Redesign of coenzyme B₁₂ dependent diol dehydratase to be resistant to the mechanism‐based inactivation by glycerol and act on longer chain 1,2‐diols

Abstract

Coenzyme B12 dependent diol dehydratase undergoes mechanism‐based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co–C bond. Bachovchin et al. [Biochemistry16, 1082–1092 (1977)] reported that glycerol bound in the GS conformation, in which the pro‐S‐CH2OH group is oriented to the hydrogen‐abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X‐ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)‐1,2‐propanediol, i.e. in the GS conformation, with its 3‐OH group hydrogen bonded to Serα301, but not to nearby Glnα336. kinact of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild‐type enzyme. kcat/kinact showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild‐type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)‐1,2‐propanediol decreased on these mutations. The substrate activities towards longer chain 1,2‐diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2‐diols. Database Structural data are available in the Protein Data Bank under the accession number http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3AUJ. Structured digital abstract http://www.uniprot.org/uniprot/Q59472, http://www.uniprot.org/uniprot/Q59471 and http://www.uniprot.org/uniprot/Q59470 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8301985)

1,2-Propanediol Dehydration in Roseburia inulinivorans

“…The starting orientation is important because during the dehydration reaction, and specifically the migration of the middle hydroxyl group of glycerol, there is an energetic penalty associated with the stronger hydrogen bonding of the G S orientation. Hence, it was proposed that undesirable side reactions leading to the suicide inhibition of the B 12 -dependent diol dehydratase occur with a much higher probability for glycerol that is bound in the G S orientation (43). Consistent with this proposal, the B 12 -dependent diol dehydratase will utilize both enantiomers of 1,2-propanediol but has a preference for the R enantiomer (44).…”

“…Interestingly, a similar mechanistic parallel is observed when comparing the activity of the GRE-dependent glycerol dehydratase, CbGD, with the GRE-dependent diol dehydratase, RiDD. Specifically, similar to what has been reported for the B 12 -dependent enzymes, the diol dehydratase may be more amendable to re-engineering (43). However, to address this thoroughly as well as the mechanism of all GREs, it will be imperative to utilize the physiological donors (i.e.…”

“…In fact, multiple binding modes have been proposed to explain the mechanism-based inactivation by glycerol that is observed for the B 12 -dependent diol dehydratase (43). Specifically, although glycerol is not a chiral compound, glycerol can bind to the active site of the B 12 -dependent diol dehydratase in two different orientations.…”

Synthesis and Properties of Formylcobalamin and Propionylcobalamin, Novel Acylcobalamins

“…We recently discussed about the mechanism of inactivation of AdoCbl-dependent diol dehydratase by glycerol (18,19) and the possible involvement of formyl-Cbl in the inactivation process (20). Theoretical calculations suggest that glycerol inactivation of this enzyme begins with an intramolecular hydrogen transfer from the glycerol 3-OH group to C1 of the substrate radical, forming an O3-centered radical that decomposes to formaldehyde and a glycol radical (20).…”

Experimental Design For 1,3-Propanediol Biosynthesis by K. Pneumoniae GLC29 Using Glycerol

“…The addition from 5 mg/l to 20 mg/l of vitamin B12, production was negatively impacted because of its interaction with glycerol. An irreversible binding of the vitamin B12 and glycerol with the enzyme, forms alkylcobalamines, and to avoid low activity of the enzyme, the amount of glycerol should be controlled [20,[29][30][31][32], but also the amount of vitamin should be controlled. As a consequence of the normal catalytic cycle with glycerol, the coenzyme B12 is occasionally rendered inactive (B12-inact).…”