Structure-function relationship within the 4-hydroxybenzoyl-CoA thioesterase enzyme superfamily

The distinguishing feature of the hotdog-fold enzyme superfamily is a small backbone scaffold consisting of a five-stranded anti-parallel beta-sheet wrapped around a long alpha-helix. The functional unit is a dimer, in which the two active sites are formed at the subunit-subunit interface. My studies have concentrated on investigating the structure-function relationship and defining divergence of physiological function within the thioesterase subfamilies.; The venue of our studies into this enzyme superfamily is the 4-hydroxybenzoyl-CoA thioesterase of the 4-chlorobenzoate degradation pathway discovered in bacteria Pseudomonas sp. strain CBS3 and Arthrobacter sp. strain SU. Despite identical function of these two thioesterases, dissimilar active site structures and catalytic mechanisms were observed. By using a combination of techniques like site-directed mutagenesis, x-ray crystallography, steady-state kinetics, transient kinetics, Raman difference and UV-vis difference spectroscopy, the catalytic mechanisms of Pseudomonas and Arthrobacter 4-HBA-CoA thioesterases were investigated. Both enzymes utilize an elongated active site alpha-helix as the carbonyl-polarizing machinery. An enzyme carboxylate group (Asp17 in the Pseudomonas thioesterase, Glu73 in the Arthrobacter thioesterase) was shown to be the major catalytic residue, and likely to function in covalent catalysis. Although these two thioesterases are highly diverged in protein sequences, they adopted the same tertiary structure as revealed by x-ray crystallographic studies. Both active site scaffolds consist of a helix-loop station derived from one subunit and the helix station of the interfaced subunit (of the functional homodimer). Remarkably the catalytic carboxylate can be positioned on either of the two structural components of the active site platform.; To study the hotdog-fold enzyme superfamily, primary family members were initially identified via x-ray structure determinations. PSI-BLAST searches have since identified several sequence subfamilies. The structures and/or functions of several representatives from the thioesterase subfamilies have been examined. The YbgC protein from the Haemophilus influenzae Tol-Pal pathway was demonstrated to be a short-chain acyl-CoA thioesterase whose structure and physiological function is still under investigation. The H. influenzae H10827 protein was shown to be an efficient short-to-medium chain length acyl-CoA thioesterase. X-ray crystallographic studies proved that the H10827 protein was indeed a hotdog-fold protein, but with a novel quaternary structure. The BH1999 protein of the Bacillus halodurans upper gentisate pathway was demonstrated to be a 2,5-dihydroxbenzoyl-CoA thioesterase. The E. coli PaaI protein in the novel phenylacetic acid degradation pathway represents a new type of thioesterase with hydroxylated phenylacetyl-CoA as substrates. Currently the physiological functions of these novel family members are not fully understood. However, based on the observed enzymatic activities and the genomic contextual information, possible physiological functions of the family members were postulated and discussed.