Lysine 2,3-Aminomutase from Clostridium subterminale SB4: Mass Spectral Characterization of Cyanogen Bromide-Treated Peptides and Cloning, Sequencing, and Expression of the Gene kamA in Escherichia coli

Abstract

Lysine 2,3-aminomutase (KAM, EC 5.4.3.2.) catalyzes the interconversion of L-lysine and L-␤-lysine, the first step in lysine degradation in Clostridium subterminale SB4. KAM requires S-adenosylmethionine (SAM), which mediates hydrogen transfer in a mechanism analogous to adenosylcobalamin-dependent reactions. KAM also contains an iron-sulfur cluster and requires pyridoxal 5-phosphate (PLP) for activity. In the present work, we report the cloning and nucleotide sequencing of the gene kamA for C. subterminale SB4 KAM and conditions for its expression in Escherichia coli. The cyanogen bromide peptides were isolated and characterized by mass spectral analysis and, for selected peptides, amino acid and N-terminal amino acid sequence analysis. PCR was performed with degenerate oligonucleotide primers and C. subterminale SB4 chromosomal DNA to produce a portion of kamA containing 1,029 base pairs of the gene. KAM from Clostridium subterminale SB4 catalyzes the interconversion of L-lysine and L-␤-lysine. In C. subterminale SB4, the production of ␤-lysine is the first step in the metabolism of lysine as the sole source of carbon and nitrogen, leading to acetate and butyrate as final products (39). Unlike other aminomutases, including D-lysine 5,6-aminomutase (1, 26), D-ornithine 2,3-aminomutase (2), and L-leucine 2,3-aminomutase (33), KAM is not adenosylcobalamin dependent. Instead, the enzyme is activated by SAM and contains iron-sulfur clusters and PLP (7,31,38).The interconversion of L-␣-lysine and L-␤-lysine is a reversible process in which an unactivated, carbon-bound hydrogen atom undergoes a 1,2-migration concomitant with the countermigration of the ␣-amino group by a novel free-radicalbased mechanism (12, 13). According to the current working hypothesis, homolytic cleavage of the S-adenosyl bond in SAM is mediated by the [4Fe-4S] center and produces the 5Ј-deoxyadenosyl radical as a transient intermediate (27,28). As illustrated in Fig. 1, the 5Ј-deoxyadenosyl radical initiates the rearrangement by abstracting the 3-pro-R hydrogen of L-lysine in the forward direction to form radical 1 or the 2-pro-R hydrogen of L-␤-lysine in the reverse direction to form radical 3.Evidence suggests that both amino acids are bound to the enzyme in the form of external aldimines with PLP. The substrate-and product-related free radicals are interconverted by way of an azacyclopropylcarbinyl, radical intermediate 2.KAM was first observed by Costilow and coworkers in crude extracts of C. subterminale SB4 (8) and later was purified and found to contain PLP and iron and to require SAM for activity (7). Enzyme activity was rapidly destroyed by dioxygen. The purified enzyme was partially inactive and could be further activated by prolonged anaerobic incubation with PLP, iron, and glutathione or dihydrolipoate, followed by addition of SAM and dithionite (7). KAM is a multisubunit protein with a subunit M r of 48,000 and an overall M r of 285,000 (7,38). Later studies demonstrated the presence of labile sulfide in the form of three [4Fe-4S]...

Lysine-2,3-Aminomutase and β-Lysine Acetyltransferase Genes of Methanogenic Archaea Are Salt Induced and Are Essential for the Biosynthesis of N ε -Acetyl-β-Lysine and Growth at High Salinity

“…This region is found in Kam (37) and AblA. Zinc is essential for the activity of Kam, and another group of three cysteines near the C terminus of Kam is suggested to present the zinc binding site (37). These three cysteines are conserved in AblA as well.…”

“…Taken together, these data indicate that ablA of M. mazei Gö1 codes for an aminomutase very similar to Kam of C. subterminale. Therefore, the mechanism for the conversion of ␣-lysine to ␤-lysine is suggested to be identical to that of Kam of C. subterminale (10) and other aminomutases (37).…”

“…␣-Lysine is proposed to be converted to N ε -acetyl-␤-lysine by the action of two enzymes, a lysine-2,3-aminomutase and an acetyltransferase (31,33). To identify the encoding genes, we did BLAST searches on the genome of M. mazei Gö1 with the lysine-2,3-aminomutase sequence from Clostridium subterminale as the query sequence (37). By using this approach, an open reading frame termed ablA whose product was very similar to the lysine-2,3-aminomutase of C. subterminale (55.8% identity) was identified.…”

Identification of the Last Unknown Genes in the Fermentation Pathway of Lysine

“…The first two steps are catalyzed by lysine-2,3-aminomutase and ␤-lysine-5,6-aminomutase. Both enzymes have been biochemically characterized (3), and their corresponding genes have been identified (4,5).…”

Basis for the equilibrium constant in the interconversion of l-lysine and l-β-lysine by lysine 2,3-Aminomutase

“…Recombinant lysine 2, 3-aminomutase was expressed and purified under anaerobic conditions as described previously (15,16). Enzyme was activated and assayed using a radiochemical method as described (17,18).…”