Structural and Mechanistic Studies on γ-Butyrobetaine Hydroxylase

Abstract

The final step in carnitine biosynthesis is catalyzed by γ-butyrobetaine (γBB) hydroxylase (BBOX), an iron/2-oxoglutarate (2OG) dependent oxygenase. BBOX is inhibited by trimethylhydrazine-propionate (THP), a clinically used compound. We report structural and mechanistic studies on BBOX and its reaction with THP. Crystallographic and sequence analyses reveal that BBOX and trimethyllysine hydroxylase form a subfamily of 2OG oxygenases that dimerize using an N-terminal domain. The crystal structure reveals the active site is enclosed and how THP competes with γBB. THP is a substrate giving formaldehyde (supporting structural links with histone demethylases), dimethylamine, malonic acid semi-aldehyde, and an unexpected product with an additional carbon-carbon bond resulting from N-demethylation coupled to oxidative rearrangement, likely via an unusual radical mechanism. The results provide a basis for development of improved BBOX inhibitors and may inspire the discovery of additional rearrangement reactions.

Structural and Evolutionary Basis for the Dual Substrate Selectivity of Human KDM4 Histone Demethylase Family

“…protein complex partners/other conditions for optimal activity), turnover numbers for the KDM4s are low compared with some other human 2-OG oxygenases, e.g. γ-butyrobetaine hydroxylase (61), and comparable with those reported for the hypoxia-inducible factor hydroxylases (62). Given the central role proposed for the hypoxia-inducible factor hydroxylases in regulating the hypoxic response in all animals (63, 64), it is possible that the demethylase activities of the KDM4 histone demethylases have roles in directly regulating gene expression, perhaps in a redox-regulated manner.…”

Oxygenase‐Catalyzed Desymmetrization of N,N‐Dialkyl‐piperidine‐4‐carboxylic Acids

“…The overall fold of BBOX in the new structure is very similar to that obtained for the human BBOX structure in complex with GBB, NOG, and Zn II ions[5] (PDB ID: 3O2G; Figure S4), with the mobile active-site loop in the “closed” conformation (as is proposed to be required for efficient substrate turnover [8a]). The active-site residues in the BBOX 1 NOG Ni II complex are positioned similarly to those in the BBOX GBB NOG Zn II complex (Figure 2).…”

Control of Histone H3 Lysine 9 (H3K9) Methylation State via Cooperative Two-step Demethylation by Jumonji Domain Containing 1A (JMJD1A) Homodimer

“…Disruption of dimerization by a single point mutation causes loss of catalytic activity (44). ␥-Butyrobetaine hydroxylase also forms a homodimer, where elements from each monomer are involved in substrate binding (45). However, with respect to the homodimers described above, substrate channeling mediated by two proximal active sites has not been identified.…”