RESULTS AND DISCUSSION
Recombinantly expressed GmCHS1 was purified using nickel affinity
chromatography with an N -terminal His6-tag, and
by using size-exclusion chromatography (Fig. S1). The purified GmCHS1
was crystallized, and the crystal diffracted to 2.5 Å resolution
(crystallographic statistics, Table 1; electron density quality, see
Fig. S2). GmCHS1 crystallized in space group
P212121 with two
molecules per asymmetric unit. The structures were solved using a
molecular replacement method, with Pinus sylvestris CHS (PDB ID:
6DXA, 85.9% sequence identity) as a search model. The refined structure
included all residues from the original sequence of GmCHS1 (residues
1-388). However, the electron densities corresponding toN- terminal extension residues including the
His6-tag were not visible owing to the highly disordered
structures of the N -terminal regions. The overall structure of
GmCHS1 exhibits the αβαβα thiolase fold with a pseudo-symmetric dimer
with 12 α-helices and 11 β-strands (Fig. 1A) per monomer, as previously
reported for the crystal structure of CHS from Medicago sativa(alfalfa, PDB ID: 1BI5).14 In fact, our size-exclusion
chromatography experiment suggests that GmCHS1 mainly exists as a
homodimer in solution (Fig. S1). The homodimeric GmCHS1 contains two
active sites in the middle of the thiolase fold (Figure 1A, one is
highlighted in a dotted circle). When viewed in surface representation,
there is a tunnel-like structure, which has been termed the CoA-binding
tunnel in previous studies, making a connection between the external
solvent environment and the active site in each monomer (Fig. 1B).
Further analysis of the interior cavity, including the Co-A binding
tunnel, indicates that there is a large cavity around the active site
wherein the condensation reactions should occur (Fig. 1C). It is
noteworthy that the large cavity forms another tunnel downward to the
external solvent (Fig. 1C, indicated by a dotted arrow). Although the
mechanism for the product export of CHS is not completely understood, it
will be of interest in the future to evaluate whether this additional
tunnel functions as a metabolic channel. A close-up view of the active
site showed that the catalytic residues (Cys164, His302 and Asn335) are
located in similar orientations to those of other CHS structures, with
the electron densities for Cys164 sidechains being in their doubly
oxidized forms (Fig. 1D). The doubly oxidized catalytic cysteine
sulfinic acid is widely observed in the crystal structures of CHS from
euphyllophytes (ferns and seed plants), but not in the CHS from either
lycophyte or moss species. Because of the clarity of electron densities
at the positions 164 and the fact that soybean belongs to
euphyllophytes, we modeled S-cysteinesulfinic acid (ligand ID: CSD)
molecules for the corresponding positions. Weng et al.hypothesized that the oxidation of catalytic Cys residues was due to the
highly reactive environment around Cys during the generation of
nucleophilic thiolate anion that was assisted by the conserved His
residue (His302 in GmCHS1).15 In the GmCHS1 structure,
distances between the sulfur atom of CSD164 and the Nεof His302 are 3.55 and 3.69 Å for molecule A and B in the asymmetric
unit, respectively (Fig. 1D). In addition to the oxidation of Cys164,
the electron densities for Met70 sidechains are observed in their
oxidized forms. Although these Met oxidations are not reported for any
other CHS structures, we modeled the methionine sulfoxide (ligand ID:
SME) for the positions 70. Further biochemical or physiological
investigations will be needed to decipher the importance of the Met
oxidation.
To better understand the catalytic mechanism of GmCHS1, newly purified
GmCHS1 was crystallized, and the apo-crystals were soaked with
naringenin, a product analog of the CHS-catalyzed reaction. The
naringenin-soaked crystals diffracted to 1.8 Å resolution, and the
structure was determined in a similar procedure to that of the
apo-structure. The refined complex structure includes a dimer of GmCHS1,
two naringenin molecules, as well as three of acetate ions and three
citrate ions, which are contained in the mother liquor. Moreover, SME
and CSD are modeled for the positions 70 and 164, respectively as well
as the apo-structure. In the complex structure, naringenin molecules are
located in the vicinity of the catalytic cysteine sulfinic acids (the
residue 164, Fig. 2A). The positions of naringenin molecules were found
to be almost equivalent to those of previously reported for the crystal
structure of M. sativa CHS (Fig. S3).14 In
addition to the CSD164, Phe215 and Phe264 are located in the vicinity of
naringenin. The importance of the conserved Phe215 was previously
proposed as an involvement in the orientation of substrates and reaction
intermediates at the active site.14 We found that one
of the citrate ions was located at the end of the CoA binding tunnel,
also in the vicinity of Phe264 (Fig. 2A, 2B). The corresponding position
of the citrate ion of our structure was occupied by the CoA moiety of
malonyl-CoA or 1,4-piperazine di-ethane sulfonic acid
(C8H18N2O6S2)
in the complex structure of the M. sativa CHS Cys164Ala mutant
(Fig. S3, PDB ID: 1CML), suggesting that a negative charge of the
citrate ion could potentially mimic that of either CoA or 1,4-piperazine
di-ethane sulfonic acid. Although citric acid has not been studied as a
potential inhibitor of CHS, our complex structure suggests the
possibility of inhibitor binding upon the existence of the product at
the active site. Superposition of apo- and naringenin-bound structures
of GmCHS1 shows that the Phe265 is likely to change its orientation
through the CHS-catalyzed reaction (Fig. 2C). This is supported by the
fact that the average temperature factor for the Phe264 sidechain is
higher than that of Phe215 (44.2 Å2 and 33.4
Å2 for the Phe264 and the Phe215, respectively). It is
an intriguing question whether the flexibility or the bulkiness of
position 264 is related to the catalytic efficiency of GmCHS1. In
conclusion, the crystal structures of GmCHS1 provide a structural basis
for further improvement of our understanding of the catalytic mechanism
of CHS. Further efforts will include structural characterization of a
complex between GmCHS1 and other components of the soybean isoflavonoid
metabolon to better understand how GmCHS1 has a distinct role in the
formation of the isoflavonoid metabolon. Coordinates and structural
factors of apo-GmCHS1 and GmCHS1/naringenin have been deposited in the
Protein Data Bank under accession codes 7BUS and 7BUR, respectively.