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

• Published in: Acta crystallographica. Section F, Structural biology communications Vol. 74; no. 11; pp. 733 - 740
• Main Authors: Takenoya, Mihoko, Taguchi, Seiichi, Yajima, Shunsuke
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England International Union of Crystallography 01.11.2018; Wiley Subscription Services, Inc
• Subjects: 3-Hydroxyacyl CoA Dehydrogenases - chemistry; 3-Hydroxyacyl CoA Dehydrogenases - genetics; 3-Hydroxyacyl CoA Dehydrogenases - metabolism; Amino Acid Substitution; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Biodegradability; Biodegradable materials; Biodegradation; bio‐based plastics; Catalysis; Catalytic Domain; Clostridium acetobutylicum; Column chromatography; Crystal structure; Crystallography, X-Ray; Crystals; Dehydrogenase; Dehydrogenases; Dimers; E coli; Enzymatic activity; enzyme kinetics; fatty‐acid metabolism; Kinetics; Models, Molecular; Mutants; Mutation; NAD; NAD - chemistry; NAD - metabolism; Oligomerization; Polymers; Protein Conformation; Protein Multimerization; Protein Subunits - chemistry; Ralstonia eutropha; Research Communications; Residues; SDR superfamily; Substrates; X‐ray protein crystallography; bio-based plastics; fatty-acid metabolism; SDR superfamily; enzyme kinetics; X-ray protein crystallography
• ISSN: 2053-230X; 2053-230X
• DOI: 10.1107/S2053230X18014814

(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. Crystal structures of (S)‐3‐hydroxybutyryl‐CoA dehydrogenase from Clostridium acetobutylicum have been determined in apo and NAD+‐bound forms. The structures, together with kinetic analyses using single‐amino‐acid substituted mutants, revealed the catalytically important residues in the enzyme.