Journal:Acta Cryst F:S2053230X18014814

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]

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, .

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