The Rate-Limiting Catalytic Steps of Hydroxymandelate Synthase from Amycolatopsis orientalis

Abstract

Hydroxymandelate synthase (HMS) catalyzes the committed step in the formation of p-hydroxyphenylglycine, a recurrent substructure of polycyclic nonribosomal peptide antibiotics such as vancomycin. HMS has the same structural fold as and uses the same substrates as 4-hydroxyphenylpyruvate dioxygenase (HPPD) (4-hydroxyphenylpyruvate (HPP) and O(2)). Moreover, HMS catalyzes a very similar dioxygenation reaction to that of HPPD, adding the second oxygen atom to the benzylic position, rather than the aromatic C1 carbon of the substrate. The dissociation constant for HPP (59 microM) was measured under anaerobic conditions by titrating substrate with enzyme and monitoring the intensity of the weak (epsilon(475nm ) approximately 250 M(-1) cm(-1)) charge-transfer absorption band of the HMS.Fe(II).HPP complex. Pre-steady-state analysis indicates that evidence exists for the accumulation of three intermediates in a single turnover and the decay of the third is rate-limiting in multiple turnovers. The rate constants used to fit the data were k(1) = 1 x 10(5) M(-1) s(-1), k(2) = 250 s(-1), k(3) = 5 s(-1), and k(4) = 0.3 s(-1). However, the values for k(1) and k(2) could not be accurately measured due to both a prolonged mixing time for the HMS system that obscures observation at the early times (<10 ms) and the apparent high relative value of k(2). The third phase, k(3), is attributed to the formation of the product complex, and no kinetic isotope effect was observed on this step when the protons of the substrate's benzylic carbon were substituted with deuteriums, suggesting that hydroxylation is fast relative to the steps observed. The final and predominantly rate-limiting step shows a 3-fold decrease in the magnitude of the rate constant in deuterium oxide solvent, and a proton inventory for this step suggests the contribution of a single proton from the solvent environment.

Structural and mechanistic comparisons of the metal-binding members of the vicinal oxygen chelate (VOC) superfamily

“…Surprisingly, no substrate isotope effect was observed when HPP's benzylic hydrogens were substituted with deuteriums, despite the fact that hydrogen abstraction at this position is a catalytic requirement. A solvent isotope effect of 3.5, however, was observed on the rate-limiting final phase that was confirmed by chemical quench and HPLC analysis to be product release, and a proton inventory suggested the contribution of a single proton by the solvent environment to aid this process [96].…”

“…HPPD and HMS share as much as 35% identity and 50% similarity in their primary sequence [71], but carry out distinct transformations of the common substrate HPP with extremely low error rates for making the product of the other enzyme [96]. The proposed mechanisms for these two enzymes within the framework of the αKAOs consist of ordered substrate binding, oxidative decarboxylation, hydroxylation, and ordered product release as shown in Scheme 4 [97].…”

“…This lack of change in coordination state upon substrate binding may be a vestige of the basic function of the VOC fold, electronic stabilization [108][109][110]. Kinetic experiments revealed that HPP-enzyme complex formation is essential in promoting O 2 reduction, increasing this rate by N10 3 -fold for both enzymes when compared to the dioxygen reduction rate by the holoenzyme [96,99]. A low energy α-keto acid n → π* transition in this complex, seen in the UV/Vis of both CD and MCD, indicates the near planar conformation that the α-keto acid adopts when coordinated bidentate to the Fe 2+ .…”

Role of Substrate Positioning in the Catalytic Reaction of 4-Hydroxyphenylpyruvate Dioxygenase—A QM/MM Study

“…14,18,19 Results of steady-state kinetics measurements indicated that HPP binds to Fe(II) prior to dioxygen, while CO 2 is the first product released from the active site, similarly to other α-keto acid dependent enzymes. [20][21][22] Respail and co-workers performed QM/MM calculations to test the possible binding modes of HPP in the HPPD active site. 23 Different models, created for an Arabidopsis thaliana HPPD structure (PDB code: 1SQD), were employed, including models of the EFe(II)-HPP and E-Fe(IV)=O-HPA complexes.…”

Chiral Hydroxylation at the Mononuclear Nonheme Fe(II) Center of 4-(S) Hydroxymandelate Synthase – A Structure-Activity Relationship Analysis

“…Taken together, these studies highlight the importance of the interaction of protein and substrate structure for an efficient, site directed oxygenation reaction. The structure of Hms from A. orientalis has been solved [10] and based on kinetic analyses, which have revealed a lack of kinetic isotope effects on the turnover number, it has recently been concluded that product release is the rate limiting step regarding the conversion of the native substrate, HPP, by A. orientalis Hms [11].…”