A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

Abstract

Lysine 5,6-aminomutase is an adenosylcobalamin and pyridoxal-5 -phosphate-dependent enzyme that catalyzes a 1,2 rearrangement of the terminal amino group of DL-lysine and of L-␤-lysine. We have solved the x-ray structure of a substrate-free form of lysine-5,6-aminomutase from Clostridium sticklandii. In this structure, a Rossmann domain covalently binds pyridoxal-5 -phosphate by means of lysine 144 and positions it into the putative active site of a neighboring triosephosphate isomerase barrel domain, while simultaneously positioning the other cofactor, adenosylcobalamin, Ϸ25 Å from the active site. In this mode of pyridoxal-5 -phosphate binding, the cofactor acts as an anchor, tethering the separate polypeptide chain of the Rossmann domain to the triosephosphate isomerase barrel domain. Upon substrate binding and transaldimination of the lysine-144 linkage, the Rossmann domain would be free to rotate and bring adenosylcobalamin, pyridoxal-5 -phosphate, and substrate into proximity. Thus, the structure embodies a locking mechanism to keep the adenosylcobalamin out of the active site and prevent radical generation in the absence of substrate.A denosylcobalamin (AdoCbl; coenzyme B 12 ) is nature's biochemical radical reservoir, capable of catalyzing challenging chemical reactions by way of H atom abstraction and the generation of free-radical intermediates (1-3). AdoCbldependent isomerases catalyze 1,2 shifts between an H atom and a functional group such as -OH, -NH 3 ϩ , -(CO)S-coenzyme A, or other carbon-based groups. The catalytic power of AdoCbl lies in the homolytic cleavage of its weak (Ϸ30 kcal͞mol) organometallic C-Co bond, formed between an octahedral Co(III) center with five N coordinations and a 5Ј-deoxyadenosyl (Ado) axial ligand. C-Co bond homolysis results in the transient formation of cob(II)alamin and 5Ј-deoxyadenosyl radical (Ado • ). Ado • abstracts an H atom from the substrate, forming a substrate radical and 5Ј-deoxyadenosine (AdoH). To close the catalytic cycle, substrate reabstracts the H atom from AdoH, and recombination of cob(II)alamin and Ado • accompanies product formation. Amazingly, the enzymatic rate of C-Co bond homolysis is enhanced by a factor of Ϸ10 12 over nonenzymatic homolysis (4, 5). AdoCbl-dependent isomerases are often present in catabolic pathways and can serve to rearrange the substrate's carbon skeleton and͞or functional groups for further degradation. One such pathway that operates in several bacterial species is the fermentation of lysine to yield acetate. Interestingly, the lysine fermentation pathway contains two analogous enzymes: lysine 5,6-aminomutase (5,6-LAM), which is AdoCbl-dependent (6, 7), and lysine 2,3-aminomutase (2,3-LAM), which is an S-adenosylmethionine (AdoMet or SAM)-dependent ironsulfur enzyme (8-10). Both enzymes require pyridoxal 5Ј-phosphate (PLP) (8, 11) in addition to AdoCbl or AdoMet, and both catalyze a 1,2 amino group shift with concomitant H atom migration (Fig. 1A). In 5,6-LAM, AdoCbl is the source of the transient Ado • , whereas, in 2,3-L...

Crystal Structures of Ethanolamine Ammonia-lyase Complexed with Coenzyme B12 Analogs and Substrates

“…1B), which is similar to other AdoCbl-dependent enzymes (32)(33)(34)(35)(36). The C-terminal side of the barrel forms a hollow that accommodates a substrate molecule.…”

“…This composition, the ␣␤ unit, is conserved in most of the other base-on AdoCbl-dependent enzymes except class II ribonucleotide reductase (31). The most striking feature of the overall structure of EAL is that this enzyme is a hexamer of the ␣␤ unit (32)(33)(34)(35)(36). In contrast, the other known AdoCbl-dependent enzymes are monomeric or dimeric with respect to the ␣␤ units.…”

Visualization of a radical B 12 enzyme with its G-protein chaperone

“…1 B and D, Fig. S3 A-C, and Table S2) (23)(24)(25)(26). As with these other enzymes, AdoCbl is bound to a Rossmann-fold domain in a "base-off/His-on" conformation with a His (His39) serving as the lower axial ligand to the cobalt (Fig.…”

Crystal structure of calcium dodecin (Rv0379), from Mycobacterium tuberculosis with a unique calcium‐binding site

“…2(a)]. Three consecutive alanines (Ala20, 21,22), along with Tyr55, complete the hydrophobic pocket around the three Glu18 residues that bind calcium at the center (Supporting Information Fig. S1).…”

“…22 The fold of the dodecin subunit is also similar to the copper-binding domain (CuBD) of an Alzheimer's disease amyloid precursor protein (PDB: 1OWT), which is a neuronal regulator of copper homeostasis 23 with no apparent sequence identity (RMSD ¼ 7.66 Å over 59 C a atoms). Based on the hits from a similarity search, a structural motif containing a three-stranded twisted b-sheet and an amphipathic a-helix is present in several proteins with varying functions, and is not restricted to riboflavin-binding or calcium-binding dodecamers.…”