Lysine 2,3-aminomutase and trans-4,5-dehydrolysine: characterization of an allylic analogue of a substrate-based radical in the catalytic mechanism.

An analogue of lysine, trans-4,5-dehydro-L-lysine (trans-4, 5-dehydrolysine), is a potent inhibitor of lysine 2,3-aminomutase from Clostridium subterminale SB4 that competes with L-lysine for binding to the active site. Inclusion of trans-4,5-dehydrolysine with activated enzyme and the coenzymes pyridoxal-5'-phosphate and S-adenosylmethionine, followed by freezing at 77 K, produces an intense signal in the electron paramagnetic resonance (EPR) spectrum at g 2.0, which is characteristic of an organic radical. A series of deuterated and (15)N-labeled samples of trans-4,5-dehydrolysine were synthesized and used to generate the EPR signal. Substitution of deuterium for hydrogen at C2, C3, C4, C5, and C6 of trans-4, 5-dehydrolysine led to significant simplifications and narrowing of the EPR signal, showing that the unpaired electron was located on the carbon skeleton of 4,5-trans-4,5-dehydrolysine. The hyperfine splitting pattern is simplified by use of 4,5-dehydro[3, 3-(2)H(2)]lysine or 4,5-dehydro[4,5-(2)H(2)]lysine, and it is dramatically simplified with 4,5-dehydro-[3,3,4,5,6,6-(2)H(6)]lysine. Spectral simulations show that the EPR signal arises from the allylic radical resulting from the abstraction of a hydrogen atom from C3 of trans-4,5-dehydrolysine. This radical is an allylic analogue of the substrate-related radical in the rearrangement mechanism postulated for this enzyme. The rate constant for formation of the 4,5-dehydrolysyl radical (2 min(-)(1)) matches that for the decrease in the concentration of [4Fe-4S](+), showing that the two processes are coupled. The cleavage of S-adenosylmethionine to 5'-deoxyadenosine and methionine takes place with a rate constant of approximately 5 min(-)(1). These kinetic correlations support the hypothesis that radical formation results from a reversible reaction between [4Fe-4S](+) and S-adenosylmethionine at the active site to form [4Fe-4S](2+), the 5'-deoxyadenosyl radical, and methionine as intermediates.

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.

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.

Large-scale SNP scoring from unamplified genomic DNA.

Discoveries from the Human Genome Project (HGP) continue to spur changes in medical technology that will lead to new diagnostic procedures in the clinical lab. As more single nucleotide polymorphisms (SNPs) are discovered and correlated to human diseases, demands for genetic tests will increase. The enormity of the number of SNPs makes developing inexpensive and reliable high-throughput methods for SNP scoring imperative. High-throughput screening (HTS) means, at a minimum, a production rate of thousands of assays per day. Ideally, the technology will be easy, inexpensive and amenable to automation. The Invader assay offers a simple diagnostic platform to detect single nucleotide changes with high specificity and sensitivity from unamplified, genomic DNA. The Invader assay uses a structure-specific 5' nuclease (or flap endonuclease) to cleave sequence-specific structures in each of two cascading reactions. The cleavage structure forms when two synthetic oligonucleotide probes hybridise in tandem to a target. One of the probes cycles on and off the target and is cut by the nuclease only when the appropriate structure forms. These cleaved probes then participate in a second Invader reaction involving a dye-labelled fluorescence resonance energy transfer (FRET) probe. Cleavage of this FRET probe generates a signal, which can be readily analysed by fluorescence microtitre plate readers. The two cascading reactions amplify the signal significantly; each original target molecule can lead to more than 10(6) cleaved signal probes in one hour. This signal amplification permits identification of single base changes directly from genomic DNA without prior target amplification. The sequences of the oligonucleotide components of the secondary reaction are independent of the target of interest and permit the development of universal secondary reaction components useful to identify any target.

S-Adenosylmethionine-dependent reduction of lysine 2,3-aminomutase and observation of the catalytically functional iron-sulfur centers by electron paramagnetic resonance.

Lysine 2,3-aminomutase catalyzes the interconversion of l-alpha-lysine and l-beta-lysine. The enzyme contains an iron-sulfur cluster with unusual properties, and it requires pyridoxal-5'-phosphate (PLP) and S-adenosylmethionine (AdoMet) for activity. The reaction proceeds by a substrate radical rearrangement mechanism, in which the external aldimine formed between PLP and lysine is initially converted into a lysyl-radical intermediate by hydrogen abstraction from C3. The present research concerns the mechanism by which a hydrogen-abstracting species is generated at the active site of lysine 2,3-aminomutase. Earlier tritium tracer experiments have implicated the 5'-deoxyadenosyl moiety of AdoMet in this process. AdoMet is here shown to interact with the iron-sulfur cluster at the active site of Clostridial lysine 2,3-aminomutase. Reduction of the iron-sulfur cluster from its EPR-silent form [4Fe-4S]2+ to the fully reduced form [4Fe-4S]1+ requires the presence of either AdoMet or S-adenosylhomocysteine (SAH) and a strong reducing agent such as dithionite or deazariboflavin and light. The reduced forms are provisionally designated E-[4Fe-4S]1+/AdoMet and E-[4Fe-4S]1+/SAH, and they display similar low-temperature EPR spectra centered at gav = 1.91. The reduced form E-[4Fe-4S]1+/AdoMet is fully active in the absence of any added reducing agent, whereas the form E-[4Fe-4S]1+/SAH is not active. It is postulated that the active form E-[4Fe-4S]1+/AdoMet is in equilibrium with a low concentration of a radical-initiating form that contains the 5'-deoxyadenosyl radical. Initiation of the radical rearrangement mechanism is postulated to take place by action of the 5'-deoxyadenosyl radical in abstracting a hydrogen atom from carbon-3 of lysine, which is bound as its external aldiminine with PLP. This process accounts for the results of tritium tracer experiments, it explains the radical rearrangement mechanism, and it rationalizes the roles of AdoMet and the [4Fe-4S] cluster in the reaction.

Observation of a second substrate radical intermediate in the reaction of lysine 2,3-aminomutase: a radical centered on the beta-carbon of the alternative substrate, 4-thia-L-lysine.

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of lysine and beta-lysine by a mechanism in which four organic radicals are postulated as intermediates. One of the intermediates has been identified as the alpha-radical of beta-lysine in imine linkage to pyridoxal phosphate (PLP) [Ballinger, M. D., Frey, P. A., & Reed, G. H. (1992) Biochemistry 31, 10782-10788]. We report here the observation of another of the four putative radical intermediates in the reaction of the alternative substrate, 4-thia-L-lysine (S-2-aminoethyl-L-cysteine). 4-Thialysine is a substrate for lysine 2,3-aminomutase. The Km of 4-thialysine is similar to that for lysine, and the Vm is approximately 3% of that for lysine. Upon mixing 4-thialysine with the activated enzyme in the presence of the required cofactor S-adenosylmethionine, followed by freeze-quenching with liquid N2 in the steady state, a strong EPR signal centered at g = 2.003 is observed. This signal exhibits strong hyperfine splitting due to the presence of 13C at carbon-3 of 4-thialysine, and the EPR pattern is narrowed upon the substitution of deuterium at carbon-3. The hyperfine interactions show that the unpaired electron is centered on carbon-3 of 4-thialysine. The hyperfine pattern in the EPR spectrum is also simplified by the use of 4-thia[5,6-2H4]lysine as the substrate, showing either that the spin is partially delocalized through the sulfur intervening between carbons-3 and -5 or that the conformation is such that protons at carbon-6 are close to carbon-3.(ABSTRACT TRUNCATED AT 250 WORDS)