The two crystal structures of Alcaligenes faecalis D-3-hydroxybutyrate dehydrogenase, before and after NAD + and acetate binding, suggest a dynamical reaction mechanism as a member of the SDR family Md. Mominul Hoque b , Satoru Shimizu b , Md. Tofazzal Hossain b§ , Tamotsu Yamamoto # , Shigeyuki Imamura # , Kaoru Suzuki $ , Masaru Tsunoda $ , Hitoshi Amano & , Takeshi Sekiguchi $ , and Akio Takénaka b$* b Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan, § Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh, # Asahi Kasei Pharma Corporation, Tagat-gun, Shizuoka 410-2323, Japan, & Fukushima National College of Technology, Taira-kamiarakawa, Iwaki 970-8034, Japan and $ College of Science and Engineering, Iwaki Meisei University, Chuodai-iino, Iwaki 970-8551, Japan *corresponding author E-mail address of the corresponding author: [email protected]TITLE RUNNING HEAD (“Alcaligenes faecalis D-3-hydroxybutyrate dehydrogenase”). Keywords: X-ray structure; hydroxybutyrate dehydrogenase; NAD + dependent enzyme; Alcaligenes faecalis; ketone bodies. Synopsis Crystal structures of D-3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis and its complex with NAD + and acetate suggest the substrate recognition and the catalytic reaction mechanism. A structural comparison with other enzymes of the short-chain dehydrogenase/reductase family suggests functional versatility developed
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The two crystal structures of Alcaligenes faecalis D-3 ... two crystal structures of Alcaligenes faecalis D-3-hydroxybutyrate dehydrogenase, before and after NAD+ and acetate binding,
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The two crystal structures of Alcaligenes faecalis D-3-hydroxybutyrate
dehydrogenase, before and after NAD+ and acetate binding, suggest a dynamical
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Figure and Table captions
Table 1. Crystal data and data collection statistics.
Table 2. Amino acid identities (%) among the related proteins which target a
hydroxyl group of the substrates.
Table 3. Structural refinement statistics.
Scheme 1. HBDH catalyzes the reversible conversion between D-3-hydroxybutyrate
and acetoacetate. Acetoacetate is spontaneously decarboxylated to acetone.
Figure 1. A stereo view of |Fo|-|Fc| omit map of NAD+ in the Af-HBDH-NAD-Acetate
complex crystal. Contours are drawn at 2.5 σ level. Figure 2. The molecular structure of HBDH from Alcaligenes faecalis. A stereo
ribbon-cartoon diagram shows the tetrameric architecture as the biological unit,
which is formed with the four subunits (each drawn in different color) with a
non-crystallographic 222 symmetry. There are two independent tetramers in the
asymmetric unit of Af-HBDH-I crystal.
Figure 3. The architecture of a subunit of the tetrameric HBDH-NAD-Acetate
complex from Alcaligenes faecalis. (a) A stereo ribbon-cartoon drawing of one of the
four subunits in the asymmetric unit. The NAD+, acetate and water molecules are
shown in ball-and-stick drawing. (b) A topological diagram of the secondary
structures. The secondary structures were assigned with the program PROCHECK
(Laskowski et al., 1993). Strands and helices are represented as arrows and cylinders,
respectively. The principal domain adopts an α/β open structure containing a typical
Rossmann fold, in which the seven β-strands are all in parallel to each other. At the
center, a topological switching point is formed between the two strands, S1 and S4,
for the catalytic site.
Figure 4. NAD+ binding geometry in Af-HBDH. Possible hydrogen bonds are
indicated by broken lines, except other hydrogen bonds with water molecules. The
bound NAD+ molecule is shown in ball-and-stick drawing. Values indicate distances
in Å for the A subunit, as well as the other subunits have.
Figure 5. A D-3-hydroxybutyrate bound model in Af-HBDH, constructed by adding
two carbon atoms around the bound acetate and water molecules. The acetate
carboxyl group forms hydrogen bonds, one oxygen atom with His144 and Lys152,
while the other oxygen atom with Gln94 and Gln196 (see the text). The bound acetate
molecule was highlighted by yellow color. Positioning of the β-carbon in the R
configuration results in a close contact with the C4 atom of the cofactor (2.8 Å) to
facilitate its hydride donation. Hydrogen bonds are shown by broken lines with
values in Å.
Figure 6. Structural comparison of the several related enzymes of the SDR family. A
stereo diagram shows the Cα traces of (S)-1-phenylethanol reductase from the
‡ (%) 27.0 23.6 Number of protein atoms 15248 7624 Water molecules 400 831 NAD+ molecules - 4 Acetate ion - 4 Ca2+ 4 2 Cl- 8 4 Root mean square deviations Bond lengths (Å) 0.007 0.005 Bond angles (degree) 1.3 1.2 Ramachandran plot Residues in most favored regions (%) 90.9 91.3 Residues in additionally allowed regions (%) 9.0 8.7 Residues in generously allowed regions (%) 0.1 0.0 Residues in disallowed regions (%) 0.0 0.0
Scheme 1
HBDH catalyzes the reversible conversion between D-3-hydroxybutyrate and
acetoacetate. Acetoacetate is spontaneously decarboxylated to acetone.
Figure 1 A stereo view of |Fo|-|Fc| omit map of NAD+ in the Af-HBDH-NAD-Acetate complex crystal. Contours are drawn at 2.5 σ level.
Figure 2 The molecular structure of HBDH from Alcaligenes faecalis. A stereo ribbon-cartoon diagram shows the tetrameric architecture as the biological unit, which is formed with the four subunits (each drawn in different color) with a non-crystallographic 222 symmetry. There are two independent tetramers in the asymmetric unit of Af-HBDH-I crystal.
Figure 3 The architecture of a subunit of the tetrameric HBDH-NAD-Acetate complex from Alcaligenes faecalis. (a) A stereo ribbon-cartoon drawing of one of the four subunits in the asymmetric unit. The NAD+, acetate and water molecules are shown in ball-and-stick drawing. (b) A topological diagram of the secondary structures. The secondary structures were assigned with the program PROCHECK (Laskowski et al., 1993). Strands and helices are represented as arrows and cylinders, respectively. The principal domain adopts an α/β open structure containing a typical Rossmann fold, in which the seven β-strands are all in parallel to each other. At the center, a topological switching point is formed between the two strands, S1 and S4, for the catalytic site.
Figure 4 NAD+ binding geometry in Af-HBDH. Possible hydrogen bonds are indicated by broken lines, except other hydrogen bonds with water molecules. The bound NAD+ molecule is shown in ball-and-stick drawing. Values indicate distances in Å for the A subunit, as well as the other subunits have.
Figure 5 A D-3-hydroxybutyrate bound model in Af-HBDH, constructed by adding two carbon atoms around the bound acetate and water molecules. The acetate carboxyl group forms hydrogen bonds, one oxygen atom with His144 and Lys152, while the other oxygen atom with Gln94 and Gln196 (see the text). The bound acetate molecule was highlighted by yellow color. Positioning of the β-carbon in the R configuration results in a close contact with the C4 atom of the cofactor (2.8 Å) to facilitate its hydride donation. Hydrogen bonds are shown by broken lines with values in Å.
Figure 6 Structural comparison of the several related enzymes of the SDR family. A stereo diagram shows the Cα traces of (S)-1-phenylethanol reductase from the denitrifying bacterium Strain EbN1 (blue, PDB code 2EWM; Hoffken et al., 2006), 3α,20β-hydroxysteroid dehydrogenase from Streptomyces hydrogenas (light blue, PDB code 2HSD; Ghosh et al., 1994), tropinone reductase I from Datura stramonium (cyan, PDB code 1AE1; Nakajima et al., 1998), sorbitol dehydrogenase from Rhodobacter sphaeroides (yellow, PDB code 1K2W; Philipsen et al., 2004), carbonyl reductase from mouse lung (orange, PDB code 1CYD; Tanaka et al., 1996), and alcohol dehydrogenase from Drosophila melanogaster (red, PDB code 1MG5; Benach et al., 2005). They are superimposed onto those of the Af-HBDH-NAD-Acetate complex (green). The principal domains are highly conserved, but the small domains differ for the generation of the wide substrate specificity.
Scheme 2 A proposed catalytic reaction mechanism of HBDH.