Kinetic and structural studies of hydroxymandelate synthase from Amycolatopsis orientalis

Hydroxymandelate synthase (HMS) catalyzes the committed step in the formation of para-hydroxyphenylglycine, a recurrent substructure of antibiotics such as vancomycin. HMS uses the same substrates as 4-hydroxyphenylpyruvate dioxygenase (HPPD), 4-hydroxyphenylpyruvate (HPP) and O2, and also conducts a dioxygenation reaction. Investigating HMS has led to further insights concerning the mechanism of the closely related HPPD, which is of significant agricultural and medical relevance, and the general mechanism of the enzyme family to which both belong.;The association of HPP to the Fe2+ center activates O 2 binding by ∼1000-fold. It also produces a charge transfer, allowing anaerobic measurement of KHPP. Evidence from pre-steady state analysis supports four phases in a single turnover. These are interpreted to correspond to the accumulation of three intermediates, followed by product release. k1 and k2 could not be accurately measured due to HMS's prolonged mixing and a large k 2. k3 decreases with increasing O2 concentration. And k4 was confirmed to be product release by chemical quench and HPLC. No isotope effect was observed when the substrate's benzylic carbons were deuterated, but the rate-limiting k4 exhibits a solvent isotope effect of 3.5 in D 2O and increases at high pH. A proton inventory suggests that the solvent contributes a single proton to assist product release.;The crystal structure of the HMS·Co2+·hydroxymandelate (HMA) determined to 2.3 A reveals HMS to have two VOC barrel domains, with the C-terminal one containing the 2H1C motif found in most Fe 2+-dependent oxygenases. It closely resembles the closed-conformation HPPD. HMS's active site is significantly smaller than HPPD's, consistent with divergent catalytic chemistries. HMA's benzylic hydroxyl and one carboxylate oxygen coordinate to Co2+. Three other H-bonding interactions to active site residues are observed. It is noted that a well-ordered water molecule is 3.2 A away from the distal carboxylate oxygen. Modeling of HPP into the active sites of both HMS and HPPD suggests different substrate binding positions. In HMS, HPP binds analogously to HMA, while in HPPD, HPP's p-hydroxyl was found to have an additional H-bonding partner. The difference in the substrate's ring orientation and corresponding intermediates may influence the hydroxylation site.