How an Enzyme Tames Reactive Intermediates: Positioning of the Active-Site Components of Lysine 2,3-Aminomutase during Enzymatic Turnover As Determined by ENDOR Spectroscopy

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Abstract
Lysine 2,3-aminomutase (LAM) utilizes a [4Fe-4S] cluster, S-adenosyl-l-methionine (SAM), and pyridoxal 5‘-phosphate (PLP) to isomerize l-α-lysine to l-β-lysine. LAM is a member of the radical-SAM enzyme superfamily in which a [4Fe-4S]+ cluster reductively cleaves SAM to produce the 5‘-deoxyadenosyl radical, which abstracts an H-atom from substrate to form 5‘-deoxyadenosine (5‘-Ado) and the α-Lys• radical (state 3 (Lys•)). This radical isomerizes to the β-Lys• radical (state 4(Lys•)), which then abstracts an H-atom from 5‘-Ado to form β-lysine and the 5‘-deoxyadenosyl radical; the latter then regenerates SAM. We use 13C, 1,2H, 31P, and 14N ENDOR to characterize the active site of LAM in intermediate states that contain the isomeric substrate radicals or analogues. With l-α-lysine as substrate, we monitor the state with β-Lys•. In parallel, we use two substrate analogues that generate stable analogues of the α-Lys• radical: trans-4,5-dehydro-l-lysine (DHLys) and 4-thia-l-lysine (SLys). This first glimpse of the motions of active-site components during catalytic turnover suggests a possible major movement of PLP during catalysis. However, the principal focus of this work is on the relative positions of the carbons involved in H-atom transfer. We conclude that the active site facilitates hydrogen atom transfer by enforcing van der Waals contact between radicals and their reacting partners. This constraint enables the enzyme to minimize and even eliminate side reactions of highly reactive species such as the 5‘-deoxyadensosyl radical.

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