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Literature summary extracted from

  • Fan, C.; Bobik, T.A.
    Functional characterization and mutation analysis of human ATP:Cob(I)alamin adenosyltransferase (2008), Biochemistry, 47, 2806-2813.
    View publication on PubMed

Application

EC Number Application Comment Organism
2.5.1.17 medicine insight in molecular mechanism of methylmalonic aciduria Homo sapiens

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.5.1.17 without mitochondrial targeting sequence from plasmid pNL166 harbouring the coding sequence, native or with C-terminal octa-His tag into pTA925 to introduce a linker region necessary for expression in Escherichia coli BL21DE3 RIL and ATR-deficient Salmonella strain BE620, hATR in pCF13 (N-terminal octa-His tag) and pCF15 (C-terminal octa-His tag) for transformation and random mutagenesis in Escherichia coli mutator strain XL1-Red followed by expression in BE620 Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
2.5.1.17 C119Y wild-type kinetics, decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression, no rescue of ATR-deficient Salmonella strain BE620 Homo sapiens
2.5.1.17 C189Y inactive in vitro (10fold excess of substrate compared to standard), decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression (but can be purified), no rescue of ATR-deficient Salmonella strain BE620 Homo sapiens
2.5.1.17 D218N substantially reduced Vmax, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620 Homo sapiens
2.5.1.17 D64G substantially reduced Vmax, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620 Homo sapiens
2.5.1.17 D90N inactive in vitro (10fold excess of substrate compared to standard) Homo sapiens
2.5.1.17 E193K inactive in vitro (10fold excess of substrate compared to standard), conserved residue, mutation found in methylmalonic aciduria patients Homo sapiens
2.5.1.17 E84K inactive in vitro (10fold excess of substrate compared to standard) Homo sapiens
2.5.1.17 E91K inactive in vitro (10fold excess of substrate compared to standard) Homo sapiens
2.5.1.17 F212S inactive in vitro (10fold excess of substrate compared to standard) Homo sapiens
2.5.1.17 F83S large change in KM for ATP and cob(I)alamin, F83 has direct contact with ATP Homo sapiens
2.5.1.17 G63E substantially reduced Vmax, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620 Homo sapiens
2.5.1.17 G87R inactive in vitro (10fold excess of substrate compared to standard) Homo sapiens
2.5.1.17 G97E wild-type kinetics, mutation distant from proposed active site, no rescue of ATR-deficient Salmonella strain BE620 possibly due to impaired reduction of cob(II)alamin to cob(I)alamin, expressed at wild-type levels Homo sapiens
2.5.1.17 G97R substantially reduced Vmax, mutation distant from proposed active site, no rescue of ATR-deficient Salmonella strain BE620 Homo sapiens
2.5.1.17 H183Y wild-type kinetics, mutation distant from proposed active site, no rescue of ATR-deficient Salmonella strain BE620 possibly due to impaired reduction of cob(II)alamin to cob(I)alamin, expressed at wild-type levels Homo sapiens
2.5.1.17 K78Q inactive in vitro (10fold excess of substrate compared to standard) Homo sapiens
2.5.1.17 K78R substantially reduced Vmax, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620 Homo sapiens
2.5.1.17 L220P inactive in vitro (10fold excess of substrate compared to standard) Homo sapiens
2.5.1.17 L223P inactive in vitro (10fold excess of substrate compared to standard) Homo sapiens
2.5.1.17 L92S inactive in vitro (10fold excess of substrate compared to standard) Homo sapiens
2.5.1.17 R186W inactive in vitro (10fold excess of substrate compared to standard), decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression (but can be purified), no rescue of ATR-deficient Salmonella strain BE620, conserved residue, mutation found in methylmalonic aciduria patients Homo sapiens
2.5.1.17 R190C inactive in vitro (10fold excess of substrate compared to standard), decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression (but can be purified), no rescue of ATR-deficient Salmonella strain BE620, conserved residue, mutation found in methylmalonic aciduria patients Homo sapiens
2.5.1.17 R191W inactive in vitro (10fold excess of substrate compared to standard), decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression (but can be purified), no rescue of ATR-deficient Salmonella strain BE620, conserved residue, mutation found in methylmalonic aciduria patients Homo sapiens
2.5.1.17 R194G inactive in vitro (10fold excess of substrate compared to standard) Homo sapiens
2.5.1.17 R215K inactive in vitro (10fold excess of substrate compared to standard), lack of activity in vitro, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620 Homo sapiens
2.5.1.17 R225K lack of activity in vitro, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620 Homo sapiens
2.5.1.17 R76G large change in KM for ATP and cob(I)alamin, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620 Homo sapiens
2.5.1.17 S126L large change in KM for ATP and cob(I)alamin, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620 Homo sapiens
2.5.1.17 S217R inactive in vitro (10fold excess of substrate compared to standard) Homo sapiens
2.5.1.17 S68F substantially reduced Vmax, residue S68 has role in ATP-binding Homo sapiens
2.5.1.17 S94L inactive in vitro (10fold excess of substrate compared to standard) Homo sapiens
2.5.1.17 T161I wild-type kinetics, decreased adenosylcobalamin production in vivo but rescues ATR-deficient Salmonella strain BE620 possibly due to impaired reduction of cob(II)alamin to cob(I)alamin, expressed at wild-type levels Homo sapiens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.5.1.17 additional information
-
 additional information elevated for either ATP or cobalamin or both substrates in case of mutants which show decreased adenosylcobalamin synthesis activity that can be partly corrected by increased hydroxycobalamin concentration Homo sapiens
2.5.1.17 additional information
-
 additional information large changes in KM for both substrates for mutants R76G, F83S and S126L Homo sapiens
2.5.1.17 additional information
-
 additional information wild-type kinetics by mutants G97E, C119Y, T161I, and H183Y Homo sapiens
2.5.1.17 0.00158
-
 cob(I)alamin N-terminally octa-His tagged wild-type, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.0016
-
 cob(I)alamin C-terminally octa-His tagged wild-type, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.0016
-
 cob(I)alamin native wild-type, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.00172
-
 cob(I)alamin mutant D218N, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.00186
-
 cob(I)alamin mutant S68F, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.00194
-
 cob(I)alamin mutant D64G, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.00224
-
 cob(I)alamin mutant G63E, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.00225
-
 cob(I)alamin mutant K78R, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.00252
-
 cob(I)alamin mutant G97E, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.00271
-
 cob(I)alamin mutant T161I, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.00287
-
 ATP mutant D218N, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.00303
-
 cob(I)alamin mutant G97R, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.005
-
 ATP mutant T161I, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.00513
-
 cob(I)alamin mutant C119Y, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.00523
-
 ATP mutant G97E, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.0069
-
 ATP native wild-type, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.00713
-
 cob(I)alamin mutant H183Y, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.00719
-
 ATP N-terminally octa-His tagged wild-type, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.00735
-
 ATP C-terminally octa-His tagged wild-type, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.00826
-
 ATP mutant K78R, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.00909
-
 cob(I)alamin mutant R76G, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.00997
-
 ATP mutant H183Y, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.0124
-
 ATP mutant G63E, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.0141
-
 ATP mutant C119Y, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.0143
-
 cob(I)alamin mutant S126L, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.0172
-
 ATP mutant S68F, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.0203
-
 ATP mutant G97R, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.0205
-
 ATP mutant D64G, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.0302
-
 cob(I)alamin mutant F83S, in presence of 0.5 mM ATP Homo sapiens
2.5.1.17 0.0459
-
 ATP mutant R76G, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.0877
-
 ATP mutant S126L, in presence of 0.05 mM cob(I)alamin Homo sapiens
2.5.1.17 0.215
-
 ATP mutant F83S, in presence of 0.05 mM cob(I)alamin Homo sapiens

Organism

EC Number Organism UniProt Comment Textmining
2.5.1.17 Homo sapiens Q96EY8
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.5.1.17 after expression at 16°C, lysis with French pressure cell, pH 7.5, and centrifugation Homo sapiens

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.5.1.17 additional information
-
 substantially reduced Vmax for mutants G63E, D64G, S68F, K78R, G97R and D218N Homo sapiens
2.5.1.17 additional information
-
 wild-type kinetics by mutants G97E, C119Y, T161I, and H183Y Homo sapiens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.5.1.17 ATP + hydroxycobalamin 37°C, pH 8, 0.5 mM ATP, 0.05 mM hydroxycobalamin, in presence of 1 mM titanium(III)citrate Homo sapiens adenosylcobalamin + phosphate + diphosphate measured by decrease in absorbance at 388 nm ?
2.5.1.17 cob(II)alamin G97, T161, and H183 possible role in stabilizing four-coordinate, cob(II)alamin C-terminal His-tagged enzyme binds cob(II)alamin base-off while N-terminal His-tagged enzyme binds it base-on (impaired base-off transition), only mutants S68F, K78Q, K78R, R186W, and R190C also bind cob(II)alamin base-off Homo sapiens cob(I)alamin
-
 ?
2.5.1.17 additional information no in vitro activity by mutants R215K, R225K as well as K78Q, E84K, G87R, D90N, E91K, L92S, S94L, R186W, C189Y, R190C, R191W, E193K, R194G, F212S, S217R, L220P, and L223P Homo sapiens ?
-
 ?
2.5.1.17 additional information no mediation of base-off transition of adenosylcobalamin (key step of catalytic mechanism) by mutants D64G, F83S, G87R, D90N, E91K, L92S, S94L, C189Y, R191W, E193K, R194G, F212S, S217R, L220P, and L223P Homo sapiens ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.5.1.17 hATR
-
 Homo sapiens
2.5.1.17 human adenosyltransferase
-
 Homo sapiens
2.5.1.17 human ATP: cob(I)alamin adenosyltransferase PduO-type enzyme, MMAB gene Homo sapiens
2.5.1.17 MMAB
-
 Homo sapiens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.5.1.17 additional information
-
 ATP C-terminally octa-His tagged wild-type, kcat/KM is 0.67 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin C-terminally octa-His tagged wild-type, kcat/KM is 3.10 per microM and min Homo sapiens
2.5.1.17 additional information
-
 additional information kcat of N-terminal octa-His tagged enzyme is 58% of native or C-terminal tagged enzyme possibly due to interference of N-terminal tag with base-off transition of adenosylcobalamin by the enzyme Homo sapiens
2.5.1.17 additional information
-
 ATP mutant C119Y, kcat/KM is 0.29 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant C119Y, kcat/KM is 0.79 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP mutant D218N, kcat/KM is 0.25 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant D218N, kcat/KM is 0.42 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP mutant D64G, kcat/KM is 0.026 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant D64G, kcat/KM is 0.27 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP mutant F83S, kcat/KM is 0.010 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant F83S, kcat/KM is 0.07 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP mutant G63E, kcat/KM is 0.039 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant G63E, kcat/KM is 0.22 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP mutant G97E, kcat/KM is 0.46 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant G97E, kcat/KM is 0.94 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP mutant G97R, kcat/KM is 0.039 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant G97R, kcat/KM is 0.26 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP mutant H183Y, kcat/KM is 0.28 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant H183Y, kcat/KM is 0.40 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP mutant K78R, kcat/KM is 0.039 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant K78R, kcat/KM is 0.14 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP mutant R76G, kcat/KM is 0.056 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant R76G, kcat/KM is 0.28 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP mutant S126L, kcat/KM is 0.007 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant S126L, kcat/KM is 0.04 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP mutant S68F, kcat/KM is 0.045 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant S68F, kcat/KM is 0.42 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP mutant T161I, kcat/KM is 0.46 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin mutant T161I, kcat/KM is 0.85 per microM and min Homo sapiens
2.5.1.17 additional information
-
 additional information mutations in conserved regions E84-S94 and R186-R194 lead to abolished enzymatic activity and support their implication in the enzymes active site Homo sapiens
2.5.1.17 additional information
-
 ATP N-terminally octa-His tagged wild-type, kcat/KM is 0.39 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin N-terminally octa-His tagged wild-type, kcat/KM is 1.75 per microM and min Homo sapiens
2.5.1.17 additional information
-
 ATP native wild-type, kcat/KM is 0.69 per microM and min Homo sapiens
2.5.1.17 additional information
-
 cob(I)alamin native wild-type, kcat/KM is 2.97 per microM and min Homo sapiens
2.5.1.17 19.2
-
 ATP mutant K78R Homo sapiens
2.5.1.17 19.2
-
 cob(I)alamin mutant K78R Homo sapiens
2.5.1.17 29.4
-
 ATP mutant G63E Homo sapiens
2.5.1.17 29.4
-
 cob(I)alamin mutant G63E Homo sapiens
2.5.1.17 31.8
-
 ATP mutant D64G Homo sapiens
2.5.1.17 31.8
-
 cob(I)alamin mutant D64G Homo sapiens
2.5.1.17 34.8
-
 ATP mutant S126L Homo sapiens
2.5.1.17 34.8
-
 cob(I)alamin mutant S126L Homo sapiens
2.5.1.17 43.8
-
 ATP mutant D218N Homo sapiens
2.5.1.17 43.8
-
 cob(I)alamin mutant D218N Homo sapiens
2.5.1.17 46.8
-
 ATP mutant S68F Homo sapiens
2.5.1.17 46.8
-
 cob(I)alamin mutant S68F Homo sapiens
2.5.1.17 48
-
 ATP mutant G97R Homo sapiens
2.5.1.17 48
-
 cob(I)alamin mutant G97R Homo sapiens
2.5.1.17 124.2
-
 ATP mutant F83S Homo sapiens
2.5.1.17 124.2
-
 cob(I)alamin mutant F83S Homo sapiens
2.5.1.17 137.4
-
 ATP mutant T161I Homo sapiens
2.5.1.17 137.4
-
 cob(I)alamin mutant T161I Homo sapiens
2.5.1.17 154.8
-
 ATP mutant R76G Homo sapiens
2.5.1.17 154.8
-
 cob(I)alamin mutant R76G Homo sapiens
2.5.1.17 166.2
-
 ATP N-terminally octa-His tagged wild-type Homo sapiens
2.5.1.17 166.2
-
 cob(I)alamin N-terminally octa-His tagged wild-type Homo sapiens
2.5.1.17 169.8
-
 ATP mutant G97E Homo sapiens
2.5.1.17 169.8
-
 cob(I)alamin mutant G97E Homo sapiens
2.5.1.17 169.8
-
 ATP mutant H183Y Homo sapiens
2.5.1.17 169.8
-
 cob(I)alamin mutant H183Y Homo sapiens
2.5.1.17 243.6
-
 ATP mutant C119Y Homo sapiens
2.5.1.17 243.6
-
 cob(I)alamin mutant C119Y Homo sapiens
2.5.1.17 285
-
 ATP native wild-type Homo sapiens
2.5.1.17 285
-
 cob(I)alamin native wild-type Homo sapiens
2.5.1.17 297.6
-
 ATP C-terminally octa-His tagged wild-type Homo sapiens
2.5.1.17 297.6
-
 cob(I)alamin C-terminally octa-His tagged wild-type Homo sapiens