Structure and proposed mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae

The formation of hydrogen peroxide (H₂O₂) by the FAD-dependent α-glycerophosphate oxidase (GlpO), is important for the pathogenesis of Streptococcus pneumoniae and Mycoplasma pneumoniae. The structurally known GlpO from Streptococcus sp. (SspGlpO) is similar to the pneumococcal protein (SpGlpO) and provides a guide for drug design against that target. However, M. pneumoniae GlpO (MpGlpO), having <20% sequence identity with structurally known GlpOs, appears to represent a second type of GlpO we designate as Type II GlpOs. Here, the recombinant His-tagged MpGlpO structure is described at ~2.5 Å resolution, solved by molecular replacement using as a search model the Bordetella pertussis protein 3253 (Bp3253) a protein of unknown function solved by structural genomics efforts. Recombinant MpGlpO is an active oxidase with a turnover number of ~580 min⁻¹ while Bp3253 showed no GlpO activity. No substantial differences exist between the oxidized and dithionite-reduced MpGlpO structures. Although, no liganded structures were determined, a comparison with the tartrate-bound Bp3253 structure and consideration of residue conservation patterns guided the construction of a model for α-glycerophosphate (Glp) recognition and turnover by MpGlpO. The predicted binding mode also appears relevant for the type I GlpOs (such as SspGlpO) despite differences in substrate recognition residues, and it implicates a histidine conserved in type I and II Glp oxidases and dehydrogenases as the catalytic acid/base. This work provides a solid foundation for guiding further studies of the mitochondrial Glp dehydrogenases as well as for continued studies of M. pneumoniae and S. pneumoniae glycerol metabolism and the development of novel therapeutics targeting MpGlpO and SpGlpO.Keywords: drug design, flavoenzyme, protein evolution, GlpA, hydride transfe

Kinetic Mechanism of L-α-Glycerophosphate Oxidase from Mycoplasma pneumoniae

L-α-glycerophosphate oxidase is an FAD-dependent enzyme that catalyzes the oxidation of L-α-glycerophosphate (Glp) by molecular oxygen to generate dihydroxyacetone phosphate (DHAP) and hydrogen peroxide (H₂O₂). The catalytic properties of the recombinant His₆-GlpO from Mycoplasma pneumoniae (His₆-MpGlpO) were investigated with transient and steady-state kinetics and ligand binding. The results indicate that the reaction mechanism of His₆-MpGlpO follows a ping-pong model. Double-mixing stopped-flow experiments show that after flavin-mediate substrate oxidation, DHAP leaves rapidly prior to the oxygen reaction. The values of the individual rate constants and k [subscript]cat (4.2 s⁻¹ at 4 °C) determined, in addition to the finding that H₂O₂ can bind to the oxidized enzyme suggest that H₂O₂ release is the rate-limiting step for the overall reaction. Results indicate that His₆-MpGlpO contains mixed populations of fast and slow reacting species. Only the fast reacting species predominantly participates in turnovers. Different from other GlpO enzymes previously reported, His₆-MpGlpO can catalyze the reverse reaction of reduced enzyme and DHAP. This result can be explained by the standard reduction potential value of His₆-MpGlpO (-167 ± 1 mV), which is lower than those of GlpO from other species. We found that DL-glyceraldehyde 3-phosphate (GAP) can be used as a substrate in the His₆-MpGlpO reaction, although it exhibited a ~100-fold lower k[subscript]cat value in comparison to the reaction of Glp. These results also imply the involvement of GlpO in glycolysis, as well as in lipid and glycerol metabolism. The kinetic models and distinctive properties of His₆-MpGlpO reported here should be useful for future studies of drug development against Mycoplasma pneumoniae infection

The enigmatic reaction of flavins with oxygen

The reaction of flavoenzymes with oxygen remains a fascinating area of research because of its relevance for reactive oxygen species (ROS) generation. Several exciting recent studies provide consistent mechanistic clues about the specific functional and structural properties of the oxidase and monooxygenase flavoenzymatic systems. Specifically, the spatial arrangement of the reacting oxygen that is in direct contact with the flavin group is emerging as a crucial factor that differentiates between oxidase and monooxygenase enzymes. A challenge for the future will be to use these emerging concepts to rationally engineer flavoenzymes, paving the way to new research avenues with far-reaching implications for oxidative biocatalysis and metabolic engineering. Copyright 2012 Elsevier Ltd. All rights reserved

Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate 3-Hydroxylase from 'Acinetobacter baumannii'

p-Hydroxyphenylacetate hydroxylase (HPAH) from 'Acinetobacter baumannii' catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA).The enzyme system is composed of two proteins: an FMN reductase (C₁) and an oxygenase that uses FMNH⁻ (C₂). We report detailed transient kinetics studies at 4°C of the reaction mechanism of C₂. C₂ binds rapidly and tightly to reduced FMN (Kd, 1.2 ± 0.2μM), but less tightly to oxidized FMN (Kd, 250 ± 50μM). The complex of C₂-FMNH⁻ reacted with oxygen to form C(4a)-hydroperoxy-FMN at 1.1 ± 0.1 x 10⁶M⁻¹S⁻¹, whereas the C₂-FMNH⁻-HPA complex reacted with oxygen to form C(4a)-hydroperoxy-FMN-HPA more slowly (k=4.8±0.2 x 10⁴M⁻¹S⁻¹).The kinetic mechanism of C₂was shown to be a preferential random order type, in which HPA or oxygen can initially bind to the C₂-FMNH⁻ complex, but the preferred path was oxygen reacting with C₂-FMNH⁻ to form the C(4a)-hydroperoxy-FMN intermediate prior to HPA binding. Hydroxylation occurs from the ternary complex with a rate constant of 20S⁻¹ to form the C₂-C(4a)-hydroxy-FMN-DHPA complex. At high HPA concentrations (&gt;0.5mM), HPA formed a dead end complex with the C₂-C(4a)-hydroxy-FMN intermediate (similar to single component flavoprotein hydroxylases), thus inhibiting the bound flavin from returning to the oxidized form. When FADH⁻ was used, C(4a)-hydroperoxy-FAD, C(4a)-hydroxy-FAD, and product were formed at rates similar to those with FMNH⁻. Thus, C₂ has the unusual ability to use both common flavin cofactors in catalysis

The Reductase of 'p'-Hydroxyphenylacetate 3-Hydroxylase from 'Acinetobacter baumannii' Requires 'p'-Hydroxyphenylacetate for Effective Catalysis

p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) from 'Acinetobacter baumannii' catalyzes hydroxylation of HPA to form 3,4-dihydroxyphenylacetate. It is a two protein system consisting of a smaller reductase component (C₁) and a larger oxygenase component (C₂). C₁ is a flavoprotein containing FMN, and its function is to provide reduced flavin for C₂ to hydroxylate HPA. We have shown here that HPA plays important roles in the reaction of C₁. The apoenzyme of C₁ binds to oxidized FMN tightly with a Kd of 0.006 μM at 4 °C, but with a Kd of 0.038 μM in the presence of HPA. Reduction of C₁ by NADH occurs in two phases with rate constants of 11.6 and 3.1 s⁻¹ and Kd values for NADH binding of 2.1 and 1.5 mM, respectively. This result indicates that C₁ exists as a mixture of isoforms. However, in the presence of HPA, the reduction of C₁ by NADH occurred in a single phase at 300 s⁻¹ with a Kd of 25 μM for NADH binding at 4 °C. Formation of the C₁-HPA complex prior to binding of NADH was required for this stimulation. The redox potentials indicate that the rate enhancement is not due to thermodynamics (E°m of the C₁-HPA complex is -245 mV compared to an E°m of C₁ of -236 mV). When the C₁-HPA complex was reduced by 4(S)-NADH, the reduction rate was changed from 300 to 30 s⁻¹, giving a primary isotope effect of 10 and indicating that C₁ is specifically reduced by the pro-(S)-hydride. In the reaction of reduced C₁ with oxygen, the reoxidation reaction is also biphasic, consistent with reduced C₁ being a mixture of fast and slow reacting species. Rate constants for both phases were the same in the absence and presence of HPA, but in the presence of HPA, the equilibrium shifted toward the faster reacting species

Stabilization of C4a-Hydroperoxyflavin in a Two-component Flavin-dependent Monooxygenase Is Achieved through Interactions at Flavin N5 and C4a Atoms

p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavin-dependent monooxygenase. Based on the crystal structure of the oxygenase component (C(2)), His-396 is 4.5 Å from the flavin C4a locus, whereas Ser-171 is 2.9 Å from the flavin N5 locus. We investigated the roles of these two residues in the stability of the C4a-hydroperoxy-FMN intermediate. The results indicated that the rate constant for C4a-hydroperoxy-FMN formation decreased ∼30-fold in H396N, 100-fold in H396A, and 300-fold in the H396V mutant, compared with the wild-type enzyme. Lesser effects of the mutations were found for the subsequent step of H(2)O(2) elimination. Studies on pH dependence showed that the rate constant of H(2)O(2) elimination in H396N and H396V increased when pH increased with pK(a) >9.6 and >9.7, respectively, similar to the wild-type enzyme (pK(a) >9.4). These data indicated that His-396 is important for the formation of the C4a-hydroperoxy-FMN intermediate but is not involved in H(2)O(2) elimination. Transient kinetics of the Ser-171 mutants with oxygen showed that the rate constants for the H(2)O(2) elimination in S171A and S171T were ∼1400-fold and 8-fold greater than the wild type, respectively. Studies on the pH dependence of S171A with oxygen showed that the rate constant of H(2)O(2) elimination increased with pH rise and exhibited an approximate pK(a) of 8.0. These results indicated that the interaction of the hydroxyl group side chain of Ser-171 and flavin N5 is required for the stabilization of C4a-hydroperoxy-FMN. The double mutant S171A/H396V reacted with oxygen to directly form the oxidized flavin without stabilizing the C4a-hydroperoxy-FMN intermediate, which confirmed the findings based on the single mutation that His-396 was important for formation and Ser-171 for stabilization of the C4a-hydroperoxy-FMN intermediate in C(2)