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

Abstract

The crystal structures of the B 12 -dependent isomerases (eliminating) diol dehydratase and ethanolamine ammonia-lyase complexed with adenosylcobalamin were solved with and without substrates.T he structures revealed that the peripheral a-acetamide side chain of the corrin ring directly interacts with the adenosyl group to maintain the group in the catalytic position, and that this side chain swings between the original and catalytic positions in asynchronized manner with the radical shuttling between the coenzyme and substrate/ product. Mutations involving key residues that cooperatively participate in the positioning of the adenosyl group,directly or indirectly through the interaction with the a-side chain, decreased the turnover rate and increased the relative rate of irreversible inactivation caused by undesirable side reactions. These findings guide the engineering of enzymes for improved catalysis and producing useful chemicals by utilizing the high reactivity of radical species.Cobalamin or vitamin B 12 has acomplex structure based on acyclic tetrapyrrolic system called the corrin ring. Thecorrin ring of cobalamin binds ac obalt atom at its center (Figure 1a,b). It carries four peripheral propionamide side chains, one of which has an ucleotide-derived tail comprising ad imethylbenzimidazole group tethered to itself,e ight methyl groups,a nd three acetamide side chains.P revious structural studies of cobalamin-dependent enzymes have suggested that the main role of the peripheral propionamide and acetamide side chains is anchoring the corrin ring into its binding site of the enzyme.O ther functional roles of the peripheral side chains of cobalamin have been unclear, although our past biochemical studies using adenosylcobalamin (AdoCbl) analogues indicated that the b-propionamide side chain plays the most important role for coenzyme activity through hydrogen-bond donation from coenzyme to apoenzyme. [1,2] AdoCbl is the coenzyme for ag roup of enzymes that catalyze carbon-skeleton rearrangements,h eteroatom eliminations,and intramolecular amino group migrations. [3] These reactions are initiated by homolysis of the cobalt-carbon (Co À C) bond of the enzyme-bound cofactor and catalyzed by aradical mechanism (Figure 1f). Thecommon feature of the AdoCbl-dependent rearrangements is a1 ,2-migration of ah ydrogen atom in exchange for ag roup Xt hat moves in the opposite direction. Diol dehydratase (DD), glycerol dehydratase (GD), and ethanolamine ammonia-lyase (EAL) belong to ac lass of AdoCbl-dependent isomerases (eliminating) which catalyze heteroatom eliminations (Figure 1c-e). GD,a ni sofunctional enzyme of DD,i sakey component for the microbial production of 1,3-propanediol from glycerol, which is ab yproduct of biodiesel fuel production. [4] Probably,t he most important industrial use of 1,3-propanediol is as am onomer to synthesize polytrimethylene terephthalate,which has excellent properties as atextile fiber and in biodegradable polyesters,p olyurethanes,a nd polyethers. [4] Since the holoenzymes of DD,G D,...

Computational Study of Glycerol Binding within the Active Site of Coenzyme B12-Dependent Diol Dehydratase

“…We would also compare our input configuration with that against the inferences from the recent crystal structure that has become available for B 12 -dDDH with AdoCbl as a cofactor. 56 This work is nearly complete and will be a companion to the work presented in the current paper.…”

“…AdoCbl and CNCbl cofactor variants were reconstructed from the cobalamin already present within the 3AUJ crystal structure. Because no crystal structure 56 was available with the biologically active form of the cofactor when this study began, AdoCbl had to be computationally reconstructed in the enzyme. Structural information, particularly the orientation of the adenine moiety of Ado/AdoCbl, was obtained from the crystal structure of DDH with the cofactor analogue adeninylpentylcobalamine [PDB code: 1EEX 25,26 ].…”

Recent Progress in the Understanding and Engineering of Coenzyme B12-Dependent Glycerol Dehydratase

“…The ring carries one nucleotide-derived tail comprising the dimethylbenzimidazole (DBI) group in addition to four propionamide, three acetamide, and eight methyl groups ( Abeles and Dolphin, 1976 ; Banerjee, 1999 ; Figure 1 ). These peripheral groups interact with the residues in the α/β interface, which maintains the cofactor in a proper position for the radical-based catalysis ( Figure 4 ; Shibata et al, 2018 ). The crystal structure of Kp GDHt reported by Yamanishi et al has cyanocobalamin, an analog of AdoCbl ( Rétey, 1990 ; Yamanishi et al, 2002 ).…”

“…The cyanocobalamin- Kp GDHt complex structure provides information regarding how the peripheral groups of the corrin ring interact with the amino acid residues of the binding pocket ( Shibata et al, 1999 ; Toraya, 2000a , 2014 ). Recently, the same group illustrated the complex structure of coenzyme B 12 -dependent diol dehydratase (DDHt) with AdoCbl ( Shibata et al, 2018 ); coenzyme B 12 -dependent DDHt has nearly the same structure and catalytic mechanism as coenzyme B 12 -dependent GDHt ( Toraya, 1994 ). This is the first complex structure including AdoCbl that shows how the native cofactor interacts with the enzyme.…”

“…The adenosyl group of AdoCbl interacts with Thr α 172 and Ser α 224 whereas the ribose moiety is stabilized by two hydrogen bonds through the acetamide group of corrin and Ser α 224. The substrate-free and substrate-bound structures exhibited the outward and inward shifts of the α-acetamide group, which was suggested to be linked to the opening and closing of a plausible channel for the passage of substrate and product ( Shibata et al, 2018 ).…”