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 Km value and a significantly lower kcat 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 .
