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Background

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Two pathways of D-lysine degradation

This enzyme belongs to the family of isomerases, specifically those intramolecular transferases transferring amino groups. The systematic name of this enzyme class is D-2,6-diaminohexanoate 5,6-aminomutase. Other names in common use include D-α-lysine mutase and adenosylcobalamin-dependent D-lysine 5,6-aminomutase, which can be abbreviated as 5,6-LAM.

 
Mutase reaction of 5,6-LAM

5,6-LAM is capable of reversibly catalyzing the migration of amino group from ε-carbon to δ-carbon in both D-lysine and L-β-lysine, and migrating hydrogen atoms from δ-carbon to ε-carbon at the same time.[1]

And it shows the best catalytic activity in 20mM Tris•HCl with pH around 9.0-9.2.[2]

In the early 1950's, 5,6-LAM was discovered that under anaerobic conditions lysine undergoes degradation to equimolar amounts of acetate and butyrate by an amino-acid-fermenting bacteria Clostridium sticklandii.[3]

Later by implementing isotopic studies two possible pathways were discovered. In pathway A both acetate and butyrate are generated from C2-C3 cleavage of D-lysine. Unlike pathway A, pathway B involves C5-C4 degradation producing the same products.

D-lysine 5,6-aminomutase (5,6-LAM) is responsible for the first conversion in pathway B to turn D-α-lysine into 2,5-diaminohexanoate. Unlike other members of the family of aminomutases (like 2,3-LAM) who are peculiar to a single substrate, 5,6-LAM can reversibly catalyze both the reaction of D-lysine to 2,5-diaminohexanoic acid and the reaction of L-β-lysinene to 3,5-diaminohexanoic acid.[3][4]

Structure

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Two units of 5,6-LAM (AdoCbl in yellow and PLP in orange)

Two subunits

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5,6-LAM is an α2β2 tetramer. The structure of the alpha subunit is predominantly a PLP-binding TIM barrel domain, with several additional alpha-helices and beta-strands at the N and C termini. These helices and strands form an intertwined accessory clamp structure that wraps around the sides of the TIM barrel and extends up toward the Ado ligand of the Cbl cofactor, which is the beta subunit providing most of the interactions observed between the protein and the Ado ligand of the Cbl, suggesting that its role is mainly in stabilising AdoCbl in the precatalytic resting state[5].The β subunit binds AdoCbl while the PLP directly binds to the α subunit. PLP also directly binds to Lys144 of the β subunit to form an internal aldimine. PLP and AdoCbl are about 24 Å away from each other.[6]

Cofactors

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  1. 5,6-LAM is pyridoxal-5'-phosphate (PLP) dependent and PLP binds to substrate with an external aldimine linkage. PLP is also important to stabilize the radical intermediate by captodative stabilization and spin delocalization.[7]
  2. Catalysis begins with 5'-deoxyadenosyl radical (Ado•) and 5'-deoxyadenosylcobalamin (AdoCbl) is an essential cofactor as a hydrogen carrier.[8]
  3. ATP, a mercaptan, and a divalent metal ion (usually Mg2+) are required to achieve the highest catalytic effect.[4]

Mechanism

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Proposed Mechanism of 5,6-LAM

Catalytic Cycle

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The catalytic cycle starts with Ado-CH2• (5'-deoxyadenosyl radical derived from adenosylcobalamine) abstracting a hydrogen atom from PLP-D-lysine adduct (substrate-related precursor SH) to generate a substrate-related radical (S•), with the radical located at carbon 5 of the lysine residue. The latter undergoes an internal cyclization/addition to the imine nitrogen producing an aziridinecarbinyl radical (I•) — a more thermodynamically stable intermediate with the radical being at a benzylic position. Rearrangement of I• produces a product-related radical (P•), which then participates in the final step of hydrogen transfer from AdoH to afford the PLP-product complex (PH).[9]

Structure-Based Catalysis

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Further understanding of the catalytic mechanism can be derived from the X-ray structure.

 
PLP (in green) maintains much interaction with enzyme in open state

First, an evident conformational change is observed after the substrate is added to the system. With a substrate-free enzyme the distance between AdoCbl and PLP is about 24 Å and PLP participates in multiple non-covalent interactions with the enzyme with 5,6-LAM presenting an “open” state.

The first step of the catalytic cycle is the enzyme accepting the substrate by forming an external aldimine with PLP replacing the PLP-Lys144β internal aldimine. With the cleavage of the internal aldimine, the β unit is able to swing towards to the top of the α unit and block the empty site. Therefore, generation of Ado-CH2• radical leads to a change in the structure of the active domain bringing AdoCbl and PLP-substrate complex closer to each other, locking the enzyme in a “closed” state. The closed state exists until the radical transfer occurs when the product is released and AdoCbl is reformed and at the same time, the closed state is transformed to the open state again to wait for the next substrate.[10]

What’s also worth mentioning is the locking mechanism to prevent radical reaction without the presence of substrate discovered by Catherine Drennan’s group. Lys144 β residue is located at a short G-rich loop highly conserved across all 5,6-LAMs, which blocks the AdoCbl from the reaction site. Based on the X-ray structure analysis, when the open structure is applied, the axes of the TIM barrel and Rossmann domains are in different directions and with the addition of the substrate, the subunits rearrange to turn the axes into each other to facilitate the catalysis.[11]

For example, in wild type 5,6-LAM, the phenol ring of Tyr263α is oriented in a slipped geometry with pyridine ring of PLP, generating a π-π stacking interaction, which is capable of modulating the electron distribution of the high-energetic radical intermediate.[12]

Mechanism research history

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Early insights into the mechanism of the catalytic reaction mainly focused on isotopic methods. Both pathways of lysine degradation and the role of 5,6-LAM were discovered in early works by Stadtman’s group during 1950s-1960s. In 1971 having a tritiated α-lysine, 2,5-diaminohexanoate and coenzyme in hand, Colin Morley and T. Stadtman discovered the role of 5'-deoxyadenosylcobalamin (AdoCbl) as a source for hydrogen migration[8]. Recently, a lot of progress has been made towards detecting the intermediates of the reaction, especially towards I•. Based on quantum-mechanical calculations, it was proposed that with 5-fluorolysine[9] as a substitute for D-lysine the 5-FS• species can be captured and analyzed. A similar approach was applied towards PLP modification, when it was modified to 4’-cyanoPLP[13] or PLP-NO[14] and the radical intermediate I• analogue is hypothesized to be easily detected to support the proposed mechanism. Other simulations can also provide some insights into the catalytic reaction[15]. 

Reference

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  1. ^ Sandala, Gregory M.; Smith, David M.; Radom, Leo (2006-12-01). "In Search of Radical Intermediates in the Reactions Catalyzed by Lysine 2,3-Aminomutase and Lysine 5,6-Aminomutase". Journal of the American Chemical Society. 128 (50): 16004–16005. doi:10.1021/ja0668421. ISSN 0002-7863.
  2. ^ Morley, C. G.; Stadtman, T. C. (1970-12-08). "Studies on the fermentation of D-alpha-lysine. Purification and properties of an adenosine triphosphate regulated B 12-coenzyme-dependent D-alpha-lysine mutase complex from Clostridium sticklandii". Biochemistry. 9 (25): 4890–4900. ISSN 0006-2960. PMID 5480154.
  3. ^ a b Stadtman, Thressa C.; White, F. H. (1954-06-01). "TRACER STUDIES ON ORNITHINE, LYSINE, AND FORMATE METABOLISM IN AN AMINO ACID FERMENTING CLOSTRIDIUM". Journal of Bacteriology. 67 (6): 651–657. ISSN 0021-9193. PMC 357300. PMID 13174491.{{cite journal}}: CS1 maint: PMC format (link)
  4. ^ a b Stadtman, T. C.; Tsai, L. (1967-09-27). "A cobamide coenzyme dependent migration of the epsilon-amino group of D-lysine". Biochemical and Biophysical Research Communications. 28 (6): 920–926. ISSN 0006-291X. PMID 4229021.
  5. ^ Berkovitch F, Behshad E, Tang KH, Enns EA, Frey PA, Drennan CL (November 2004). "A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase". Proceedings of the National Academy of Sciences of the United States of America. 101 (45): 15870–5. doi:10.1073/pnas.0407074101. PMC 528771. PMID 15514022.
  6. ^ Lo, Hsin-Hsi; Lin, Hsin-Hua; Maity, Amarendra Nath; Ke, Shyue-Chu (2016-05-03). "The molecular mechanism of the open–closed protein conformational cycle transitions and coupled substrate binding, activation and product release events in lysine 5,6-aminomutase". Chem. Commun. 52 (38): 6399–6402. doi:10.1039/c6cc01888b. ISSN 1364-548X.
  7. ^ Chen, Yung-Han; Maity, Amarendra N.; Pan, Yu-Chiang; Frey, Perry A.; Ke, Shyue-Chu (2011-11-02). "Radical Stabilization Is Crucial in the Mechanism of Action of Lysine 5,6-Aminomutase: Role of Tyrosine-263α As Revealed by Electron Paramagnetic Resonance Spectroscopy". Journal of the American Chemical Society. 133 (43): 17152–17155. doi:10.1021/ja207766c. ISSN 0002-7863.
  8. ^ a b Morley, C. G.; Stadtman, T. C. (1971-06-08). "Studies on the fermentation of p-alpha-lysine. On the hydrogen shift catalyzed by the B 12 coenzyme dependent D-alpha-lysine mutase". Biochemistry. 10 (12): 2325–2329. ISSN 0006-2960. PMID 5114991.
  9. ^ a b Maity, Amarendra Nath; Ke, Shyue-Chu (2013-10-15). "5-Fluorolysine as alternative substrate of lysine 5,6-aminomutase: A computational study". Computational and Theoretical Chemistry. 1022: 1–5. doi:10.1016/j.comptc.2013.08.007.
  10. ^ Chen, Yung-Han; Maity, Amarendra N.; Frey, Perry A.; Ke, Shyue-Chu (2013-01-16). "Mechanism-based Inhibition Reveals Transitions between Two Conformational States in the Action of Lysine 5,6-Aminomutase: A Combination of Electron Paramagnetic Resonance Spectroscopy, Electron Nuclear Double Resonance Spectroscopy, and Density Functional Theory Study". Journal of the American Chemical Society. 135 (2): 788–794. doi:10.1021/ja309603a. ISSN 0002-7863.
  11. ^ Berkovitch, Frederick; Behshad, Elham; Tang, Kuo-Hsiang; Enns, Eva A.; Frey, Perry A.; Drennan, Catherine L. (2004-11-09). "A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase". Proceedings of the National Academy of Sciences of the United States of America. 101 (45): 15870–15875. doi:10.1073/pnas.0407074101. ISSN 0027-8424. PMC 528771. PMID 15514022.{{cite journal}}: CS1 maint: PMC format (link)
  12. ^ Wetmore, Stacey D.; Smith, David M.; Radom, Leo (2001-09-01). "Enzyme Catalysis of 1,2-Amino Shifts:  The Cooperative Action of B6, B12, and Aminomutases". Journal of the American Chemical Society. 123 (36): 8678–8689. doi:10.1021/ja010211j. ISSN 0002-7863.
  13. ^ Maity, Amarendra Nath; Ke, Shyue-Chu (2015-02-06). "4′-CyanoPLP presents better prospect for the experimental detection of elusive cyclic intermediate radical in the reaction of lysine 5,6-aminomutase". Biochemical and Biophysical Research Communications. 457 (2): 161–164. doi:10.1016/j.bbrc.2014.12.076.
  14. ^ Maity, Amarendra Nath; Lin, Hsin-Hua; Chiang, Hsiang-Sheng; Lo, Hsin-Hsi; Ke, Shyue-Chu (2015-05-01). "Reaction of Pyridoxal-5′-phosphate-N-oxide with Lysine 5,6-Aminomutase: Enzyme Flexibility toward Cofactor Analog". ACS Catalysis. 5 (5): 3093–3099. doi:10.1021/acscatal.5b00671.
  15. ^ Sandala, Gregory M.; Smith, David M.; Radom, Leo (2006-12-01). "In Search of Radical Intermediates in the Reactions Catalyzed by Lysine 2,3-Aminomutase and Lysine 5,6-Aminomutase". Journal of the American Chemical Society. 128 (50): 16004–16005. doi:10.1021/ja0668421. ISSN 0002-7863.
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