title: 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-beta-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. The complete gene was obtained from a genomic library of C. subterminale SB4 chromosomal DNA by use of DNA probe analysis based on the 1,029-base pair fragment. The full-length gene consisted of 1,251 base pairs specifying a protein of 47,030 Da, in reasonable agreement with 47, 173 Da obtained by electrospray mass spectrometry of the purified enzyme. N- and C-terminal amino acid analysis of KAM and its cyanogen bromide peptides firmly correlated its amino acid sequence with the nucleotide sequence of kamA. A survey of bacterial genome databases identified seven homologs with 31 to 72% sequence identity to KAM, none of which were known enzymes. An E. coli expression system consisting of pET 23a(+) plus kamA yielded unsatisfactory expression and bacterial growth. Codon usage in kamA includes the use of AGA for all 29 arginine residues. AGA is rarely used in E. coli, and arginine clusters at positions 4 and 5, 25 and 27, and 134, 135, and 136 apparently compound the barrier to expression. Coexpression of E. coli argU dramatically enhanced both cell growth and expression of KAM. Purified recombinant KAM is equivalent to that purified from C. subterminale SB4.
authors.affiliation: Institute for Enzyme Research, The Graduate School, Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.
authors.forename: F J
authors.initials: FJ
authors.lastname: Ruzicka
authors.forename: K W
authors.initials: KW
authors.lastname: Lieder
authors.forename: P A
authors.initials: PA
authors.lastname: Frey
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D003488:Cyanogen Bromide / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D019751:Intramolecular Transferases / Q000235:genetics* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry*; D012347:RNA, Transfer, Arg / Q000235:genetics; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D010455:Peptides; D012347:RNA, Transfer, Arg; D019751:Intramolecular Transferases; C021518:lysine 2,3-aminomutase; D003488:Cyanogen Bromide