Cation–π Interactions Contribute to Substrate Recognition in γ‐Butyrobetaine Hydroxylase Catalysis

Abstract

Abstractγ‐Butyrobetaine hydroxylase (BBOX) is a non‐heme FeII‐ and 2‐oxoglutarate‐dependent oxygenase that catalyzes the stereoselective hydroxylation of an unactivated C−H bond of γ‐butyrobetaine (γBB) in the final step of carnitine biosynthesis. BBOX contains an aromatic cage for the recognition of the positively charged trimethylammonium group of the γBB substrate. Enzyme binding and kinetic analyses on substrate analogues with P and As substituting for N in the trimethylammonium group show that the analogues are good BBOX substrates, which follow the efficiency trend N+>P+>As+. The results reveal that an uncharged carbon analogue of γBB is not a BBOX substrate, thus highlighting the importance of the energetically favorable cation–π interactions in productive substrate recognition.

19F NMR studies on γ-butyrobetaine hydroxylase provide mechanistic insights and suggest a dual inhibition mode

“…(C) Overlaid g-butyrobetaine (GBB, 3; green sticks) and N-oxalylglycine (NOG; salmon sticks) binding residues of hBBOX (blue/orange cartoon (dimer view)) blue lines (PDB: 3O2G) 1 and a psBBOX model 23 (yellow lines). psBBOX Tyr201 (Y194 hBBOX, 1 pink sticks) is involved in GBB quaternary ammonium ion recognition; 24 it is located in a 'flexible-loop' region. (D) Titrations of (4) manifest only B60% GBB displacement using a 1 H NMR reporter assay; 25 by contrast (5) apparently displaces B90% GBB.…”

Adventures in Defining Roles of Oxygenases in the Regulation of Protein Biosynthesis

“…Substrate analogue studies on BBOX have provided insights into its selectivity . Notably, BBOX has been shown to accept the trimethylphosphine analogue of carnitine, as well as various cyclic carnitine analogues (Figure B) ,.…”

“…Substrate analogue studies on BBOX have provided insights into its selectivity . Notably, BBOX has been shown to accept the trimethylphosphine analogue of carnitine, as well as various cyclic carnitine analogues (Figure B) ,. The latter work is of interest from the perspective of asymmetric catalysis by oxygenases, e. g. an achiral meso ‐cyclic analogue was shown to undergo BBOX catalysed desymmetrisation to give a product with 3 chiral centres in a single enzyme‐catalysed step .…”

Substrate scope for trimethyllysine hydroxylase catalysis

“…This result is in agreement with mechanistic studies on other non-haem Fe(II) and 2OG oxygenases, including BBOX. 5,7,9 Control experiments under standard conditions, but in the absence of TMLH, showed no formation of 3-hydroxytrimethyllysine, indicating that the hydroxylation is TMLH-catalysed ( Fig. S4, ESI †).…”

“…This result is consistent with the catalytic cycle and the oxygen labelling studies on several other members of Fe(II)/2OG oxygenases. 7,19 Although there is some evidence that trimethyllysine that is required for carnitine biosynthesis in mammals can be obtained via food (e.g. vegetables), 20 the most likely source of trimethyllysine in cells are endogeneous trimethyllysine-containing proteins, most commonly histones, calmodulin, cytochrome C and myosine.…”