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PDBsum entry 1s9c
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248 a.a.
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271 a.a.
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276 a.a.
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250 a.a.
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255 a.a.
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256 a.a.
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References listed in PDB file
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Key reference
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Title
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Crystal structure of 2-Enoyl-Coa hydratase 2 from human peroxisomal multifunctional enzyme type 2.
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Authors
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K.M.Koski,
A.M.Haapalainen,
J.K.Hiltunen,
T.Glumoff.
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Ref.
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J Mol Biol, 2005,
345,
1157-1169.
[DOI no: ]
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PubMed id
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Abstract
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2-Enoyl-CoA hydratase 2 is the middle part of the mammalian peroxisomal
multifunctional enzyme type 2 (MFE-2), which is known to be important in the
beta-oxidation of very-long-chain and alpha-methyl-branched fatty acids as well
as in the synthesis of bile acids. Here, we present the crystal structure of the
hydratase 2 from the human MFE-2 to 3A resolution. The three-dimensional
structure resembles the recently solved crystal structure of hydratase 2 from
the yeast, Candida tropicalis, MFE-2 having a two-domain subunit structure with
a C-domain complete hot-dog fold housing the active site, and an N-domain
incomplete hot-dog fold housing the cavity for the aliphatic acyl part of the
substrate molecule. The ability of human hydratase 2 to utilize such bulky
compounds which are not physiological substrates for the fungal ortholog, e.g.
CoA esters of C26 fatty acids, pristanic acid and di/trihydroxycholestanoic
acids, is explained by a large hydrophobic cavity formed upon the movements of
the extremely mobile loops I-III in the N-domain. In the unliganded form of
human hydratase 2, however, the loop I blocks the entrance of fatty enoyl-CoAs
with chain-length >C8. Therefore, we expect that upon binding of substrates
bulkier than C8, the loop I gives way, contemporaneously causing a secondary
effect in the CoA-binding pocket and/or active site required for efficient
hydration reaction. This structural feature would explain the inactivity of
human hydratase 2 towards short-chain substrates. The solved structure is also
used as a tool for analyzing the various inactivating mutations, identified
among others in MFE-2-deficient patients. Since hydratase 2 is the last
functional unit of mammalian MFE-2 whose structure has been solved, the
organization of the functional units in the biologically active full-length
enzyme is also discussed.
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Figure 3.
Figure 3. Ribbon representations of the HsMFE-2(dDhSCP-2LD)
dimer. (a) The upper image shows the four-helix bundle
dimerization motif of hydratase 2 formed by the a-helices a1,
a5, a1' and a5' as well as the two salt bridges formed between
Glu366 and Arg506 (side-chains shown as ball-and-stick
representations). The arrows point to the active sites with
catalytic Asp510 and His515 also shown as magenta. The N and
C-domains are colored as in Figure 2(b). Note that the flexible
loops I-III are fragmented in the lower subunit (subunit J in
the crystal structure of HsMFE-2(dDhSCP-2LD)). The lower image
shows a close-up of the salt bridge Glu366-Arg506. (b) The upper
image shows the dimer after a 90° rotation around the
vertical axis of the upper image of (a). The side-chains of
Asn457 and Tyr347 are shown as ball-and-stick representations,
and the active sites of the human hydratase 2 dimer are
indicated by black arrows. The lower image shows in detail the
interactions of a2 with a1 and with the N-domain b-sheet layer.
The connections shown are described in detail in Discussion.
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Figure 4.
Figure 4. Comparison of the ligand-binding pockets of the
human and C. tropicalis hydratase 2 subunits. (a) Electrostatic
surface potentials of the HsMFE-2(dDhSCP-2LD) ligand-binding
pocket. The positively and negatively charged regions are
colored blue and red, respectively. The residues suggested to
interact with the CoA moiety of the fatty enoyl-CoA substrate
are illustrated. The C-domain overhanging segment is labelled as
the H2-motif. (b) Electrostatic surface potentials of
CtMfe2p(dh[a+b]D) ligand-binding pocket with the bound
(3R)-hydroxydecanoyl-CoA (Protein Data Bank accession code ID
1PN417). The ligand binds to the positively charged CoA-binding
pocket in a bent conformation, where the adenine ring of the
3'-phosphate-ADP moiety points toward the protein, while the
phosphate groups are solvent-exposed. The stronger positive
charge at the surface of the ligand-binding pocket of
CtMfe2p(dh[a+b]D) is created by the side-chains of two lysine
residues (Lys820 and Lys823 of the C-domain overhanging segment)
and an arginine (Arg760 of b-strand b5), which are not found in
HsMFE-2(dDhSCP-2LD). Nevertheless, those residues are not
directly involved in substrate binding. (c) A close-up view of
the CoA-binding pocket after superimposing the apo form of
HsMFE-2(dDhSCP-2LD) (magenta) with the holo form of
CtMfe2p(dh[a+b]D) (light gray). The salt bridge between the
Lys729 of CtMfe2p(dh[a+b]D) and 3'-phosphate of the substrate as
well as the stacking interaction between Arg855 of
CtMfe2p(dh[a+b]D) and adenine ring of the substrate are shown
with black lines. (d) The differences in the region of the
flexible loop I of hydratase 2s from human (green), C.
tropicalis apoenzyme (gray) and C. tropicalis holoenzyme (red)
after superimposition of the three structures. The
(3R)-hydroxydecanoyl-CoA molecule of the C. tropicalis
holoenzyme is also shown. The b-strands b2 and b5 and the
C-domain are only partially shown for clarity. The side-chains
of Met386 and Val404 of HsMFE-2(dDhSCP-2LD) (in pink) as well as
Leu697 of CtMfe2p(dh[a+b]D) (in yellow) are shown. The black
arrow points to the position of the a-methyl group of
branched-chain fatty acids.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2005,
345,
1157-1169)
copyright 2005.
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