Two NADH-dependent (S)-3-hydroxyacyl-CoA dehydrogenases from polyhydroxyalkanoate-producing Ralstonia eutropha

Area of Science:

• Microbiology
• Biochemistry
• Metabolic Engineering

Background:

• Ralstonia eutropha H16 possesses both NADH- and NADPH-dependent pathways for acetoacetyl-CoA reduction.
• The NADPH-dependent pathway yields (R)-3-hydroxybutyryl-CoA for polyhydroxybutyrate synthesis.
• The gene for the NADH-dependent enzyme, crucial for understanding competing metabolic routes, remained unidentified.

Purpose of the Study:

• To identify and characterize the NADH-dependent dehydrogenase in R. eutropha H16 with (S)-stereospecificity.
• To investigate the roles of PaaH1 and Had in acetoacetyl-CoA reduction and polyester biosynthesis.
• To explore the function of Had in the context of copolyester synthesis.

Main Methods:

• Gene deletion analysis of PaaH1 homologs.
• Partial purification and identification of NADH-dependent enzymes.
• Kinetic analysis of recombinant enzymes and gene disruption in engineered strains.

Main Results:

• Deletion of H16_A0282 (PaaH1) reduced NADH-dependent activity by half.
• H16_A0602 (Had) was identified as the enzyme responsible for the remaining activity.
• PaaH1 and Had are NADH-dependent 3-hydroxyacyl-CoA dehydrogenases with broad substrate specificity.
• Had deletion in a copolyester strain decreased the C6 component, indicating a role in (S)-specific reduction.

Conclusions:

• PaaH1 and Had are the primary enzymes mediating NADH-dependent acetoacetyl-CoA reduction in R. eutropha H16.
• These enzymes exhibit (S)-stereospecificity and can compete with (R)-3HB-CoA synthesis.
• Had plays a role in the reverse β-oxidation pathway during copolyester synthesis.