Enantiomeric free radicals and enzymatic control of stereochemistry in a radical mechanism: the case of lysine 2,3-aminomutases - PubMed (original) (raw)

Enantiomeric free radicals and enzymatic control of stereochemistry in a radical mechanism: the case of lysine 2,3-aminomutases

E Behshad et al. Biochemistry. 2006.

Abstract

The product of yjeK in Escherichia coli is a homologue of lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4, and both enzymes catalyze the isomerization of (S)- but not (R)-alpha-lysine by radical mechanisms. The turnover number for LAM from E. coli is 5.0 min(-1), 0.1% of the value for clostridial LAM. The reaction of E. coli LAM with (S)-alpha-[3,3,4,4,5,5,6,6-(2)H8]lysine proceeds with a kinetic isotope effect (kH/kD) of 1.4, suggesting that hydrogen transfer is not rate-limiting. The product of the E. coli enzyme is (R)-beta-lysine, the enantiomer of the clostridial product. Beta-lysine-related radicals are observed in the reactions of both enzymes by electron paramagnetic resonance (EPR). The radical in the reaction of clostridial LAM has the (S)-configuration, whereas that in the reaction of E. coli LAM has the (R)-configuration. Moreover, the conformations of the beta-lysine-related radicals at the active sites of E. coli and clostridial LAM are different. The nuclear hyperfine splitting between the C3 hydrogen and the unpaired electron at C2 shows the dihedral angle to be 6 degrees, unlike the value of 77 degrees reported for the analogous radical bound to the clostridial enzyme. Reaction of (S)-4-thialysine produces a substrate-related radical in the steady state of E. coli LAM, as in the action of the clostridial enzyme. While (S)-beta-lysine is not a substrate for E. coli LAM, it undergoes hydrogen abstraction to form an (S)-beta-lysine-related radical with the same stereochemistry of hydrogen transfer from C2 of (S)-beta-lysine to the 5'-deoxyadenosyl radical as in the action of the clostridial enzyme. The resulting beta-lysyl radical has a conformation different from that at the active site of clostridial LAM. All evidence indicates that the opposite stereochemistry displayed by E. coli LAM is determined by the conformation of the lysine side chain in the active site. Stereochemical models for the actions of LAM from C. subterminale and E. coli are presented.

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Figures

Fig. 1

UV circular dichroism spectra for PITC derivatives of (S)-β-lysine (—) and (R)-β-lysine produced in the reaction of E. coli LAM (–X–X–).

Fig. 2. EPR spectra of the radical from (S)-α-lysine at the active site of E. coli LAM

The EPR samples were prepared under anaerobic conditions with 200 μM E. coli LAM, 1.1 mM SAM, 2.5 mM dithionite, and 200 mM EPPS buffer at pH 8.0. The EPR spectra were recorded at 77K. A, 40 mM α-(S)-lysine; B, 40 mM (S)-α-[3,3,4,4,5,5,6,6-2H8]lysine; C, 40 mM (S)-α-[2-2H]lysine; D, 40 mM [2-13C]lysine. The spectra show that the dominant radical in the steady state is the β-lysyl radical, 3 in Scheme 1.

Figure 3. Simulated EPR spectra of β-lysyl radicals at the active site of E. coli LAM

Experimental and simulated spectra of the β-lysyl radical are shown for the species generated with (S)-α-[2-2H]lysine, (RS)-α-[2-15N,2,3,3,4,4,5,5,6,6-2H9]lysine and unlabeled (S)-α-lysine in the active site of E. coli LAM. A. The spectrum (—) and simulation (- - -) of the radical generated upon addition of 40 mM (S)-α-lysine to 180 μM enzyme. B. The spectrum (—) and simulation (- - -) of the radical generated upon addition of 40 mM (S)-α-[2-2H]lysine. The samples were frozen within 30–40 seconds after addition of substrate. C. The spectrum (—) and simulation (- - -) of the radical generated upon addition of 80 mM (RS)-α-[2-15N,- 2,3,3,4,4,5,5,6,6-2H9]lysine. Parameters used for the calculation of simulated spectra are given in Table 1.

Figure 4. EPR spectra of the radical induced at the active site of E. coli LAM by (S)-4- thialysine

The spectrum (—) is generated upon addition of 80 mM (RS)-4-thialysine and the spectrum (- - -) is the radical formed upon addition of 80 mM (RS)-4-thia[3,3-2H2]lysine. The samples contain 2.5 mM dithionite, 1.5 mM SAM, and 200 mM EPPS at pH 8. Samples were frozen within one minute after addition of the substrate. The spectra indicate that the dominant radical in the steady state is the 4-thia analogue of radical 1 in Scheme 1.

Figure 5. EPR spectra of the (S)-β-lysyl radical at the active site of E. coli LAM

A. The radical generated upon addition of 70 mM (S)-β-lysine. B. Radical generated upon addition of 27 mM (S)-β-[2-13C]lysine. EPR samples were prepared with 200 μM enzyme, 2.5 mM dithionite, and 1.3 mM SAM in 200 mM EPPS at pH 8.0 and were frozen with 20–25 seconds after addition of substrate. C. The radical formed upon addition of 1 mM (S)-β-[2-2H]lysine. The samples contain 40 μM enzyme, 2.5 mM dithionite, 0.3 mM SAM, and 200 mM EPPS at pH 8.0 and were frozen within 20 seconds after addition of the substrate. The modulation amplitude was set at 8 G.

Figure 6. Effects of [5′-13C]SAM and [5′-2H2]SAM on the EPR spectra of β-lysyl radicals at the active site of E. coli LAM

A. The upper spectra are of the (R)-β-lysyl radical formed with (S)-α-lysine in the presence of SAM (—) or of [5′-2H2]SAM (- - -). The lower spectra are of the (S)-β-lysyl radical formed with (S)-β-lysine in the presence of SAM (—) or [5′-2H2]SAM (- - -). B. The upper spectra are of the (R)-β-lysyl radical formed with (S)-α-lysine in the presence of SAM (—) or of [5′-13C]SAM (- - -). The lower spectra are of the (S)-β-lysyl radical formed with (Σ)-β-lysine in the presence of SAM (—) or [5′-13C]SAM (- - -).

Figure 6. Effects of [5′-13C]SAM and [5′-2H2]SAM on the EPR spectra of β-lysyl radicals at the active site of E. coli LAM

Fig. 7. Stereochemical models for the mechanisms of reactions of LAM from C. subterminale and E. coli

The sequence on the left depicts the mechanism and stereochemistry for the reaction of C. subterminale LAM, and that on the right depicts the hypothetical mechanism and stereochemistry of the action of E. coli LAM. Available evidence indicates that the basic chemistry is the same for the two enzymes, but the stereochemistry is different for the initial hydrogen abstraction by the 5′-deoxyadenosyl radical and cyclization to the azacyclopropylcarbinyl radical intermediate. Stereochemistry of hydrogen transfer to C2 of β-lysine is postulated, but not proven, to be the same for the two enzymes.

Scheme 1

Scheme 2

References

1. Ruzicka FJ, Lieder KW, Frey PA. 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. J Bacteriol. 2000;182:469–76. - PMC - PubMed
1. Chirpich TP, Zappia V, Costilow RN, Barker HA. Lysine 2,3-aminomutase. Purification and properties of a pyridoxal phosphate and S-adenosylmethionine-activated enzyme. J Biol Chem. 1970;245:1778–1789. - PubMed
1. Aberhart DJ, Gould SJ, Lin HJ, Thiruvengadam TK, Weiller BH. Stereochemistry of Lysine 2,3-Aminomutase Isolated from Clostridium subterminale Strain SB4. J Am Chem Soc. 1983;105:5461–5470.
1. Grammel N, Pankevych K, Demydchuk J, Lambrecht K, Saluz HP, Krugel H. A beta-lysine adenylating enzyme and a beta-lysine binding protein involved in poly beta-lysine chain assembly in nourseothricin synthesis in Streptomyces noursei. Eur J Biochem. 2002;269:347–357. - PubMed
1. Singh K, Sehgal SN, Rakhit S, Vezina C. Isolation of a Streptomyces rochei idiotroph requiring beta-lysine for production of streptothricin. J Antibiot (Tokyo) 1983;36:1770–1773. - PubMed

Enantiomeric free radicals and enzymatic control of stereochemistry in a radical mechanism: the case of lysine 2,3-aminomutases - PubMed (original) (raw)