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

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)-3hydroxybutyryl-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: P2 1 2 1 2 1 and P2 1 . 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.

Self-sufficient asymmetric reduction of β-ketoesters catalysed by a novel and robust thermophilic alcohol dehydrogenase co-immobilised with NADH

“…To better understand the function/structure relationships for this enzyme, we created a structure model based on the X-ray structure of the 3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum (PDB: ; 54% homology). 31 This model confirmed the roles of the conserved residues according to those predicted by the conservation plots ( Fig. 1 ).…”

“…54% homology). 31 This model confirmed the roles of the conserved residues according to those predicted by the conservation plots (Fig. 1).…”

“…The gene ttc0898 from Thermus thermophilus HB27 encodes a protein annotated as a 3-hydroxybutyryl-CoA dehydrogenase (EC:1.1.1.157). This enzyme, here named as Tt27-HBDH, is 54–50% identical to the previously described bacterial ( S )-3-hydroxybutyryl-CoA dehydrogenases from Clostridium butyricum , 30,41 Clostridium acetobutylicum , 31 and Ralstonia eutropha , 42 and 45% identical to the human 3-hydroxyacyl-CoA dehydrogenase. 43 The multi-sequence alignment (MSA) of Tt27-HBDH revealed two conserved motifs; the catalytic triad (S119, H140 and N190) and the nucleotide-binding motif formed by both GXGXXG sequence and the polar residues D33, D92, K97 and N117 (Fig.…”

“…As found for other HBDH, the pantothenic moiety binds strongly the substrate through a conserved asparagine duplex (Asn143 and Asn223 in Tt27-HBDH) that establish a hydrogen-bond interaction with the CoA molecule. 30,31,41,42,53 This enzyme was 8.7 fold more catalytically effective for the reduction than for the oxidation under neutral pH according to the data shown in Table 2. This trend aligns with the behaviour of other dehydrogenases from the same microorganism.…”

“…This enzyme belongs to the large family of NAD(P)H dependent dehydrogenases (EC 1.1.1.X) that catalyse the asymmetric reduction of acetoacetyl-CoA and its reverse oxidative reaction with exquisite enantioselectivity. [30][31][32] Having this enzyme in hand, we screened different immobilization and postimmobilization protocols to further stabilise both tertiary and quaternary enzyme structures under high temperatures, low pH and high concentration of co-solvents like 2-propanol. Furthermore, the most stable immobilization protocol was also exploited to co-immobilise both Tt27-HBDH and NADH, resulting in a self-sufficient heterogeneous biocatalyst where enzyme and cofactor interplay within the porous environment of the carrier.…”