Crystallographic studies of ACCO obtained from P. hybrida provide the following insights into the structure of the protein. The structure consists of the conserved, distorted double stranded β helix (DSBH) or jelly-roll motif (Figure. 3A) found in all other 2-OG oxygenases for which crystal structures have been characterized (DAOCS, ANS – Anthocyanidine synthase). The polypeptide backbone is folded into eleven α helices and thirteen β strands, of which eight β strands (β4- β11) are involved in the formation of the DBSH. α helices 1-6 are located at the N-terminal while helices 8-11 are located at the C-terminal end of the enzyme.
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The active site of ACCO is situated at one end of the DSBH and the DBSH is stabilized by the hydrophobic interactions of residues many of which are well conserved in IPNS, DAOCS, and ANS. Despite a lack of sequence similarity, the structure of the DSBH core of all the three enzymes is remarkably identical. However, certain differences in some secondary structures of ACCO provide a basis for the explanation of the unusual properties of the enzyme.
IPNS, DAOCS and ANS are thought to be monomers in their catalytically active forms. The C – terminus of these proteins is in close proximity to the active site of the enzyme providing a “cover” for the active site. For example, the penultimate residue in the C-terminus of IPNS, Gln330 projects into the active site and the side chain of the Gln330 lies close to the metal (Figure 4D). However, substrate binding displaces this residue from the active site (Figure 4E). Crystal structures of ACCO indicate that it exists as a tetramer (Figure 2A). This can be explained on the basis of the structure of the C-terminus of ACCO.
The C-terminus is comprised of β-13, a short 310 helix (α-9) and two longer helices α-10 and α-11 (Figure 3). Akin to IPNS and ANS, the penultimate helix, α 10 helps in enclosing the active site, but the final helix – α 11 points away from the DBSH/active site thus impeding any interactions between the helix and the
Page 2 Crystal Structure And Mechanistic Implications Of 1-Aminocyclopropane-1-Carboxylic Acid Oxidase – The Ethylene-Forming Enzyme Essay
active site of the same monomer leaving the active site open. In the crystal structure, the C-terminus of one monomer interlocks with the C-terminus of an adjacent monomer resulting in tetramer formation (Figure 2A). The monomeric – monomeric interactions probably aid in the catalytic activity of ACCO oxidase. Mutation of some residues in the C terminus results in a significant decrease in the catalytic activity. The C terminus may play a role in the reduction of the oxidized form of the enzyme resulting from the oxidation of ACC.
The most distinguishing character of ACCO is the presence of the unusually long helix α-3. The α-3 helix along with the β strand-3 and a turn forms an extension that projects about 25 – 35Å from the surface of the monomer. The extension is stabilized by the hydrophobic interactions of the residues on the helix and the β strand. The projection of α-3 allows the side chains of the residues present on the loop after the helix to come into the proximity of the active site of the adjacent monomer (Figure 2b). The interactions include Gln78 of monomer 1 (m1) with His177 and Tyr289 of monomer 2 (m2), Asp83 (m1) and Lys158 (m2) and Glu80 (m1) and Fe (II) of m2. These interactions are significant because Glu80 is involved in binding the iron.
The active site of ACCO is open compared to that of IPNS and ANS. The extended nature of the α-3 helix and the orientation of the final helix (α-11) away from the active site lead to the active site being open. As a result of the α -3 helix extension in ACCO, the two large loops that are responsible for covering the active site in ANS (Figure 3B) can no longer face the active site. Interestingly, in IPNS, only one of the two loops covers the active site; the inward movement of the C-terminal α helix (α-10) and another loop from the N-terminal region compensate for the absence of the second loop in enclosing the active site (Figure 3C).
The iron atom in the active site is ligated to the side chains of His177, Asp179 and His234. Together, these three ligands form a “triad” and the conformation of their side chains is similar to those of the related enzymes (Figure 6). In this arrangement, Glu80 of located between the extended α helix and β strand of an adjacent ACCO monomer also approaches the metal (Figure 4A). The presence of Asn252 close to the metal suggests that it may serve to stabilize the iron binding site and also involved in oxygen binding. Two of the tyrosine residues located on α-10, Tyr289 and Try255 may come in close proximity of the active site if the conformational changes allow α-10 to approach the active site. Tyr289 may be involved in mediating electron transfer to Fe (II).
It is believed that the conserved residue Lys158 is involved in the binding of the substrate, ACC. Lys158 in ACCO assumes a similar conformation as the conserved Lys213 of ANS. Lys213 of ANS is probably involved in substrate binding. The reduction in the activity of ACCO observed after the mutation of Lys158 and the affect on the Km of ACC and Vmax upon the mutation of the adjacent Thr157 residue suggest that Lys158 may bind to the ACC. This may precede or occur simultaneously during ACC ligation to iron.
The 2-OG oxygenases also contain an RXS motif. The RXS motif consisting of Arg244 and Ser246 is completely conserved in all ACCOs. These residues are typically involved in the binding of the co-substrate as seen in ANS and DAOCS or to the substrate as in IPNS. However, due to the direction of Arg244 pointing away from the active site in ACCO, it is not possible for this residue to be involved in substrate binding.
The analogous Arg279 in ANS although directed away from the active site rotates about its Cα – Cβ bond into the active site after substrate binding. Based on this observation, even if Arg244 of ACCO rotates into the active site after substrate binding, it is not close enough to bind to the carboxylate of ACC. On the other hand, the conformational change of Arg244 attained after substrate binding enables binding of bicarbonate which in turn binds to an iron bound dioxygen derived intermediate, facilitating correct proton and electron transfer.See More on Mechanism, Structure