger by the presence of a sequence motif in the EtaA sequence we a range of ketones that have been to by It was found that several and by with ketones of the or by the of the of a of a Baeyer-Villiger to was found that a range of ketones was by EtaA several long-chain 2-hexanone to ketones also via a Baeyer-Villiger the corresponding With the exception of the long-chain and of of the ketones in the of the the of and of the also the of Baeyer-Villiger the is by of the of the identified the of the in the range for ethionamide. the to a that is the found of the of the of in a found for of the that the of is by a the the of the enzyme the was the for the presence of the enzyme with of the of EtaA of and was of the was found that the enzyme is in the indicating that a in the is the of the a is the to the the of the the is to the of the by and the of EtaA was of indicating that the a the of with of the the of the and EtaA the for EtaA is the for and to for and is also in the range to of the by of EtaA and the activity of EtaA with of the is several for or the enzyme to the of several activity was of of the in sequence have been found the and EtaA was found of the activity by of was found that of EtaA activity was a in the also was the of that the of the of in the of the is in the and the and is of the is of is in with the in and a is found for of EtaA in a of EtaA by activity of EtaA was in the presence of of and in the of the of a was in the It was found that the a of for to was the of of that EtaA is in by or with is to in by been by also been with the of a was also was that the is for the is that is of EtaA for and was also and found to in the and a activity of the activity was with a low that to the the the of the of to the the of EtaA in the presence of was It was found that of in a of of by EtaA sequence with a monooxygenase. the sequence also to the activity of monooxygenase with was It was found that the enzyme converts the corresponding to EtaA monooxygenase was also found to the of the in the of the for and for that monooxygenase is in ethionamide. the corresponding is in the range found for to monooxygenase is also able to the in activity with indicating that the of the activity of monooxygenase with that monooxygenase represents a enzyme to the of and of a and the was purified in the presence of of of purified enzyme a in corresponding to a of is to the of the of the the in low with the of that the enzyme is of EtaA by been identified in the EtaA sequence that a EtaA sequence with a number of that to or was found that EtaA to by in that the enzyme a of large to with purified EtaA and the activity of enzyme is EtaA been for of the EtaA was purified a indicating that a been identified of the purified enzyme a and in and for the purified was was found to the for of the indicating that is the of the enzyme by in a of the of the the and that the purified enzyme of the for EtaA the of EtaA of of the in the enzyme was with of It was found that the enzyme was by the the with with the by with that the enzyme is that the enzyme is able to and with that the enzyme is and is in with the that of the for with that the enzyme a of low activity also been with and a of the thioamide the of of by the of the was the of the of EtaA for that is a remarkably for the prodrug a low of and a of a low of to of for FAD-containing range to is to that prodrug activation the in enzyme was found to in a of is to found for sequence been identified by It is that with the exception of also the of a the FAD-containing the a of to the of the for is a was to the by EtaA we the of a that is by It was found that EtaA of the of to the the that the is in the of EtaA by with the of the a of EtaA in EtaA Baeyer-Villiger by the presence of a sequence motif in the EtaA sequence we a range of ketones that have been to by It was found that several and by with ketones of the or by the of the of a of a Baeyer-Villiger to was found that a range of ketones was by EtaA several long-chain 2-hexanone to ketones also via a Baeyer-Villiger the corresponding With the exception of the long-chain and of of the ketones in the of the the of and of the also the of Baeyer-Villiger the is by of the of the identified the of the in the range for ethionamide. the to a that is the found found for of the that the of is by a the the of the enzyme the was the for the presence of the enzyme with of the of EtaA of and was of the was found that the enzyme is in the indicating that a in the is the of the a is the to the the of the the is to the of the by and the of EtaA was of indicating that the a the of with of the the of the and EtaA the for EtaA is the for and to for and is also in the range to of the by of EtaA and the activity of EtaA with of the is several for or the enzyme to the of several activity was of of the in sequence have been found the and EtaA was found of the activity by of was found that of EtaA activity was a in the also was the of that the of the of in the of the is in the and the and is of the is of is in with the in and a is found for the of the of a was in the It was found that the a of for to was the of of that EtaA is in by or with is to in by been by also been with the of a was also was that the is for the is that is of EtaA for and was also and found to in the and a activity of the activity was with a low that to the the the of the of to the the of EtaA in the presence of was It was found that of in a of of by EtaA sequence with a monooxygenase. the sequence also to the activity of monooxygenase with was It was found that the enzyme converts the corresponding to EtaA monooxygenase was also found to the of the in the of the for and for that monooxygenase is in ethionamide. the corresponding is in the range found for to monooxygenase is also able to the in activity with indicating that the of the activity of monooxygenase with that monooxygenase represents a enzyme to the of have been to been that in the of the activation by a prodrug represents a FAD-containing monooxygenase that the monooxygenase is of a newly identified of we have been able to that EtaA is able to Baeyer-Villiger to and the of sequence with that in EtaA is a have that the low of the enzyme of a low of EtaA by It was found that by that the activity of EtaA in is by with a EtaA ketones the of the to identified in the of ketones to ketones have that of a to the by the is of been found to and by a of the sequence of and Mycobacterium and the is in the identified in the sequence identified in EtaA is the that in the in of of the in of the that in EtaA to of for the been that the of EtaA of EtaA to by or of the in the of in the of is of and the sequence motif that a large number of the been by with been that a large of for of in is It been that activity of a large number of in the to in the that EtaA a in the of of EtaA is in with a of the have been to in a the is in EtaA also a in a that is for of the It been that the of to the a Baeyer-Villiger responsible for the of esters been also been that a in the of is by a of EtaA to and with a in the of EtaA and in in of It been found that a of a number sequence the for of With the exception of a of in have also been found in a and have been identified in or that of of to prodrug in the of a wide range of of (e.g. that the EtaA prodrug a and corresponding for thioamide for for the activity of EtaA or of have been to been that in the of the activation by a prodrug represents a FAD-containing monooxygenase that the monooxygenase is of a newly identified of we have been able to that EtaA is able to Baeyer-Villiger to and the of sequence with that in EtaA is a have that the low of the enzyme of a low of EtaA by It was found that by that the activity of EtaA in is by with a EtaA ketones the of the to identified in the of ketones to ketones have that of a to the by the is of been found to and by a of the sequence of and Mycobacterium and the is in the identified in the sequence identified in EtaA is the that in the in of of the in of the that in EtaA to of for the been that the of EtaA of EtaA to by or of the in the of in the of is of and the sequence motif that a large number of the been by with been that a large of for of in is It been that activity of a large number of in the to in the that EtaA a in the of of EtaA is in with a of the have been to in a the is in EtaA also a in a that is for of the It been that the of to the a Baeyer-Villiger responsible for the of esters been also been that a in the of is by a of EtaA to and with a in the of EtaA and in in of It been found that a of a number sequence the for of With the exception of a of in have also been found in a and have been identified in or that of of to prodrug in the of a wide range of of (e.g. that the EtaA prodrug a and corresponding for thioamide for for the activity of EtaA or of