In enzymology, D-lysine 5,6-aminomutase () is an enzyme that catalyzes the chemical reaction
D-lysine 2,5-diaminohexanoate
Hence, this enzyme has one substrate, D-lysine, and one product, 2,5-diaminohexanoate.
This enzyme participates in lysine degradation. It employs one cofactor, cobamide.
Background
D-lysine 5,6-aminomutase belongs to the isomerase family of enzymes, specifically intramolecular transferases, which transfers amino groups. Its systematic name is D-2,6-diaminohexanoate 5,6-aminomutase. Other names in common use include D-α-lysine mutase and adenosylcobalamin-dependent D-lysine 5,6-aminomutase, which can be abbreviated as 5,6-LAM.
5,6-LAM is capable of reversibly catalyzing the migration of an amino group from ε-carbon to δ-carbon in both D-lysine and L-β-lysine, and catalyzing the migration of hydrogen atoms from δ-carbon to ε-carbon at the same time. It demonstrates greatest catalytic activity in 20mM Tris•HCl at pH 9.0-9.2.
In the early 1950s, 5,6-LAM was discovered in the amino-acid-fermenting bacteria Clostridium sticklandii, in which lysine undergoes degradation under anaerobic conditions to equimolar amounts of acetate and butyrate.
Later, isotopic studies uncovered two possible pathways. In pathway A, both acetate and butyrate are generated from C2-C3 cleavage of D-lysine. Unlike pathway A, pathway B involves C5-C4 degradation, producing the same products.
D-lysine 5,6-aminomutase (5,6-LAM) is responsible for the first conversion in pathway B to convert D-α-lysine into 2,5-diaminohexanoate. Unlike other members of the family of aminomutases (like 2,3-LAM), which are peculiar to a single substrate, 5,6-LAM can reversibly catalyze both the reaction of D-lysine to 2,5-diaminohexanoic acid and the reaction of L-β-lysine to 3,5-diaminohexanoic acid.
Structure
Subunits
5,6-LAM is an α2β2 tetramer. The structure of the alpha subunit is predominantly a PLP-binding TIM barrel domain, with several additional alpha-helices and beta-strands at the N and C termini. These helices and strands form an intertwined accessory clamp structure that wraps around the sides of the TIM barrel and extends up toward the Ado ligand of the Cbl cofactor, which is the beta subunit providing most of the interactions observed between the protein and the Ado ligand of the Cbl, suggesting that its role is mainly in stabilizing AdoCbl in the precatalytic resting state. The β subunit binds AdoCbl while the PLP directly binds to α subunit. PLP also directly binds to Lys144 of the β subunit to form an internal aldimine. PLP and AdoCbl are separated by a distance of 24Å.
Cofactors
5,6-LAM is pyridoxal-5'-phosphate (PLP) dependent. PLP binds to its substrate with an external aldimine linkage. PLP is also important for stabilizing the radical intermediate by captodative stabilization and spin delocalization. Catalysis begins with a 5'-deoxyadenosyl radical (Ado-CH2•), and 5'-deoxyadenosylcobalamin (AdoCbl) is an essential cofactor as a hydrogen carrier. ATP, a mercaptan, and a divalent metal ion (usually Mg2+) are required to achieve the highest catalytic effect.
Mechanism
Catalytic cycle
The catalytic cycle starts with Ado-CH2• (5'-deoxyadenosyl radical derived from adenosylcobalamine) abstracting a hydrogen atom from PLP-D-lysine adduct (substrate-related precursor SH) to generate a substrate-related radical (S•), with the radical located at carbon 5 of the lysine residue. The latter undergoes an internal cyclization/addition to the imine nitrogen producing an aziridinecarbinyl radical (I•) — a more thermodynamically stable intermediate with the radical being at a benzylic position. Rearrangement of I• produces a product-related radical (P•), which then participates in the final step of hydrogen transfer from AdoH to afford the PLP-product complex (PH).
Structure-based catalysis
Further understanding of the catalytic mechanism can be derived from the X-ray structure.
First, an evident conformational change is observed after the substrate is added to the system. With a substrate-free enzyme, the distance between AdoCbl and PLP is about 24 Å. PLP participates in multiple non-covalent interactions with the enzyme with 5,6-LAM presenting an “open” state.