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A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase.

ABSTRACT: Lysine 5,6-aminomutase is an adenosylcobalamin and pyridoxal-5'-phosphate-dependent enzyme that catalyzes a 1,2 rearrangement of the terminal amino group of dl-lysine and of l-beta-lysine. We have solved the x-ray structure of a substrate-free form of lysine-5,6-aminomutase from Clostridium sticklandii. In this structure, a Rossmann domain covalently binds pyridoxal-5'-phosphate by means of lysine 144 and positions it into the putative active site of a neighboring triosephosphate isomerase barrel domain, while simultaneously positioning the other cofactor, adenosylcobalamin, approximately 25 A from the active site. In this mode of pyridoxal-5'-phosphate binding, the cofactor acts as an anchor, tethering the separate polypeptide chain of the Rossmann domain to the triosephosphate isomerase barrel domain. Upon substrate binding and transaldimination of the lysine-144 linkage, the Rossmann domain would be free to rotate and bring adenosylcobalamin, pyridoxal-5'-phosphate, and substrate into proximity. Thus, the structure embodies a locking mechanism to keep the adenosylcobalamin out of the active site and prevent radical generation in the absence of substrate.

SUBMITTER: Berkovitch F

PROVIDER: S-EPMC528771 | biostudies-literature | 2004 Nov

REPOSITORIES: biostudies-literature

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A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase.

Berkovitch Frederick F Behshad Elham E Tang Kuo-Hsiang KH Enns Eva A EA Frey Perry A PA Drennan Catherine L CL

Proceedings of the National Academy of Sciences of the United States of America 20041028 45

Lysine 5,6-aminomutase is an adenosylcobalamin and pyridoxal-5'-phosphate-dependent enzyme that catalyzes a 1,2 rearrangement of the terminal amino group of dl-lysine and of l-beta-lysine. We have solved the x-ray structure of a substrate-free form of lysine-5,6-aminomutase from Clostridium sticklandii. In this structure, a Rossmann domain covalently binds pyridoxal-5'-phosphate by means of lysine 144 and positions it into the putative active site of a neighboring triosephosphate isomerase barre ...[more]

PMID: 15514022

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Project description:Lysine 5,6-aminomutase (5,6-LAM) catalyzes the interconversions of D- or L-lysine and the corresponding enantiomers of 2,5-diaminohexanoate, as well as the interconversion of L-beta-lysine and l-3,5-diaminohexanoate. The reactions of 5,6-LAM are 5'-deoxyadenosylcobalamin- and pyridoxal-5'-phosphate (PLP)-dependent. Similar to other 5'-deoxyadenosylcobalamin-dependent enzymes, 5,6-LAM is thought to function by a radical mechanism. No free radicals can be detected by electron paramagnetic resonance (EPR) spectroscopy in reactions of 5,6-LAM with D- or L-lysine or with L-beta-lysine. However, the substrate analogues 4-thia-L-lysine and 4-thia-D-lysine undergo early steps in the mechanism to form two radical species that are readily detected by EPR spectroscopy. Cob(II)alamin and 5'-deoxyadenosine derived from 5'-deoxyadenosylcobalamin are also detected. The radicals are proximal to and spin-coupled with low-spin Co(2+) in cob(II)alamin and appear as radical triplets. The radicals are reversibly formed but do not proceed to stable products, so that 4-thia-D- and L-lysine are suicide inhibitors. Inhibition attains equilibrium between the active Michaelis complex and the inhibited radical triplets. The structure of the transient 4-thia-L-lysine radical is analogous to that of the first substrate-related radical in the putative isomerization mechanism. The second, persistent radical is more stable than the transient species and is assigned as a tautomer, in which a C6(H) of the transient radical is transferred to the carboxaldehyde carbon (C4') of PLP. The persistent radical blocks the active site and inhibits the enzyme, but it decomposes very slowly at </=1% of the rate of formation to regenerate the active enzyme. Fundamental differences between reversible suicide inactivation by 4-thia-D- or L-4-lysine and irreversible suicide inactivation by D- or L-lysine are discussed. The observation of the transient radical supports the hypothetical isomerization mechanism.

Project description:The yodO gene product of Bacillus subtilis has been cloned and overexpressed in Escherichia coli and purified. The nucleotide sequence encodes a protein of 471 amino acids with a calculated molecular mass of 54071 Da. The translated amino acid sequence is more than 60% identical to that of the lysine 2,3-aminomutase from Clostridium subterminale SB4. Analytical HPLC gel-permeation chromatography leads to an estimate of an over all molecular mass of 224000+/-21000 Da, which corresponds to a tetrameric protein. The purified protein contains iron, sulphide and pyridoxal 5'-phosphate (PLP) and displays an optical absorption band extending to 700 nm, suggesting the presence of an iron-sulphide cluster. After reductive incubation with L-cysteine anaerobically, the protein catalyses the transformation of L-lysine into beta-lysine in the presence of S-adenosylmethionine (AdoMet) and sodium dithionite. The K(m) value for L-lysine is estimated to be 8.0+/-2.2 mM. The iron-sulphur centre is stable in air,allowing aerobic purification. EPR spectroscopy at 10 K of the purified enzyme revealed an EPR signal similar to that of the [4Fe-4S](3+) cluster observed in the clostridial lysine 2, 3-aminomutase. Incubation with cysteine under anaerobic conditions converts the iron-sulphur centre into the EPR-silent [4Fe-4S](2+). Unlike the clostridial enzyme, the fully reduced [4Fe-4S](+) could not be characterized by further reduction with dithionite in the presence of AdoMet, although both dithionite and AdoMet were required to activate the enzyme. Upon addition of L-lysine, dithionite and AdoMet to the reduced enzyme and freezing the solution to 77 K, the EPR spectrum revealed the presence of an organic free-radical signal (g=2.0023), which displayed multiple hyperfine transitions very similar to the spectrum of the beta-lysine-related radical in the mechanism of the clostridial lysine 2,3-aminomutase. Experiments with isotopically substituted L-lysine and lysine analogues verified the association of spin density with the carbon skeleton of lysine. The data indicate that the protein encoded by the yodO gene of B. subtilis is a novel lysine 2,3-aminomutase. The E. coli homologue of clostridial lysine 2,3-aminomutase was also expressed in E. coli and purified. This protein contained ironand sulphide but not PLP, it did not display lysine 2,3-aminomutase activity, and addition of PLP did not induce 2,3-aminomutase activity.

Dataset Information

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase.

Publications

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase.

Similar Datasets

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