Mechanistic and Spectroscopic Studies of Lysine 2,3-Aminomutase

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine to L-β-lysine using a [4Fe4S] cluster, S-adenosyl-L-methionine (SAM), and pyridoxal 5’-phosphate (PLP). LAM is a member of the radicalSAM superfamily of proteins which use iron-sulfur clusters and SAM to initiate H atom abstraction reactions. Included in this unusual chemistry is the reductive cleavage of SAM to generate the highly reactive 5’deoxyadenosyl radical species. This paper will focus on how physical methods, including X-ray absorption spectroscopy (XAS), electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopies, have provided important information about the structure and mechanism of this remarkable enzyme. Introduction: The primary function of iron sulfur clusters is electron transport, as most types of iron sulfur clusters possess at least two readily accessible redox states. One of the most interesting roles of iron sulfur clusters is their role in radical catalysis in the radical SAM superfamily. This protein superfamily is proposed to contain over 600 different enzymes and includes proteins such as lysine 2,3-aminomutase (LAM), biotin synthase (BioB), lipoate synthase (LipA), pyruvate formate lyase activating enzyme (PFL-AE), anaerobic nucleotide reductase activating enzyme (anRNR), and spore photoproduct lyase (SPL). One of the characteristics of the radical SAM superfamily is a conserved cysteine motif, CX3CX2C, which coordinates an iron-sulfur cluster. With only 3 cysteinal ligands that coordinate the iron sulfur cluster, these proteins can coordinate either a [3Fe-4S] cluster or a [4Fe-4S] cluster containing one non-cysteinal ligand. The iron sulfur clusters of these enzymes are oxygen sensitive and most are purified under strict anaerobic conditions to maintain activity. These enzymes use S-adenosylmethionine (SAM) to catalyze reactions involving radical intermediates. LAM was first purified and characterized from Clostridium subterminale SB4 to study lysine metabolism in Clostridia. Lysine 2,3-aminomutase catalyzes the interconversion of L-αlysine and L-β-lysine. This is the initial step in the catabolism of the amino acid to acetyl-CoA and ammonia, which are usable carbon and nitrogen sources for the bacterium. LAM is a homohexamer (MW 285 kDa) and functions by a r closely resembles those catalyzed by adenosylcobalamin-dependent reactions activated by AdoCbl, is the participation of the 5’-deoxya initiates free radical formation. However, in LAM, S-adenosylmethi deoxyadenosyl that acts in mediating hydrogen transfer from carbon-3 to initiator occurs from a reversible chemical reaction between SAM and leading to the cleavage of the C5’-S bond in SAM. Spectroscopic re results from reductive cleavage of SAM through electron transfer from leading to the [4Fe-4S] cluster and the 5’deoxyadenosyl radical. The catalytic mechanism of LAM is shown in Scheme 1. The ly in an imine linkage with pyridoxal 5’-phosphate (PLP) and isomer abstraction of the 3-pro-R hydrogen of lysine by the 5’deoxyadenosyl rad substrate radical of α-lysine (radical 1). Proceeding through an aza-cycl intermediate (radical 2), the α-lysine-3-yl radical rearranges to the β-lys 3). Finally, the 5’deoxyadenosine donates a methyl hydrogen atom ba (1) adical mechanism that enzymes. Similar to denosyl radical, which onine supplies the 5’carbon-2. This radical the [4Fe-4S] cluster, sults indicate that this the [4Fe-4S] cluster, sine substrate is bound ization begins by the ical. This produces the opropylcarbinyl radical ine-2-yl radical (radical ck to the β-lysine-2-yl radical and the pyridoxal-5’-phosphate imine of β-lysine is formed. The regenerated 5’deoxyadenosyl radical is either ready for the next turnover or recombines with methionine coordinated to the [4Fe-4S] cluster to reform SAM.