The Structural Basis for Methylmalonic Aciduria

ATP:cobalamin adenosyltransferase MMAB was recently identified as the gene responsible for a disorder of cobalamin metabolism in humans (cblB complementation group). The crystal structure of the MMAB sequence homologue from Thermoplasma acidophilum (TA1434; GenBank™ identification number gi|16082403) was determined to a resolution of 1.5 Å. TA1434 was confirmed to be an ATP:cobalamin adenosyltransferase, which depended absolutely on divalent metal ions (Mg2+ > Mn2+ > Co2+) and only used ATP or dATP as adenosyl donors. The apparent Km of TA1434 was 110 μm (kcat = 0.23 s-1) for ATP, 140 μm (kcat = 0.11 s-1) for dATP, and 3 μm (kcat = 0.18 s-1) for cobalamin. TA1434 is a trimer in solution and in the crystal structure, with each subunit composed of a five-helix bundle. The location of disease-related point mutations and other residues conserved among the homologues of TA1434 suggest that the active site lies at the junctions between the subunits. Mutations in TA1434 that correspond to the disease-related mutations resulted in proteins that were inactive for ATP:cobalamin adenosyltransferase activity in vitro, confirming that these mutations define the molecular basis of the human disease. ATP:cobalamin adenosyltransferase MMAB was recently identified as the gene responsible for a disorder of cobalamin metabolism in humans (cblB complementation group). The crystal structure of the MMAB sequence homologue from Thermoplasma acidophilum (TA1434; GenBank™ identification number gi|16082403) was determined to a resolution of 1.5 Å. TA1434 was confirmed to be an ATP:cobalamin adenosyltransferase, which depended absolutely on divalent metal ions (Mg2+ > Mn2+ > Co2+) and only used ATP or dATP as adenosyl donors. The apparent Km of TA1434 was 110 μm (kcat = 0.23 s-1) for ATP, 140 μm (kcat = 0.11 s-1) for dATP, and 3 μm (kcat = 0.18 s-1) for cobalamin. TA1434 is a trimer in solution and in the crystal structure, with each subunit composed of a five-helix bundle. The location of disease-related point mutations and other residues conserved among the homologues of TA1434 suggest that the active site lies at the junctions between the subunits. Mutations in TA1434 that correspond to the disease-related mutations resulted in proteins that were inactive for ATP:cobalamin adenosyltransferase activity in vitro, confirming that these mutations define the molecular basis of the human disease. Cobalamin (vitamin B12) is an important cofactor in both prokaryotes and eukaryotes. A wide variety of cobalamin-dependent metabolic reactions are known, including acetyl-CoA synthesis, generation of deoxyribonucleotides for DNA synthesis, methane production in methanogenic archaea, and fermentation of The metabolic for cobalamin among as the other for cobalamin on and is that and cobalamin A that cobalamin from other is that only and the for to in the cobalamin be of active used used and these are to be used in The to from The as a to to which the The and of is important in the of and and of a that for a The of cobalamin to and the of the from a to and the of the the of the is from ATP and to the an ATP:cobalamin adenosyltransferase are of and on and the with which are are mutations in cobalamin metabolic mutations in The and of complementation are and to synthesis, and and and of both on the complementation with or or of are with and The and of MMAB a ATP:cobalamin adenosyltransferase, was recently identified as responsible for with the complementation with and metabolic The and of mutations in gene and the between the mutations in MMAB and a and of an MMAB sequence homologue from Thermoplasma acidophilum that sequence to MMAB and to the adenosyltransferase in confirmed that TA1434 is an ATP:cobalamin adenosyltransferase, with activity that for or The crystal structure for TA1434 that is a with each subunit composed of a five-helix bundle. disease-related mutations are in a conserved at the subunit which is the active mutations that are to mutations in the human MMAB resulted in a inactive for the of these mutations in was from was as and and the of cobalamin adenosyltransferase was in a of and the as ATP:cobalamin adenosyltransferase with the of in The of of ATP:cobalamin adenosyltransferase is and the in as the The and for were in and at The to the with the a = = = Å. The was determined as in a of with a in each and was at on and from a crystal of ATP:cobalamin adenosyltransferase were on and were and with the of are in was used to the and the and was used to the and a of the and were with and were and of were with in of both and are in is the and is the from of of of of is the and is the is the and is the from of of of is the and is the in a in a of ATP:cobalamin adenosyltransferase was with a with and and was at at a of were a of a The were at at in and with for The ATP, and were with for and a to the The of and were and of the were at for of to of at a the reactions were the of an solution of the of and the in at was The activity was = for ATP, dATP, and were determined the as for activity were for in from to for ATP the of μm to 1.5 for dATP the of μm and to μm for the of were in and the are were with the and were the = of TA1434 was The used to of the TA1434 are in DNA TA1434 was used as a for of DNA was with the and DNA the in the the was to each and at for of the was from the the and mutations were DNA adenosyltransferase in a of of the between and of proteins of of the were in the as ATP:cobalamin adenosyltransferase and as sequence of the conserved TA1434 was used to the for sequence A number of as were in archaea, and with sequence from to The of TA1434 with homologues is in TA1434 sequence to and to which are ATP:cobalamin from and that TA1434 are residues that are absolutely conserved among the The conserved residues and in TA1434 correspond to and in the human which are residues in of the sequence of TA1434 with human confirmed that TA1434 ATP:cobalamin adenosyltransferase activity an adenosyltransferase to TA1434 as ATP:cobalamin of TA1434 with sequence are for the from Thermoplasma from the from from from MMAB MMAB and a adenosyltransferase from The are conserved in an that homologues and structure of ATP:cobalamin adenosyltransferase was determined the as the was to of and the of was The was of and the of to and of from and and were The of from to and The and the residues were and be in the The was to resolution with an of and an of to of the residues are in the and are residues in the of the adenosyltransferase of a five-helix and from to from to from residues to from residues 110 to and from to The of the is is a in the and and The between and are the between and are A of and are between and are between and and are between and of ATP:cobalamin of subunit of ATP:cobalamin The is composed of a with a five-helix The structure are of ATP:cobalamin The and were used in the production of the adenosyltransferase was to be is with of the crystal The is the of from subunit with of subunit and is a of and The that are between A and of the of and of of and of and of and of are between and as as between and at the are A is between residues and is between and and between and active site in ATP:cobalamin adenosyltransferase was identified the location of the conserved of the conserved residues are at the of are in subunit a conserved Mutations in of these residues are in the MMAB gene in with the of cobalamin a for these is at the of and is of and from subunit and and from the other The of ATP:cobalamin adenosyltransferase that is which the ATP to be The and to location of the The active site in the of ATP:cobalamin adenosyltransferase, was in the of The residues important for ATP and to be determined with these of ATP:cobalamin from to The active site is and ATP was used in the production of number of homologues of ATP:cobalamin adenosyltransferase were identified from to The of homologues and The homologues to the a structure as of or the structure the of the and are composed of at the of a of the of ATP:cobalamin adenosyltransferase with in the of the the of the as as the the that the is to ATP:cobalamin adenosyltransferase as of the of the conserved residues is to as an the location of the site in the is in the of the the active site of ATP:cobalamin adenosyltransferase is on the of the at the of with on sequence are of cobalamin the the and the The recently determined crystal structure of a ATP:cobalamin adenosyltransferase a five-helix bundle. and ATP:cobalamin adenosyltransferase are both that and ATP:cobalamin adenosyltransferase for and to the of cobalamin adenosyltransferase is a adenosyltransferase for in structure to the of the for of the are responsible for of are of the of with and and from are used an TA1434 was for ATP:cobalamin adenosyltransferase activity the with and The of resulted in the of to which as an in the adenosyltransferase The activity of ATP:cobalamin adenosyltransferase was with an of The activity of was at at at at and at is was for the of other and MMAB of adenosyltransferase was The activity was with at was of the in the of Mn2+ and with was activity with or from that of which the activity in the of and the of of ATP and ATP:cobalamin adenosyltransferase the for both and Mn2+ = = 0.11 and for = metal ATP:cobalamin adenosyltransferase activity in the of is the of ATP:cobalamin adenosyltransferase to other as the was that a for the and ATP, and as the of the for activity to ATP:cobalamin adenosyltransferase a for the and was only active with ATP or dATP and with or The Km for dATP was with that for ATP, the was as the with dATP in between and ATP:cobalamin adenosyltransferase with that the proteins of with a and ATP:cobalamin adenosyltransferase is between the and cobalamin adenosyltransferase between the and and are for and in and metal a in both proteins the production of the used to TA1434 for ATP:cobalamin adenosyltransferase The for ATP, dATP, and were between ATP:cobalamin adenosyltransferase activity and between ATP:cobalamin adenosyltransferase activity and between TA1434 activity and for and in a ATP:cobalamin adenosyltransferase and were with ATP, dATP, and The are in to both ATP and dATP, with ATP activity and that ATP is the in The Km for was with that of the Km for ATP was μm for TA1434 a activity a to point mutations identified in the MMAB in with the of cobalamin metabolism of these are in ATP:cobalamin adenosyltransferase and are among the conserved residues the point mutations in ATP:cobalamin adenosyltransferase that were identified in MMAB and the for ATP:cobalamin adenosyltransferase and resulted in proteins with activity these residues with other and only with were in to the ATP:cobalamin and were and activity of ATP:cobalamin adenosyltransferase conserved residues are and residues are other conserved activity of and the mutations identified in MMAB in with mutations identified in MMAB in with mutations identified in MMAB in with the mutations identified in MMAB in with in a the and residues are for the activity of that and in the subunit are of the active site of ATP:cobalamin is the and the The of that is in in the the trimer is in a Km for ATP that was that of the an Km for that be in the with The of these in activity to be crystal structure of ATP:cobalamin adenosyltransferase a with each composed of a five-helix bundle. is the acidophilum homologue of the human MMAB that in the cobalamin metabolic of residues in ATP:cobalamin adenosyltransferase in inactive that MMAB is inactive in with these at with the cobalamin and ATP, to Cobalamin (vitamin B12) is an important cofactor in both prokaryotes and eukaryotes. A wide variety of cobalamin-dependent metabolic reactions are known, including acetyl-CoA synthesis, generation of deoxyribonucleotides for DNA synthesis, methane production in methanogenic archaea, and fermentation of The metabolic for cobalamin among as the other for cobalamin on and is that and cobalamin A that cobalamin from other is that only and the for to in the cobalamin be of active used used and these are to be used in The to from The as a to to which the The and of is important in the of and and of Cobalamin a that for a The of cobalamin to and the of the from a to and the of the the of the is from ATP and to the an ATP:cobalamin adenosyltransferase are of and on and the with which are are mutations in cobalamin metabolic mutations in The and of complementation are and to synthesis, and and and of both on the complementation with or or of are with and The and of The MMAB a ATP:cobalamin adenosyltransferase, was recently identified as responsible for with the complementation with and metabolic The and of mutations in gene and the between the mutations in MMAB and a and of an MMAB sequence homologue from Thermoplasma acidophilum that sequence to MMAB and to the adenosyltransferase in confirmed that TA1434 is an ATP:cobalamin adenosyltransferase, with activity that for or The crystal structure for TA1434 that is a with each subunit composed of a five-helix bundle. disease-related mutations are in a conserved at the subunit which is the active mutations that are to mutations in the human MMAB resulted in a inactive for the of these mutations in was from was as and and the of cobalamin adenosyltransferase was in a of and the as ATP:cobalamin adenosyltransferase with the of in The of of ATP:cobalamin adenosyltransferase is and the in as the The and for were in and at The to the with the a = = = Å. The was determined as in a of with a in each and was at on and from a crystal of ATP:cobalamin adenosyltransferase were on and were and with the of are in was used to the and the and was used to the and a of the and were with and were and of were with in of both and are in is the and is the from of of of of is the and is the is the and is the from of of of is the and is the in a in a of ATP:cobalamin adenosyltransferase was with a with and and was at at a of were a of a The were at at in and with for The ATP, and were with for and a to the The of and were and of the were at for of to of at a the reactions were the of an solution of the of and the in at was The activity was = for ATP, dATP, and were determined the as for activity were for in from to for ATP the of μm to 1.5 for dATP the of μm and to μm for the of were in and the are were with the and were the = of TA1434 was The used to of the TA1434 are in DNA TA1434 was used as a for of DNA was with the and DNA the in the the was to each and at for of the was from the the and mutations were DNA adenosyltransferase in a of of the between and of proteins of of the were in the as ATP:cobalamin adenosyltransferase and as was from was as and and the of cobalamin adenosyltransferase was in a of and the as ATP:cobalamin adenosyltransferase with the of in The of of ATP:cobalamin adenosyltransferase is and the in as the The and for were in and at The to the with the a = = = Å. The was determined as in a of with a in each and was at on and from a crystal of ATP:cobalamin adenosyltransferase were on and were and with the of are in was used to the and the and was used to the and a of the and were with and were and of were with in of both and are in of ATP:cobalamin adenosyltransferase was with a with and and was at at a of were a of a The were at at in and with for The ATP, and were with for and a to the The of and were and of the were at for of to of at a the reactions were the of an solution of the of and the in at was The activity was = The for ATP, dATP, and were determined the as for activity were for in from to for ATP the of μm to 1.5 for dATP the of μm and to μm for the of were in and the are were with the and were the = of TA1434 was The used to of the TA1434 are in DNA TA1434 was used as a for of DNA was with the and DNA the in the the was to each and at for of the was from the the and mutations were DNA of of the between and of proteins of of the were in the as ATP:cobalamin adenosyltransferase and as sequence of the conserved TA1434 was used to the for sequence A number of as were in archaea, and with sequence from to The of TA1434 with homologues is in TA1434 sequence to and to which are ATP:cobalamin from and that TA1434 are residues that are absolutely conserved among the The conserved residues and in TA1434 correspond to and in the human which are residues in of the sequence of TA1434 with human confirmed that TA1434 ATP:cobalamin adenosyltransferase activity an adenosyltransferase to TA1434 as ATP:cobalamin structure of ATP:cobalamin adenosyltransferase was determined the as the was to of and the of was The was of and the of to and of from and and were The of from to and The and the residues were and be in the The was to resolution with an of and an of to of the residues are in the and are residues in the of the adenosyltransferase of a five-helix and from to from to from residues to from residues 110 to and from to The of the is is a in the and and The between and are the between and are A of and are between and are between and and are between and of ATP:cobalamin of subunit of ATP:cobalamin The is composed of a with a five-helix The structure are of ATP:cobalamin The and were used in the production of the adenosyltransferase was to be is with of the crystal The is the of from subunit with of subunit and is a of and The that are between A and of the of and of of and of and of and of are between and as as between and at the are A is between residues and is between and and between and active site in ATP:cobalamin adenosyltransferase was identified the location of the conserved of the conserved residues are at the of are in subunit a conserved Mutations in of these residues are in the MMAB gene in with the of cobalamin a for these is at the of and is of and from subunit and and from the other The of ATP:cobalamin adenosyltransferase that is which the ATP to be The and to location of the The active site in the of ATP:cobalamin adenosyltransferase, was in the of The residues important for ATP and to be determined with these of ATP:cobalamin from to The active site is and ATP was used in the production of number of homologues of ATP:cobalamin adenosyltransferase were identified from to The of homologues and The homologues to the a structure as of or the structure the of the and are composed of at the of a of the of ATP:cobalamin adenosyltransferase with in the of the the of the as as the the that the is to ATP:cobalamin adenosyltransferase as of the of the conserved residues is to as an the location of the site in the is in the of the the active site of ATP:cobalamin adenosyltransferase is on the of the at the of with on sequence are of cobalamin the the and the The recently determined crystal structure of a ATP:cobalamin adenosyltransferase a five-helix bundle. and ATP:cobalamin adenosyltransferase are both that and ATP:cobalamin adenosyltransferase for and to the of cobalamin adenosyltransferase is a adenosyltransferase for in structure to the of the for of the are responsible for of are of the of with and and from are used an TA1434 was for ATP:cobalamin adenosyltransferase activity the with and The of resulted in the of to which as an in the adenosyltransferase The activity of ATP:cobalamin adenosyltransferase was with an of The activity of was at at at at and at is was for the of other and MMAB of adenosyltransferase was The activity was with at was of the in the of Mn2+ and with was activity with or from that of which the activity in the of and the of of ATP and ATP:cobalamin adenosyltransferase the for both and Mn2+ = = 0.11 and for = metal ATP:cobalamin adenosyltransferase activity in the of is the of ATP:cobalamin adenosyltransferase to other as the was that a for the and ATP, and as the of the for activity to ATP:cobalamin adenosyltransferase a for the and was only active with ATP or dATP and with or The Km for dATP was with that for ATP, the was as the with dATP in between and ATP:cobalamin adenosyltransferase with that the proteins of with a and ATP:cobalamin adenosyltransferase is between the and cobalamin adenosyltransferase between the and and are for and in and metal a in both proteins the production of the used to TA1434 for ATP:cobalamin adenosyltransferase The for ATP, dATP, and were between ATP:cobalamin adenosyltransferase activity and between ATP:cobalamin adenosyltransferase activity and between TA1434 activity and for and in a ATP:cobalamin adenosyltransferase and were with ATP, dATP, and The are in to both ATP and dATP, with ATP activity and that ATP is the in The Km for was with that of the Km for ATP was μm for TA1434 a activity a to point mutations identified in the MMAB in with the of cobalamin metabolism of these are in ATP:cobalamin adenosyltransferase and are among the conserved residues the point mutations in ATP:cobalamin adenosyltransferase that were identified in MMAB and the for ATP:cobalamin adenosyltransferase and resulted in proteins with activity these residues with other and only with were in to the ATP:cobalamin and were and activity of ATP:cobalamin adenosyltransferase conserved residues are and residues are other conserved activity of and the mutations identified in MMAB in with mutations identified in MMAB in with mutations identified in MMAB in with the mutations identified in MMAB in with in a the and residues are for the activity of that and in the subunit are of the active site of ATP:cobalamin is the and the The of that is in in the the trimer is in a Km for ATP that was that of the an Km for that be in the with The of these in activity to be crystal structure of ATP:cobalamin adenosyltransferase a with each composed of a five-helix bundle. is the acidophilum homologue of the human MMAB that in the cobalamin metabolic of residues in ATP:cobalamin adenosyltransferase in inactive that MMAB is inactive in with these at with the cobalamin and ATP, to sequence of the conserved TA1434 was used to the for sequence A number of as were in archaea, and with sequence from to The of TA1434 with homologues is in TA1434 sequence to and to which are ATP:cobalamin from and that TA1434 are residues that are absolutely conserved among the The conserved residues and in TA1434 correspond to and in the human which are residues in of the sequence of TA1434 with human confirmed that TA1434 ATP:cobalamin adenosyltransferase activity an adenosyltransferase to TA1434 as ATP:cobalamin structure of ATP:cobalamin adenosyltransferase was determined the as the was to of and the of was The was of and the of to and of from and and were The of from to and The and the residues were and be in the The was to resolution with an of and an of to of the residues are in the and are residues in the of the adenosyltransferase of a five-helix and from to from to from residues to from residues 110 to and from to The of the is is a in the and and The between and are the between and are A of and are between and are between and and are between and The adenosyltransferase was to be is with of the crystal The is the of from subunit with of subunit and is a of and The that are between A and of the of and of of and of and of and of are between and as as between and at the are A is between residues and is between and and between and active site in ATP:cobalamin adenosyltransferase was identified the location of the conserved of the conserved residues are at the of are in subunit a conserved Mutations in of these residues are in the MMAB gene in with the of cobalamin a for these is at the of and is of and from subunit and and from the other The of ATP:cobalamin adenosyltransferase that is which the ATP to be The and to location of the The active site in the of ATP:cobalamin adenosyltransferase, was in the of The residues important for ATP and to be determined with these number of homologues of ATP:cobalamin adenosyltransferase were identified from to The of homologues and The homologues to the a structure as of or the structure the of the and are composed of at the of a of the of ATP:cobalamin adenosyltransferase with in the of the the of the as as the the that the is to ATP:cobalamin adenosyltransferase as of the of the conserved residues is to as an the location of the site in the is in the of the the active site of ATP:cobalamin adenosyltransferase is on the of the at the of subunits. with on sequence are of cobalamin the the and the The recently determined crystal structure of a ATP:cobalamin adenosyltransferase a five-helix bundle. and ATP:cobalamin adenosyltransferase are both that and ATP:cobalamin adenosyltransferase for and to the of cobalamin adenosyltransferase is a adenosyltransferase for in structure to the of the for of the are responsible for of are of the of with and and from are used TA1434 an TA1434 was for ATP:cobalamin adenosyltransferase activity the with and The of resulted in the of to which as an in the adenosyltransferase The activity of ATP:cobalamin adenosyltransferase was with an of The activity of was at at at at and at is was for the of other and MMAB of ATP:cobalamin adenosyltransferase was The activity was with at was of the in the of Mn2+ and with was activity with or from that of which the activity in the of and the of of ATP and ATP:cobalamin adenosyltransferase the for both and Mn2+ = = 0.11 and for = the of ATP:cobalamin adenosyltransferase to other as the was that a for the and ATP, and as the of the for activity to ATP:cobalamin adenosyltransferase a for the and was only active with ATP or dATP and with or The Km for dATP was with that for ATP, the was as the with dATP in between and ATP:cobalamin adenosyltransferase with that the proteins of with a and ATP:cobalamin adenosyltransferase is between the and cobalamin adenosyltransferase between the and and are for and in and metal a in both proteins the production of the used to ATP:cobalamin adenosyltransferase and were with ATP, dATP, and The are in to both ATP and dATP, with ATP activity and that ATP is the in The Km for was with that of the Km for ATP was μm for TA1434 a activity a to point mutations identified in the MMAB in with the of cobalamin metabolism of these are in ATP:cobalamin adenosyltransferase and are among the conserved residues the point mutations in ATP:cobalamin adenosyltransferase that were identified in MMAB and the for ATP:cobalamin adenosyltransferase and resulted in proteins with activity these residues with other and only with were in to the ATP:cobalamin and were and activity the and residues are for the activity of that and in the subunit are of the active site of ATP:cobalamin is the and the The of that is in in the the trimer is in a Km for ATP that was that of the an Km for that be in the with The of these in activity to be The crystal structure of ATP:cobalamin adenosyltransferase a with each composed of a five-helix bundle. is the acidophilum homologue of the human MMAB that in the cobalamin metabolic of residues in ATP:cobalamin adenosyltransferase in inactive that MMAB is inactive in with these at with the cobalamin and ATP, to of the of the at and the of the for for in

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