Roles of adenine anchoring and ion pairing at the coenzyme B₁₂‐binding site in diol dehydratase catalysis

Abstract

Adenosylcobalamin (AdoCbl) is a cofactor for enzymatic radical reactions, including carbon skeleton rearrangements, heteroatom eliminations, and intramolecular amino group migrations [1][2][3]. These reactions involve the migration of a hydrogen atom from one carbon atom of the substrate to the adjacent carbon atom [4,5] in exchange for group X, which moves in the opposite direction [6]. The reactions are initiated by abstraction of a hydrogen atom from substrates with an adenosyl radical that is generated in the active site through homolysis of the cobalt-carbon (Co-C) bond of AdoCbl [1][2][3]7,8]. The activation and homolysis of the Co-C bond upon coenzyme binding to apoenzyme is therefore considered to be a key step for all the AdoCbldependent reactions. Diol dehydratase (dl-1,2-propanediol hydrolyase; EC 4.2.1.28) is an enzyme that catalyzes the AdoCbl-dependent conversion of 1,2-diols and glycerol to the corresponding aldehydes [9,10]. The X-ray structure of the diol dehydratase-adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Sera224, and of 6-NH 2 , N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the e-NH 2 group of Lysb135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Sera224 and Lysb135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Sa224A, Sa224N and Kb135E mutants were 19-2% as active as the wild-type enzyme, whereas the Kb135A, Kb135Q and Kb135R mutants retained 58-76% of the wild-type activity. The presence of a positive charge at the b135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme-cobalamin interaction. The Sa224A and Sa224N mutants showed a k cat ⁄ k inact value that was less than 2% that of the wild-type, whereas for Lysb135 mutants this value was in the range 25-75%, except for the Kb135E mutant (7%). Unlike the wild-type holoenzyme, the Sa224N and Sa224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Sera224 is important for cobalt-carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Sa224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5¢-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one.Abbreviations AdePeCbl, adeninylpentylcobalamin; AdoCbl, adenosylcobalamin or coenzyme B 12 ; aqCbl, aquacobalamin; CN-Cbl, cyanocobalamin; OH-Cbl, hydroxocobalamin.

Direct Participation of a Peripheral Side Chain of a Corrin Ring in Coenzyme B12 Catalysis

“…Thep resent structures of DD complexed with AdoCbl indicated that Thra172 and Sera224 are key residues for stabilizing the post-homolysis state of the adenosyl group in DD.W en ext investigated the importance of the hydrogen bonds involving these two residues for catalytic properties by site-directed mutagenesis.A mong all of the present and reported mutants of the residues, [23] the Ta172A mutant displayed the lowest k cat value and k cat /k inact ratio ( Table 1). The k cat /k inact ratio indicates the average number of catalytic turnovers before inactivation.…”

“…Consistently,t he lower k cat /k inact value indicates that the mutant enzyme is more prone to be inactivated than the wild type,s uggesting that the positionally-unstable adenosyl radical in the Ta172A mutant causes undesirable side reactions at greater probability than does the wild type.T he importance of the hydrogen bond for catalysis is also supported by kinetic studies using the Ta172S mutant. [23] Previous biochemical study using AdoCbl monocarboxylate isomers,i nw hich one of the propionamide side chains was hydrolyzed, indicated that coenzyme activity in DD decreases in the order d-isomer > e-isomer > b-isomer. Indeed, the k cat value of the mutant decreased but still retained 10 %that of the wild type (Table 1).…”

Direct Participation of a Peripheral Side Chain of a Corrin Ring in Coenzyme B12 Catalysis

“…Indeed, the k cat value of the mutant decreased but still retained 10 %that of the wild type (Table 1). TheS era224 mutants,i ncluding previously reported ones, [23] showed relative k cat values ranging from 3% to 19 %a gainst the wild type enzyme.O ne explanation for the decrease of the k cat values upon mutation is due to complete or partial loss of their hydrogen bond interactions with 2'-OH of the ribose group and/or improper positioning of the adenosyl radical caused by the decrease or increase in steric hindrance by the volume change of the side chain. Another possible explanation is that the reduction of the anchoring effect on the adenine moiety is caused by the loss of the hydrogen bond interaction and destabilizes the post-homolysis state of the adenosyl group.…”

“…Indeed, the structure of the mutant DD(Ta172A)/1,2-PDO reveals that electron density corresponding to the ribose moiety of the adenosyl group is not visible (Supporting Information, Figure S2c), suggesting that the hydrogen bonds between Thra172 and the a-side chain of the corrin ring and between the a-side chain and 3'-OH of the ribose group in the post-homolysis state are crucial for holding the adenosyl [a] From ref. [23].…”

“…Another possible explanation is that the reduction of the anchoring effect on the adenine moiety is caused by the loss of the hydrogen bond interaction and destabilizes the post-homolysis state of the adenosyl group. [23] Previous biochemical study using AdoCbl monocarboxylate isomers,i nw hich one of the propionamide side chains was hydrolyzed, indicated that coenzyme activity in DD decreases in the order d-isomer > e-isomer > b-isomer. [1,2] At the b-site modification, hydrogen-bond donation from coenzyme to apoenzyme is important, whereas inhibition by steric effects is suggested at the d-site.F unctions of the acetamide side chains remain unclear.…”