TY - JOUR
T1 - Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase
AU - Toraya, Tetsuo
AU - Tanokuchi, Aya
AU - Yamasaki, Ai
AU - Nakamura, Takehiro
AU - Ogura, Kenichi
AU - Tobimatsu, Takamasa
N1 - Funding Information:
This work was supported in part by Grants-in-Aid for Scientific Research [(B) 13480195, (B) 17370038, (C) 22570143, and (Priority Area 753) 13125205 to T. Toraya and (C) 23560943, (C) 24570161 and (Challenging Exploratory Research) 15K14237 to T. Tobimatsu] from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a Grant-in-Aid for Natural Sciences Research (to T. Toraya) from the Asahi Glass Foundation (Tokyo, Japan).
Publisher Copyright:
© 2015 American Chemical Society.
PY - 2016/1/19
Y1 - 2016/1/19
N2 - Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg2+ (or Mn2+) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg2+, diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO showed the lowest adenosyltransfease activity toward free cob(I)alamin. These results suggest that (1) diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of DD-R, PduO adenosyltransferase, and a reducing system, (2) the releasing factor DD-R is essential for the recycling of adenosycobalamin, a tightly bound, prosthetic group-type coenzyme, and (3) PduO is a specific adenosylating enzyme for the DD reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme. Although FMNH2 was mainly used as a reductant in this study, a natural reducing system might consist of PduS cobalamin reductase and NADH.
AB - Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg2+ (or Mn2+) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg2+, diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO showed the lowest adenosyltransfease activity toward free cob(I)alamin. These results suggest that (1) diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of DD-R, PduO adenosyltransferase, and a reducing system, (2) the releasing factor DD-R is essential for the recycling of adenosycobalamin, a tightly bound, prosthetic group-type coenzyme, and (3) PduO is a specific adenosylating enzyme for the DD reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme. Although FMNH2 was mainly used as a reductant in this study, a natural reducing system might consist of PduS cobalamin reductase and NADH.
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U2 - 10.1021/acs.biochem.5b01023
DO - 10.1021/acs.biochem.5b01023
M3 - Article
C2 - 26704729
AN - SCOPUS:84954436224
SN - 0006-2960
VL - 55
SP - 69
EP - 78
JO - Biochemistry
JF - Biochemistry
IS - 1
ER -