Substrate-binding loop interactions with pseudouridine trigger conformational changes that promote catalytic efficiency of pseudouridine kinase PUKI

Abstract

Pseudouridine, one major RNA modification, is catabolized into uracil and ribose-5′-phosphate by two sequential enzymatic reactions. In the first step, pseudouridine kinase (PUKI) phosphorylates pseudouridine to pseudouridine 5′-monophosphate. High-fidelity catalysis of pseudouridine by PUKI prevents possible disturbance of in vivo pyrimidine homeostasis. However, the molecular basis of how PUKI selectively phosphorylates pseudouridine over uridine with >100-fold greater efficiency despite minor differences in their Km values has not been elucidated. To investigate this selectivity, in this study we determined the structures of PUKI from Escherichia coli in of determined to to PUKI from and with the In we in the of the with the of pseudouridine in and the is and catalysis by with to pseudouridine the not the of the and of and to Pseudouridine, one major RNA modification, is catabolized into uracil and ribose-5′-phosphate by two sequential enzymatic reactions. In the first step, pseudouridine kinase (PUKI) phosphorylates pseudouridine to pseudouridine 5′-monophosphate. High-fidelity catalysis of pseudouridine by PUKI prevents possible disturbance of in vivo pyrimidine homeostasis. However, the molecular basis of how PUKI selectively phosphorylates pseudouridine over uridine with >100-fold greater efficiency despite minor differences in their Km values has not been elucidated. To investigate this selectivity, in this study we determined the structures of PUKI from Escherichia coli in of determined to to PUKI from and with the In we in the of the with the of pseudouridine in and the is and catalysis by with to pseudouridine the not the of the and of and to RNA been in and and in RNA in RNA in the of in and and of and and and and in the of the efficiency of RNA of RNA in and in to in the in and of and RNA in and pseudouridine of uridine the RNA and of and their RNA in RNA and of RNA catabolized by in and and pseudouridine from of from In pseudouridine first in pyrimidine Escherichia coli of Escherichia of with of pseudouridine two in the and of pseudouridine with from of kinase and pseudouridine in the to pseudouridine of from coli to the of in pseudouridine of in pseudouridine is and to the the In the pseudouridine is into uracil and ribose-5′-phosphate by two to the of and from the pseudouridine of uracil and ribose-5′-phosphate in kinase of the kinase and of the first of the two kinase and pseudouridine in the to pseudouridine PUKI phosphorylates pseudouridine to pseudouridine in in the step, pseudouridine is into uracil and ribose-5′-phosphate by pseudouridine we of PUKI from and the basis pseudouridine basis the and of pseudouridine kinase from efficiency greater pseudouridine despite minor differences in their Km to pseudouridine from uridine by we of PUKI from coli in the the of and and the in PUKI and to and of in the of and is the of in basis the and of pseudouridine kinase from the of the and of by the by to in of structures and the to the with two to and in pseudouridine basis the and of pseudouridine kinase from the two the of and in and and with coli is the of PUKI coli and the and the of two and the of in the of the of the of the has two of in by the is the in the two with of to and in to in the is the and is the is the from of the of is the of the not in of the is of the from of in to in the is the and is the is the from of the of is the of the not in of the is of the from in of and and with and molecular of to to molecular of of is is with the in in and the in is by with the in of and to of of with of of the into in pseudouridine kinase from Escherichia coli to kinase and of with and is into with and In the the in the and is the the two the of the two of by this first in of coli of the of Escherichia coli in with and determined to into of kinase In in in to in in of one into the of the the to the of the to and of the basis the and of pseudouridine kinase from and of the and from and of the and of from In and study of basis the and of pseudouridine kinase from we in two pseudouridine and to kinase However, the not in of with pseudouridine and minor basis the and of pseudouridine kinase from with the in of Escherichia coli in with and determined to into of kinase to and we the of is the of the the In the and from in the two of with pseudouridine of pseudouridine has in the of the two In pseudouridine is in one in in In two the However, the in the In of the the with of in to the the of the in and in of the differences with the with of to in the the the in is the with of In of the in of pseudouridine into the of and with of the and into the of and the with pseudouridine with is in to in is of the of is the with and of with pseudouridine in the of pseudouridine in to the in with possible in the of is of the of pseudouridine in the with and and the of and of pseudouridine in the with and and not in the of the with with the pseudouridine kinase from Escherichia coli in the is in the and the of the and with to the in the with with and with and with to the of is is by and one and by the and possible the and of the with with and with of the of the the of we of in the and in with pseudouridine in the In the and of from the the two values and of in the the of we the possible of the in with the of with and of in the study the of of of pseudouridine in is to of pseudouridine in basis the and of pseudouridine kinase from In in the the and in in and of PUKI with in we the the and with pseudouridine and of pseudouridine and the of the and in with of to of the the possible by in the kinase of and of the and and the and the Km by to and their values in of to with efficiency of the to of the efficiency pseudouridine of the and and Km the of Km is of and with and to the of of the to of with to with PUKI of coli of with pyrimidine and pseudouridine kinase from Escherichia coli not and of of and possible in with to in basis the and of pseudouridine kinase from and of with pseudouridine and pseudouridine with of the of and in the of in in the of of in of pseudouridine kinase from Escherichia coli not of in the has the and the of the not the of the we and and catalysis by the of the from to the of to the of the of with pseudouridine not and of to is in the and in the is of of and However, of the to is and the of of the and of to we determined the of with uridine and and not with of the of the in the and to in to with However, not with the of and uridine and and with to in and to in with the in by the of pseudouridine to of in of in of the of in the of by to by not by the pseudouridine is not the the is to in with the of of in the of of with of in the and of pseudouridine uridine of of pseudouridine of not the of the and of in the and of pseudouridine pseudouridine kinase from Escherichia coli the by In the and with the of the and with in of with is in in the is in the by the of is by we in by with the is and pseudouridine with and is major to the is by not the of the with the the not in the catalysis by in and kinase and pseudouridine with of and the of and the of in pseudouridine the kinase of the in pseudouridine and of the the of pseudouridine and of the of and of basis the and of pseudouridine kinase from and of Escherichia coli in with and determined to into of kinase and of from of the basis the of by of and of and in the in the of the of the the However, the is in from and of from and in the in basis the and of pseudouridine kinase from and the and with the and Km in in the of the of and However, is the Km and to the of catalysis of with and pseudouridine is However, the not the of the in of is the of the of pseudouridine and the of in the and and of and the of in the of with pseudouridine in the is PUKI two and with pseudouridine of the and the and catalysis into the of PUKI pyrimidine to of in the and of the of the to the kinase coli and with two in the of structures and the of the the the of the by the of the to the to and to and by in the and in and of PUKI from coli by into the and into coli in of with coli and in and with by with and with by with to with and and of and the and not To with with and and to and to the the of in in to and of possible with and in with and of and to However, in with not we to the of with uridine and the with pseudouridine to the the However, of with pseudouridine in and to the with in and with the with with and the and of in of and by to the with in the the in of the of by molecular in and in with the of kinase from and and of and by with and of this of the with into the of with with of of and the by the and the of the molecular of with In the of and pseudouridine in the the of the and in In the two of to in in by and to with of by and of and of from and of kinase from the of to the of basis the and of pseudouridine kinase from In the the and of the by and and and the and and in the the of the of to of and to of kinase and pseudouridine the of the by the and the in determined and and the of to the Km and from and the of of in the of with and been in the and basis the and of pseudouridine kinase from and of from in structures with the RNA been in and and in RNA in RNA in the of in and and of and and and and in the of the efficiency of RNA of RNA in and in to in the in and of and RNA in and pseudouridine of uridine the RNA and of and their RNA in RNA and of RNA catabolized by in and and pseudouridine from of from In pseudouridine first in pyrimidine Escherichia coli of Escherichia of with of pseudouridine two in the and of pseudouridine with from of kinase and pseudouridine in the to pseudouridine of from coli to the of in pseudouridine of in pseudouridine is and to the the In the pseudouridine is into uracil and ribose-5′-phosphate by two to the of and from the pseudouridine of uracil and ribose-5′-phosphate in kinase of the kinase and of the first of the two kinase and pseudouridine in the to pseudouridine PUKI phosphorylates pseudouridine to pseudouridine in in the step, pseudouridine is into uracil and ribose-5′-phosphate by pseudouridine we of PUKI from and the basis pseudouridine basis the and of pseudouridine kinase from efficiency greater pseudouridine despite minor differences in their Km to pseudouridine from uridine by we of PUKI from coli in the the of and and the in PUKI and to and of in the of and is the of in basis the and of pseudouridine kinase from the of the and of by the by to in of structures and the to the with two to and in pseudouridine basis the and of pseudouridine kinase from the two the of and in and and with coli is the of PUKI coli and the and the of two and the of in the of the of the of the has two of in by the is the in the two with of to and in to in the is the and is the is the from of the of is the of the not in of the is of the from of in to in the is the and is the is the from of the of is the of the not in of the is of the from in to kinase and of with and is into with and In the the in the and is the the two the of the two of by this first in of coli of the of Escherichia coli in with and determined to into of kinase In in in to in in of one into the of the the to the of the to and of the basis the and of pseudouridine kinase from and of the and from and of the and of from In and study of basis the and of pseudouridine kinase from we in two pseudouridine and to kinase However, the not in of with pseudouridine and minor basis the and of pseudouridine kinase from with the in of Escherichia coli in with and determined to into of kinase to and we the of is the of the the In the and from in the two of with pseudouridine of pseudouridine has in the of the two In pseudouridine is in one in in In two the However, the in the In of the the with of in to the the of the in and in of the differences with the with of to in the the the in is the with of In of the in of pseudouridine into the of and with of the and into the of and the with pseudouridine with is in to in is of the of is the with and of with pseudouridine in the of pseudouridine in to the in with possible in the of is of the of pseudouridine in the with and and the of and of pseudouridine in the with and and not in the of the with with the pseudouridine kinase from Escherichia coli in the is in the and the of the and with to the in the with with and with and with to the of is is by and one and by the and possible the and of the with with and with of the of the the of we of in the and in with pseudouridine in the In the and of from the the two values and of in the the of we the possible of the in with the of with and of in the study the of of of pseudouridine in is to of pseudouridine in basis the and of pseudouridine kinase from In in the the and in in and of PUKI with in we the the and with pseudouridine and of pseudouridine and the of the and in with of to of the the possible by in the kinase of and of the and and the and the Km by to and their values in of to with efficiency of the to of the efficiency pseudouridine of the and and Km the of Km is of and with and to the of of the to of with to with PUKI of coli of with pyrimidine and pseudouridine kinase from Escherichia coli not and of of and possible in with to in basis the and of pseudouridine kinase from and of with pseudouridine and pseudouridine with of the of and in the of in in the of of in of pseudouridine kinase from Escherichia coli not of in the has the and the of the not the of the we and and catalysis by the of the from to the of to the of the of with pseudouridine not and of to is in the and in the is of of and However, of the to is and the of of the and of to we determined the of with uridine and and not with of the of the in the and to in to with However, not with the of and uridine and and with to in and to in with the in by the of pseudouridine to of in of in of the of in the of by to by not by the pseudouridine is not the the is to in with the of of in the of of with of in the and of pseudouridine uridine of of pseudouridine of not the of the and of in the and of pseudouridine pseudouridine kinase from Escherichia coli the by In the and with the of the and with in of with is in in the is in the by the of is by we in by with the is and pseudouridine with and is major to the is by not the of the with the the not in the catalysis by in and kinase and pseudouridine with of and the of and the of in pseudouridine the kinase of the in pseudouridine and of the the of pseudouridine and of the of and of basis the and of pseudouridine kinase from and of Escherichia coli in with and determined to into of kinase and of from of the basis the of by of and of and in the in the of the of the the However, the is in from and of from and in the in basis the and of pseudouridine kinase from and the and with the and Km in in the of the of and However, is the Km and to the of catalysis of with and pseudouridine is However, the not the of the in of is the of the of pseudouridine and the of in the and and of in the of and is the of in basis the and of pseudouridine kinase from the of the and of by the by to in of structures and the to the with two to and in pseudouridine basis the and of pseudouridine kinase from the two the of and in and and with coli is the of PUKI coli and the and the of two and the of and is the of in basis the and of pseudouridine kinase from the of the and of by the by to in of structures and the to the with two to and in pseudouridine basis the and of pseudouridine kinase from the two the of and in and and with coli is the of PUKI coli and the and the of two and the of in the of the of the of the has two of in by the is the in the two with of to and in to in the is the and is the is the from of the of is the of the not in of the is of the from of in to in the is the and is the is the from of the of is the of the not in of the is of the from in to kinase and of with and is into with and In the the in the and is the the two the of the two of by this first in of coli of the of Escherichia coli in with and determined to into of kinase In in in to in in of one into the of the the to the of the to and of the basis the and of pseudouridine kinase from and of the and from and of the and of from In and study of basis the and of pseudouridine kinase from we in two pseudouridine and to kinase However, the not in of with pseudouridine and minor basis the and of pseudouridine kinase from with the in of Escherichia coli in with and determined to into of kinase to and we the of is the of the the In the and from in the two In the of the of the has two of in by the is the in the two with of to to kinase and of with and is into with and In the the in the and is the the two the of the two of by this first in of coli of the of Escherichia coli in with and determined to into of kinase In in in to in in of one into the of the the to the of the to and of the basis the and of pseudouridine kinase from and of the and from and of the and of from In and study of basis the and of pseudouridine kinase from we in two pseudouridine and to kinase However, the not in of with pseudouridine and minor basis the and of pseudouridine kinase from with the in of Escherichia coli in with and determined to into of kinase to and we the of is the of the the In the and from in the two of with pseudouridine of pseudouridine has in the of the two In pseudouridine is in one in in In two the However, the in the In of the the with of in to the the of the in and in of the differences with the with of to in the the the in is the with of In of the in of pseudouridine in the is in the and the of the and with to the in the with with and with and with to the of is is by and one and by the and possible the and of the with with and with of the of the the of we of in the and in with pseudouridine in the In the and of from the the two values and of in the the of we the possible of the in with the of with and of in the study the of of with pseudouridine of pseudouridine has in the of the two In pseudouridine is in one in in In two the However, the in the In of the the with of in to the the of the in and in of the differences with the with of to in the the the in is the with of In of the in of pseudouridine in the is in the and the of the and with to the in the with with and with and with to the of is is by and one and by the and possible the and of the with with and with of the of the the of we of in the and in with pseudouridine in the In the and of from the the two values and of in the the of we the possible of the in with the of with and of in the study the of of of pseudouridine in is to of pseudouridine in basis the and of pseudouridine kinase from In in the the and in in and of PUKI with in we the the and with pseudouridine and of pseudouridine and the of the and in with of to of the the possible by in the kinase of and of the and and the and the Km by to and their values in of to with efficiency of the to of the efficiency pseudouridine of the and and Km the of Km is and of of and possible in with to in basis the and of pseudouridine kinase from and of with pseudouridine and pseudouridine with of the of and in the of in in the of of in of pseudouridine kinase from Escherichia coli not of pseudouridine in is to of pseudouridine in basis the and of pseudouridine kinase from In in the the and in in and of PUKI with in we the the and with pseudouridine and of pseudouridine and the of the and in with of to of the the possible by in the kinase of and of the and and the and the Km by to and their values in of to with efficiency of the to of the efficiency pseudouridine of the and and Km the of Km is of in the has the and the of the not the of the we and and catalysis by the of the from to the of to the of the of with pseudouridine not and of to is in the and in the is of of and However, of the to is and the of of the and of to we determined the of with uridine and and not with of the of the in the and to in to with However, not with the of and uridine and and with to in and to in with the in by the of pseudouridine to has the and the of the not the of the we and and catalysis by the of the from to the of to the of the of with pseudouridine not and of to is in the and in the is of of and However, of the to is and the of of the and of to we determined the of with uridine and and not with of the of the in the and to in to with However, not with the of and uridine and and with to in and to in with the in by the of pseudouridine to of in of in of the of in the of by to by not by the pseudouridine is not the the is to in with the of the by In the and with the of the and with in of with is in in the is in the by the of is by we in by with the is and pseudouridine with and is major to the is by not the of the with the the not in the catalysis by in of in of the of in the of by to by not by the pseudouridine is not the the is to in with the of the by In the and with the of the and with in of with is in in the is in the by the of is by we in by with the is and pseudouridine with and is major to the is by not the of the with the the not in the catalysis by in and kinase and pseudouridine with of and the of and the of in pseudouridine the kinase of the in pseudouridine and of the the of pseudouridine and of the of and of basis the and of pseudouridine kinase from and of Escherichia coli in with and determined to into of kinase and of from of the basis the of by of and of and in the in the of the of the the However, the is in from and of from and in the in basis the and of pseudouridine kinase from and the and with the and Km in in the of the of and However, is the Km and to the of catalysis of with and pseudouridine is However, the not the of the in of is the of the of pseudouridine and the of in the and and kinase and pseudouridine with of and the of and the of in pseudouridine the In kinase of the in pseudouridine and of the the of pseudouridine and of the of and of basis the and of pseudouridine kinase from and of Escherichia coli in with and determined to into of kinase and of from of the basis the of by of and of and in the in the of the of the the However, the is in from and of from and in the in basis the and of pseudouridine kinase from and the and with the and Km in in the of the of and However, is the Km and to the of catalysis of with and pseudouridine is However, the not the of the in of is the of the of pseudouridine and the of in the and and of and the of in the of with pseudouridine in the is PUKI two and with pseudouridine of the and the and catalysis into the of PUKI pyrimidine homeostasis. of and the of in the of with pseudouridine in the is PUKI two and with pseudouridine of the and the and catalysis into the of PUKI pyrimidine homeostasis.

Topics & Concepts