Journal:Acta Cryst F:S2053230X18014814

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Crystal structure and kinetic analyses of a hexameric form of (S)-3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum

Mihoko Takenoya, Seiichi Taguchi and Shunsuke Yajima ^[1]

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Molecular Tour

Abstract

(S)-3-Hydroxybutyryl-CoA dehydrogenase (HBD) has been gaining increased attention recently as it is a key enzyme in the enantiomeric formation of (S)-3-hydroxybutyryl-CoA [(S)-3HB-CoA]. It converts acetoacetyl-CoA to (S)-3HB-CoA in the synthetic metabolic pathway. (S)-3HB-CoA is further modified to form (S)-3-hydroxybutyrate, which is a source of biodegradable polymers. During the course of a study to develop biodegradable polymers, attempts were made to determine the crystal structure of HBD from Clostridium acetobutylicum (CacHBD), and the crystal structures of both apo and NAD⁺-bound forms of CacHBD were determined. The crystals belonged to different space groups: P2₁2₁2₁ and P2₁. However, both structures adopted a hexamer composed of three dimers in the asymmetric unit, and this oligomerization was additionally confirmed by gel-filtration column chromatography. Furthermore, to investigate the catalytic residues of CacHBD, the enzymatic activities of the wild type and of three single-amino-acid mutants were analyzed, in which the Ser, His and Asn residues that are conserved in the HBDs from C. acetobutylicum, C. butyricum and Ralstonia eutropha, as well as in the L-3-hydroxyacyl-CoA dehydrogenases from Homo sapiens and Escherichia coli, were substituted by alanines. The S117A and N188A mutants abolished the activity, while the H138A mutant showed a slightly lower K_m value and a significantly lower k_cat value than the wild type. Therefore, in combination with the crystal structures, it was shown that His138 is involved in catalysis and that Ser117 and Asn188 may be important for substrate recognition to place the keto group of the substrate in the correct position for reaction.

Overall structure

The crystal structures of the apo (6acq) and NAD⁺-bound (6aa8) forms of CacHBD were determined at 2.5 and 2.1 A˚ resolution, respectively. . The N- and C-terminal domains are colored cyan and magenta, respectively. NAD⁺ is shown as a wheat ball-and-stick model with N, O, and P atoms are in CPK. The consisted of a Rossmann fold, which binds to , and the in the crystal structure (the second monomer is shown in yellow). The apo (6acq) and NAD⁺-bound (6aa8) CacHBD crystals belonged to space groups P2₁2₁2₁ and P2₁, respectively. In the asymmetric unit, both crystals contained three dimers of CacHBD forming a hexamer, which belonged to point-group symmetry D3 with a and . The dimers are shown in yellow, green and purple.

Comparison of monomer subunits of hexameric CacHBD

The crystal structures obtained in this study were composed of two types of dimers: a homodimer of two apo subunits and a heterodimer of NAD⁺-bound and apo subunits. No homodimers of NAD+-bound subunits were obtained. Therefore, three types of subunits existed: and an . When the C-terminal domains of the 12 monomers of apo and NAD⁺-bound CacHBD in the asymmetric units were superposed, (only N-terminal domains are shown). .

, types A, B and C are partly colored cyan, blue and magenta, respectively. However, one difference was observed for Glu90 in loop 1 containing Ala87–Arg91. . These two subunits were type C. Among the 12 subunits in a hexamer, only two subunits bound to NAD⁺, and their counterpart subunits in the dimers did not bind to NAD⁺. The difference was caused by the mode of interaction of Glu90. , forming hydrogen bonds with distances of 2.6 and 2.7 A˚, respectively. ⁺ at distances of 2.5, 3.0, 2.9 and 2.7 A˚, respectively. On the other hand, . Furthermore, the temperature factors of loop 1 (consisting of Ala87–Arg91) and loop 2 (consisting of Asn115–Ser120) were examined. The B factor is shown in color. The highest and lowest values are shown in red and blue, respectively, with a gradient of colors in between

min temp

max temp

. When loop 1 interacted with loop 2 in , the temperature factors of loops 1 and 2 had high and low values, respectively. In , the temperature factors of both loops were lower. In , which exhibited no interactions between loop 1 and loop 2, the temperature factors of both loops were higher. Although the corresponding Glu residues are located at the nonflipped position in the case of the crystal structures of CbuHBD, the results suggest that loop 1 retains the flexibility to accept NAD⁺. Upon the binding of NAD⁺, loop 1 becomes more stable than that in apo CacHBD.

Comparison of monomer subunits between CacHBD and L-3-hydroxyacyl-CoA dehydrogenase from Homo sapiens

l-3-Hydroxyacyl-CoA dehydrogenase from H. sapiens (HuHAD) has acetoacetyl-CoA reductase activity, and the crystal structure was determined as a ternary complex with NAD⁺ and acetoacetyl-CoA (PDB entry 1f0y^[2]). Furthermore, crystal structures of HuHAD in the apo form (PDB entry 1f14) and in a binary form with NADH (PDB entry 1f17) have been determined^[2]). These three crystal structures of HuHAD and the apo and NAD⁺-bound CacHBD structures were superposed. A significant difference was observed between apo CacHBD and the ternary complex of HuHAD. The other structures were similar to that of apo CacHBD. In this study, the N-terminal domains of the ternary complex of HuHAD and apo CacHBD were superposed. The r.m.s.d. value (calculated for Cα atoms) of the N-terminal domain was 0.77 A˚ (residues 1–188 in the N-terminus of CacHBD and residues 15–208 in the N-terminus of HuHAD). In this case, the r.m.s.d. value of the C-terminal domains was 7.50 A˚ over 93 residues (residues 189–281 in the C-terminus of CacHBD and residues 209–301 in the C-terminus of HuHAD). Since the N- and C-terminal domains of the ternary complex of HuHAD adopted a closed conformation compared with those of apo CacHBD, the distances between the N-terminal and C-terminal domains were measured using the amino acids located at the corresponding positions in the crystal structures. The distance between Thr11 in the N-terminal domain and Lys272 in the C-terminal domain of apo CacHBD was 14.2 A˚. Meanwhile, in the ternary complex of HuHAD the distance between Leu25 in the N-terminal domain and Lys293 in the C-terminal domain was 8.0 A˚. A large domain movement therefore seemed to be induced upon the binding of acetoacetyl-CoA rather than upon that of NAD⁺. (PDB entry 1f0y). .

The NAD⁺-bound form of CacHBD and the ternary complex of HuHAD are superposed on the N-terminal domains and are colored cyan and yellow, respectively. Dotted lines indicate the distances (labeled) between the N-terminal and C-terminal domains in each structure.

The NAD⁺-binding site of CacHBD

In the CacHBD structure from the P2₁ crystal, only two subunits in the hexamer were observed to bind to NAD⁺. In the binding mode for NAD⁺, in both or either of the subunits. On the other hand, the same residues as in CacHBD, except for Arg39, are involved in hydrogen bonds in CbuHBD (PDB entry 4kug^[3]). When the crystal structures were superposed, they fitted well with an r.m.s.d. value of 0.88 A˚ (calculated on Cα atoms), and the binding modes for NAD⁺ in both proteins were almost identical, with a slight difference in the directions of the side chains of the amino-acid residues. These differences may reflect the flexibility of the side chains of the residues and/or the differences in the resolutions of the crystal structures: 2.3 and 2.1 A˚ for CbuHBD and CacHBD, respectively. In addition to the hydrogen bonds formed, the adenine moiety of NAD⁺ was positioned in a hydrophobic pocket formed by Leu7, Ile32, Ala88, Val89, Ile94 and Ile98 in CacHBD. This pocket was also observed in CbuHBD^[3], with a single difference of Val89 in CacHBD compared with Ile89 in CbuHBD.

References

1. ↑ Takenoya M, Taguchi S, Yajima S. Crystal structure and kinetic analyses of a hexameric form of (S)-3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum. Acta Crystallogr F Struct Biol Commun. 2018 Nov 1;74(Pt 11):733-740. doi:, 10.1107/S2053230X18014814. Epub 2018 Oct 31. PMID:30387779 doi:http://dx.doi.org/10.1107/S2053230X18014814
2. ↑ ^{2.0 2.1} Barycki JJ, O'Brien LK, Strauss AW, Banaszak LJ. Sequestration of the active site by interdomain shifting. Crystallographic and spectroscopic evidence for distinct conformations of L-3-hydroxyacyl-CoA dehydrogenase. J Biol Chem. 2000 Sep 1;275(35):27186-96. PMID:10840044 doi:http://dx.doi.org/10.1074/jbc.M004669200
3. ↑ ^{3.0 3.1} Kim EJ, Kim J, Ahn JW, Kim YJ, Chang JH, Kim KJ. Crystal structure of (S)-3-hydroxybutyryl-CoA dehydrogenase from Clostridium butyricum and its mutations that enhance reaction kinetics. J Microbiol Biotechnol. 2014 Dec 28;24(12):1636-43. PMID:25112316