The hta Gene of Thermus thermophilus HB8 Encodes a Protein with Homoserine Transacetylase and PHB Synthase Activity

A genomic survey of Thermus thermophilus HB8 for loci containing an /-hydrolase fold and the conserved lipase box motif identified the TTHA0759 locus.This gene was previously annotated as encoding a homoserine transacetylase (HTA), based on sequence homology to related proteins in other microorganisms.The hta gene was amplified from Thermus thermophilus genome and overexpressed in the mesophylic bacterium of Escherichia coli.The resulting recombinant protein, rHTA, exhibits dual functionality as both a homoserine transacetylase and a PHA synthase, as both activities involve the transfer of an acyl moiety.Kinetic studies showed that rHTA had a K m value of 0.11 mM for acetyl-CoA and a K m for synthase of 0.25 mM with 3HBCoA as substrate.Both K m values are very close to the native enzymes.Synthase activity is activated by acetyl-CoA, acetylphosphate and inhibited by CoA, NAD, NADP, similarly to the native enzyme.Treatment of rHTA with alkaline phosphatase leads to loss of its enzymic activity.In addition, synthase activity is inactivated by iodoacetamide suggesting that a cysteine moiety was involved in the catalytic reaction.Interestingly, the recombinant protein was found to catalyze the in vitro polymerization of (R)-3-hydroxybutyryl-CoA into polyhydroxybutyrate (PHB), demonstrating a high affinity for PHB granules.Notably, deletion of its transmembrane domain abolished this granule-binding capability, indicating that membrane association is critical for its interaction with PHB.These findings suggest that T. thermophilus HB8 utilize the homoserine transacetylase as a key component in the biosynthetic pathway of polyhydroxyalkanoates, facilitating their accumulation as intracellular storage biomolecules.