Hydroxymandelate Synthase Research Articles

A Four-Step Enzymatic Cascade for Efficient Production of L-Phenylglycine from Biobased L-Phenylalanine.

Enantiopure amino acids are of particular interest in the agrochemical and pharmaceutical industries. Here, we report a multi-enzyme cascade for efficient production of L-phenylglycine (L-Phg) from biobased L-phenylalanine (L-Phe). We first attempted to engineer Escherichia coli for expressing L-amino acid deaminase (LAAD) from Proteus mirabilis, hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis, (S)-mandelate dehydrogenase (SMDH) from Pseudomonas putida, the endogenous aminotransferase (AT) encoded by ilvE and L-glutamate dehydrogenase (GluDH) from E. coli. However, 10 mM L-Phe only afforded the synthesis of 7.21±0.15 mM L-Phg. The accumulation of benzoylformic acid suggested that the transamination step might be rate-limiting. We next used leucine dehydrogenase (LeuDH) from Bacillus cereus to bypass the use of L-glutamate as amine donor, and 40 mM L-Phe gave 39.97±3.84 mM (6.04±0.58 g/L) L-Phg, reaching 99.9 % conversion. In summary, this work demonstrates a concise four-step enzymatic cascade for L-Phg synthesis from biobased L-Phe, with a potential for future industrial applications.

Microbial synthesis of 4-hydroxybenzoic acid from renewable feedstocks

4-Hydroxybenzoic acid (4HBA) and its esterified forms can be used as preservatives in the pharmaceutical and food industries. Here, we reported the establishment of a coenzyme-A (CoA) free multi-enzyme cascade in Escherichia coli to utilize biobased L-tyrosine for efficient synthesis of 4HBA. The multi-enzyme cascade contains L-amino acid deaminase from Proteus mirabilis, hydroxymandelate synthase from Amycolatopsis orientalis, (S)-mandelate dehydrogenase and benzoylformate decarboxylase from Pseudomonas putida, and aldehyde dehydrogenase from Saccharomyces cerevisiae. The whole-cell biocatalysis afforded the synthesis of 128 ± 1 mM of 4HBA (17.7 ± 0.1 g/L) from 150 mM L-tyrosine with > 85% conversion within 96 h. In addition, the artificial enzymatic cascade also allowed the synthesis of benzoic acid from 100 mM L-phenylalanine with a conversion ∼ 90%. In summary, our research offers a sustainable alternative for synthesizing 4HBA and benzoic acid from renewable feedstocks.

One-Pot Cascade Biotransformation for Efficient Synthesis of Benzyl Alcohol and Its Analogs.

Benzyl alcohol is a naturally occurring aromatic alcohol and has been widely used in the cosmetics and flavor/fragrance industries. The whole-cell biotransformation for synthesis of benzyl alcohol directly from bio-based L-phenylalanine (L-Phe) was herein explored using an artificial enzyme cascade in Escherichia coli. Benzaldehyde was first produced from L-Phe via four heterologous enzymatic steps that comprises L-amino acid deaminase (LAAD), hydroxymandelate synthase (HmaS), (S)-mandelate dehydrogenase (SMDH) and benzoylformate decarboxylase (BFD). The subsequent reduction of benzaldehyde to benzyl alcohol was achieved by a broad substrate specificity phenylacetaldehyde reductase (PAR) from Solanum lycopersicum. We found the designed enzyme cascade could efficiently convert L-Phe into benzyl alcohol with conversion above 99%. In addition, we also examined L-tyrosine (L-Tyr) and m-fluoro-phenylalanine (m-f-Phe) as substrates, the cascade biotransformation could also efficiently produce p-hydroxybenzyl alcohol and m-fluoro-benzyl alcohol. In summary, the developed biocatalytic pathway has great potential to produce various high-valued fine chemicals.

In vivo cascade catalysis of aromatic amino acids to the respective mandelic acids using recombinant E. coli cells expressing hydroxymandelate synthase (HMS) from Amycolatopsis mediterranei

Mandelic acids are valuable products which are used in a broad field of applications. The enzyme hydroxymandelate synthase (HMS) is a non-heme iron dioxygenase which converts para-hydroxyphenylpyruvate and other 3-aryl pyruvates by decarboxylation to the corresponding mandelates. In the present work, the gene hms encoding the hydroxymandelate synthase from Amycolatopsis mediterranei was cloned and overexpressed in Escherichia coli BL21(DE3) for in vivo cascade catalysis taking advantage of resident aromatic amino acid transaminases. The resulting recombinant cells were used for whole cell biotransformations. We successfully accomplished the production of para-hydroxymandelate exclusively by using the aromatic amino acid l-tyrosine in biotransformation. Furthermore, by utilizing different phenylalanine derivatives (including chloro-, fluoro- and hydroxylated amino acids), the corresponding S-mandelic acids were obtained with high conversion (21–87 %) and high ee (38–97%). This process is an alternative and attractive way to get access to a variety of mandelic acids.

Engineering of hydroxymandelate synthases and the aromatic amino acid pathway enables de novo biosynthesis of mandelic and 4-hydroxymandelic acid with Saccharomyces cerevisiae

Mandelic acid (MA) and 4-hydroxymandelic acid (HMA) are valuable specialty chemicals used as precursors for flavors as well as for cosmetic and pharmaceutical purposes. Today they are mainly synthesized chemically. Their synthesis through microbial fermentation would allow for environmentally sustainable production. In this work, we engineered the yeast Saccharomyces cerevisiae for high-level production of MA and HMA. Expressing the hydroxymandelate synthase from Amycolatopsis orientalis in a yeast wild type strain resulted in the production of 119mg/L HMA from glucose. As the enzyme also accepts phenylpyruvate as a substrate aside from its native substrate 4-hydroxyphenylpyruvate, 0.7mg/L MA was also produced. Preventing binding of 4-hydroxyphenylpyruvate to the hydroxymandelate synthase by introducing a S201V replacement in its substrate binding site nearly completely prevented HMA production but increased MA production only 3.5-fold. To further increase HMA and MA production, the aromatic amino acid pathway was engineered. We increased the precursor supply by introducing modifications in the shikimic acid pathway (ARO1↑, ARO3K222L↑, ARO4K220L↑) and reducing flux into the Ehrlich pathway (aro10Δ), and thereby enhanced the HMA titer to 465mg/L and the MA titer to 2.9mg/L. A further increase in HMA and MA titers was achieved by replacing the hydroxymandelate synthase from A. orientalis with the corresponding enzyme from Nocardia uniformis. Subsequently, we introduced additional deletions to block the competing tryptophan branch (trp2Δ), to further decrease flux into the Ehrlich pathway (pdc5Δ) and to avoid transamination of phenylpyruvate and 4-hydroxyphenylpyruvate (aro8Δ, aro9Δ). We achieved more than 1g/L 4-hydroxymandelate when additionally preventing formation of phenylpyruvate by deleting PHA2. When deleting TYR1 to prevent formation of 4-hydroxyphenylpyruvate instead, an MA titer of 236mg/L was achieved. This is a more than 200-fold increase in MA production compared to the wild type strain expressing the hydroxymandelate synthase from A. orientalis. Finally, we showed that S. cerevisiae tolerates HMA and MA to concentrations as high as 3g/L and 7.5g/L, respectively. Our results demonstrate that S. cerevisiae is a promising host for sustainable MA and HMA production.

A Theoretical Study on the Mechanism of Decarboxylations for Hydroxymandelate Synthase

Hydroxymandelate synthase is one type of alpha‐keto acid‐dependent oxygenases and plays an important role in the biosynthesis of hydroxyphenyl‐pyruvate. The ferrous and ferryl intermediates in the reaction were considered to be very important in the DNA repair process. The mechanism of hydroxymandelate synthase was investigated by a theoretical method based on B3LYP/LACVP*. The data indicated that each decarboxylation went through a process of direct dioxygen insertion. The first oxidative decarboxylation experienced only one transition state, while there were two potential pathways for the second decarboxylation process. The calculation data showed that the stepwise one was favored to the concerted one due to the lower energy barrier. The process to form the iron‐superoxide was the rate‐limiting step. After two consecutive decarboxylations, a proton transfer reaction was needed to obtain the target products. Two possible transition states were found out to achieve this step. The one with a six‐membered ring structure was preferred because of the lower activation energy. In addition, several ferryl species with high spins were captured in the whole process, which could be supplied as requisite substrates for DNA repair reaction.

Engineering Escherichia coli for renewable benzyl alcohol production

Benzyl alcohol is an aromatic hydrocarbon used as a solvent and an intermediate chemical in the pharmaceutical, cosmetics, and flavor/fragrance industries. The de novo biosynthesis of benzyl alcohol directly from renewable glucose was herein explored using a non-natural pathway engineered in Escherichia coli. Benzaldehyde was first produced from endogenous phenylpyruvate via three heterologous steps, including hydroxymandelate synthase (encoded by hmaS) from Amycolatopsis orientalis, followed by (S)-mandelate dehydrogenase (encoded by mdlB) and phenylglyoxylate decarboxylase (encoded by mdlC) from Pseudomonas putida ATCC 12633. The subsequent rapid and efficient reduction of benzaldehyde to benzyl alcohol occurred by the combined activity and native regulation of multiple endogenous alcohol dehydrogenases and/or aldo-keto reductases. Through systematic deletion of competing aromatic amino acid biosynthesis pathways to promote endogenous phenylpyruvate availability, final benzyl alcohol titers as high as 114±1mg/L were realized, representing a yield of 7.6±0.1mg/g on glucose and a ~5-fold improvement over initial strains.

Dioxygen activation and two consecutive oxidative decarboxylations of phenylpyruvate by nonheme iron(ii) complexes: functional models of hydroxymandelate synthase (HMS) and CloR

Two mononuclear iron(ii)-phenylpyruvate complexes of monoanionic facial N3 ligands are reported to react with dioxygen to undergo two consecutive oxidative decarboxylation steps via an iron-mandelate complex mimicking the function of HMS and CloR.

Role of substrate positioning in the catalytic reaction of 4-hydroxyphenylpyruvate dioxygenase-A QM/MM Study.

Ring hydroxylation and coupled rearrangement reactions catalyzed by 4-hydroxyphenylpyruvate dioxygenase were studied with the QM/MM method ONIOM(B3LYP:AMBER). For electrophilic attack of the ferryl species on the aromatic ring, five channels were considered: attacks on the three ring atoms closest to the oxo ligand (C1, C2, C6) and insertion of oxygen across two bonds formed by them (C1-C2, C1-C6). For the subsequent migration of the carboxymethyl substituent, two possible directions were tested (C1→C2, C1→C6), and two different mechanisms were sought (stepwise radical, single-step heterolytic). In addition, formation of an epoxide (side)product and benzylic hydroxylation, as catalyzed by the closely related hydroxymandelate synthase, were investigated. From the computed reaction free energy profiles it follows that the most likely mechanism of 4-hydroxyphenylpyruvate dioxygenase involves electrophilic attack on the C1 carbon of the ring and subsequent single-step heterolytic migration of the substituent. Computed values of the kinetic isotope effect for this step are inverse, consistent with available experimental data. Electronic structure arguments for the preferred mechanism of attack on the ring are also presented.

Intermediate Partitioning Kinetic Isotope Effects for the NIH Shift of 4-Hydroxyphenylpyruvate Dioxygenase and the Hydroxylation Reaction of Hydroxymandelate Synthase Reveal Mechanistic Complexity

4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) are similar enzymes that catalyze complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. Both enzymes decarboxylate HPP and then hydroxylate the resulting hydroxyphenylacetate (HPA). The hydroxylation reaction catalyzed by HPPD displaces the aceto substituent of HPA in a 1,2-shift to form 2,5-dihydroxyphenylacetate (homogentisate, HG), whereas the hydroxylation reaction of HMS places a hydroxyl on the benzylic carbon forming 3'-hydroxyphenylacetate (S-hydroxymandelate, HMA) without ensuing chemistry. The wild-type form of HPPD and variants of both enzymes uncouple to form both native and non-native products. We have used intermediate partitioning to probe bifurcating steps that form these products by substituting deuteriums for protiums at the benzylic position of the HPP substrate. These substitutions result in altered ratios of products that can be used to calculate kinetic isotope effects (KIE) for the formation of a specific product. For HPPD, secondary normal KIEs indicate that cleavage of the bond in the displacement reaction prior to the shift occurs by a homolytic mechanism. NMR analysis of HG derived from HPPD reacting with enantiomerically pure R-3'-deutero-HPP indicates that no rotation about the bond to the radical occurs, suggesting that collapse of the biradical intermediate is rapid. The production of HMA was observed in HMS and HPPD variant reactions. HMS hydroxylates to form exclusively S-hydroxymandelate. When HMS is reacted with R-3'-deutero-HPP, the observed kinetic isotope effect represents geometry changes in the initial transition state for the nonabstracted proton. These data show evidence of sp(3) hybridization in a HPPD variant and sp(2) hybridization in HMS variants, suggesting that HMS stabilizes a more advanced transition state in order to catalyze H-atom abstraction.

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

(S)-Hydroxymandelate synthase (Hms) is a nonheme Fe(II) dependent dioxygenase that catalyzes the oxidation of 4-hydroxyphenylpyruvate to (S)-4-hydroxymandelate by molecular oxygen. In this work, the substrate promiscuity of Hms is characterized in order to assess its potential for the biosynthesis of chiral α-hydroxy acids. Enzyme kinetic analyses, the characterization of product spectra, quantitative structure activity relationship (QSAR) analyses and in silico docking studies are used to characterize the impact of substrate properties on particular steps of catalysis. Hms is found to accept a range of α-oxo acids, whereby the presence of an aromatic substituent is crucial for efficient substrate turnover. A hydrophobic substrate binding pocket is identified as the likely determinant of substrate specificity. Upon introduction of a steric barrier, which is suspected to obstruct the accommodation of the aromatic ring in the hydrophobic pocket during the final hydroxylation step, the racemization of product is obtained. A steady state kinetic analysis reveals that the turnover number of Hms strongly correlates with substrate hydrophobicity. The analysis of product spectra demonstrates high regioselectivity of oxygenation and a strong coupling efficiency of C-C bond cleavage and subsequent hydroxylation for the tested substrates. Based on these findings the structural basis of enantioselectivity and enzymatic activity is discussed.

Mechanism of Benzylic Hydroxylation by 4-Hydroxymandelate Synthase. A Computational Study

Hydroxymandelate synthase (HMS) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) are highly related enzymes using the same substrates but catalyzing hydroxylation reactions yielding different products. The first steps of the HMS and HPPD catalytic reactions are believed to proceed in the same way and lead to an Fe(IV)═O-hydroxyphenylacetate (HPA) intermediate. Further down the catalytic cycles, HMS uses Fe(IV)═O to perform hydroxylation of the benzylic carbon, whereas in HPPD, the reactive oxoferryl intermediate attacks the aromatic ring of HPA. This study focuses on this part of the HMS catalytic cycle that starts from the oxoferryl intermediate and aims to identify interactions within the active site that are responsible for enzyme specificity. To this end, a HMS-Fe(IV)═O-HPA complex was modeled with molecular dynamics simulations. On the basis of the molecular dynamics-equilibrated structure, an active site model suitable for quantum chemical investigations was constructed and used for density functional theory (B3LYP) calculations of the mechanism of the native reaction of HMS, i.e., benzylic hydroxylation, and the alternative electrophilic attack on the ring, which is a step of the HPPD catalytic cycle. The most important result of this study is the finding that the conformation of the Ser201 side chain in the second coordination shell has a key role in directing the reaction of Fe(IV)═O into either the HMS or the HPPD channel.

Metabolic engineering of the L-phenylalanine pathway in Escherichia coli for the production of S- or R-mandelic acid

BackgroundMandelic acid (MA), an important component in pharmaceutical syntheses, is currently produced exclusively via petrochemical processes. Growing concerns over the environment and fossil energy costs have inspired a quest to develop alternative routes to MA using renewable resources. Herein we report the first direct route to optically pure MA from glucose via genetic modification of the L-phenylalanine pathway in E. coli.ResultsThe introduction of hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis into E. coli led to a yield of 0.092 g/L S-MA. By combined deletion of competing pathways, further optimization of S-MA production was achieved, and the yield reached 0.74 g/L within 24 h. To produce R-MA, hydroxymandelate oxidase (Hmo) from Streptomyces coelicolor and D-mandelate dehydrogenase (DMD) from Rhodotorula graminis were co-expressed in an S-MA-producing strain, and the resulting strain was capable of producing 0.68 g/L R-MA. Finally, phenylpyruvate feeding experiments suggest that HmaS is a potential bottleneck to further improvement in yields.ConclusionsWe have constructed E. coli strains that successfully accomplished the production of S- and R-MA directly from glucose. Our work provides the first example of the completely fermentative production of S- and R-MA from renewable feedstock.

Evidence for the Mechanism of Hydroxylation by 4-Hydroxyphenylpyruvate Dioxygenase and Hydroxymandelate Synthase from Intermediate Partitioning in Active Site Variants

4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) each catalyze similar complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. The reactions differ in that HPPD hydroxylates at the ring C1 and HMS at the benzylic position. The HPPD reaction is more complex in that hydroxylation at C1 instigates a 1,2-shift of an aceto substituent. Despite that multiple intermediates have been observed to accumulate in single turnover reactions of both enzymes, neither enzyme exhibits significant accumulation of the hydroxylating intermediate. In this study we employ a product analysis method based on the extents of intermediate partitioning with HPP deuterium substitutions to measure the kinetic isotope effects for hydroxylation. These data suggest that, when forming the native product homogentisate, the wild-type form of HPPD produces a ring epoxide as the immediate product of hydroxylation but that the variant HPPDs tended to also show the intermediacy of a benzylic cation for this step. Similarly, the kinetic isotope effects for the other major product observed, quinolacetic acid, showed that either pathway is possible. HMS variants show small normal kinetic isotope effects that indicate displacement of the deuteron in the hydroxylation step. The relatively small magnitude of this value argues best for a hydrogen atom abstraction/rebound mechanism. These data are the first definitive evidence for the nature of the hydroxylation reactions of HPPD and HMS.

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

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.

We two alone will sing: the two-substrate α-keto acid-dependent oxygenases

4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) are the only two known members of the alpha-keto acid-dependent oxygenases that adopt the fold common to the vicinal oxygen chelate superfamily of enzymes. All other oxygenases of this type have a jellyroll fold. Despite a clearly different ancestry, the salient details of HPPD and HMS catalysis are the same as all alpha-keto acid-dependent oxygenases. All alpha-keto acid-dependent oxygenases use reducing equivalents from an alpha-keto acid to reduce dioxygen via an active site ferrous ion mediator and then catalyze decarboxylation of the alpha-keto acid to promote the formation of a high valence iron-oxo species. The most common purpose of which is to then hydroxylate the substrate. This mini-review summarizes the structural and mechanistic data assembled in recent years for HPPD and HMS in the context of both their roles in nature and the vicinal oxygen chelate and alpha-keto acid-dependent oxygenases superfamilies.

Two Roads Diverged: The Structure of Hydroxymandelate Synthase from Amycolatopsis orientalis in Complex with 4-Hydroxymandelate,

The crystal structure of the hydroxymandelate synthase (HMS).Co2+.hydroxymandelate (HMA) complex determined to a resolution of 2.3 A reveals an overall fold that consists of two similar beta-barrel domains, one of which contains the characteristic His/His/acid metal-coordination motif (facial triad) found in the majority of Fe2+-dependent oxygenases. The fold of the alpha-carbon backbone closely resembles that of the evolutionarily related enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) in its closed conformation with a root-mean-square deviation of 1.85 A. HPPD uses the same substrates as HMS but forms instead homogentisate (HG). The active site of HMS is significantly smaller than that observed in HPPD, reflecting the relative changes in shape that occur in the conversion of the common HPP substrate to the respective HMA or HG products. The HMA benzylic hydroxyl and carboxylate oxygens coordinate to the Co2+ ion, and three other potential H-bonding interactions to active site residue side chains are observed. Additionally, it is noted that there is a buried well-ordered water molecule 3.2 A from the distal carboxylate oxygen. The p-hydroxyl group of HMA is within hydrogen-bonding distance of the side chain hydroxyl of a serine residue (Ser201) that is conserved in both HMS and HPPD. This potential hydrogen bond and the known geometry of iron ligation for the substrate allowed us to model 4-hydroxyphenylpyruvate (HPP) in the active sites of both HMS and HPPD. These models suggest that the position of the HPP substrate differs between the two enzymes. In HMS, HPP binds analogously to HMA, while in HPPD, the p-hydroxyl group of HPP acts as a hydrogen-bond donor and acceptor to Ser201 and Asn216, respectively. It is suggested that this difference in the ring orientation of the substrate and the corresponding intermediates influences the site of hydroxylation.

The interaction of hydroxymandelate synthase with the 4-hydroxyphenylpyruvate dioxygenase inhibitor: NTBC

Hydroxymandelate synthase (HMS) catalyzes the committed step in the formation of para-hydroxyphenylglycine, a recurrent substructure of polycyclic non-ribosomal peptide antibiotics such as vancomycin. HMS uses the same substrates as 4-hydroxyphenylpyruvate dioxygenase (HPPD), 4-hydroxyphenylpyruvate (HPP) and O 2, and also conducts a dioxygenation reaction. The difference between the two lies in the insertion of the second oxygen atom, HMS directing this atom onto the benzylic carbon of the substrate while HPPD hydroxylates the aromatic C1 carbon. We have shown that HMS will bind NTBC, a herbicide/therapeutic whose mode of action is based on the inhibition of HPPD. This occurs despite residue differences at the active site of HMS from those known to contact the inhibitor in HPPD. Moreover, the minimal kinetic mechanism for association of NTBC to HMS differs only slightly from that observed with HPPD. The primary difference is that three charge-transfer species are observed to accumulate during association. The first reversible complex forms with a weak dissociation constant of 520 μM, the subsequent two charge-transfer complexes form with rate constants of 2.7 s −1 and 0.67 s −1. As was the case for HPPD, the final complex has the most intense charge-transfer, is not observed to dissociate, and is unreactive towards dioxygen.

Conversion of hydroxyphenylpyruvate dioxygenases into hydroxymandelate synthases by directed evolution

Hydroxymandelate synthase (HmaS) and hydroxyphenylpyruvate dioxygenase (HppD) are non-heme iron-dependent dioxygenases, which share a common substrate and first catalytic step. The catalytic pathways then diverge to yield hydroxymandelate for secondary metabolism, or homogentisate in tyrosine catabolism. To probe the differences between these related active sites that channel a common intermediate down alternative pathways, we attempted to interconvert their activities by directed evolution. HmaS activity was readily introduced to HppD by just two amino acid changes. A parallel attempt to engineer HppD activity in HmaS was unsuccessful, suggesting that homogentisate synthesis places greater chemical and steric demands on the active site.

Engineering p-hydroxyphenylpyruvate dioxygenase to a p-hydroxymandelate synthase and evidence for the proposed benzene oxide intermediate in homogentisate formation.

p-Hydroxyphenylpyruvate dioxygenase (HPD) plays a key role in the normal catabolism of tyrosine. An Fe2+/oxygen-dependent enzyme, it converts p-hydroxyphenylpyruvate into homogentisate and is part of the superfamily of alpha-ketoglutarate-dependent enzymes that couples oxidative decarboxylation of an alpha-ketoacid cofactor to oxidative modification of its substrate. In this case, the alpha-ketoacid is part of the substrate side chain. HPD shows strong homology to p-hydroxymandelate synthase (HMS), an enzyme that catalyzes the formation of p-hydroxymandelate from p-hydroxyphenylpyruvate, an early step in the biosynthesis of p-hydroxyphenylglycine, which is a nonproteinogenic amino acid incorporated into several biologically active secondary metabolites. Sequence alignment between the HPD and the HMS enzyme families and analysis of the Pseudomonas fluorescens HPD crystal structure highlighted four residues within each active site that may play roles in catalytic differentiation between the two products. We attempted to convert Streptomyces avermitilis HPD into an engineered S. avermitilis HMS by site-directed mutagenesis of these four residues individually and in combination. HPLC assay analysis of each His6-tagged mutant indicated that F337I successfully produced p-hydroxymandelate, along with homogentisate and an unknown compound. The structure of the latter was determined to be an oxepinone derived from the benzene-oxide intermediate long hypothesized in HPD catalysis.