Genetic redundancy of 4-hydroxybenzoate 3-hydroxylase genes ensures the catabolic safety of Pigmentiphaga sp. H8 in 3-bromo-4-hydroxybenzoate-contaminated habitats.

Genetic redundancy is prevalent in organisms and plays important roles in the evolution of biodiversity and adaptation to environmental perturbation. However, selective advantages of genetic redundancy in overcoming metabolic disturbance due to structural analogues have received little attention. Here, functional divergence of the three 4-hydroxybenzoate 3-hydroxylase (PHBH) genes (phbh1~3) was found in Pigmentiphaga sp. strain H8. The genes phbh1/phbh2 were responsible for 3-bromo-4-hydroxybenzoate (3-Br-4-HB, an anthropogenic pollutant) catabolism, whereas phbh3 was primarily responsible for 4-hydroxybenzoate (4-HB, a natural intermediate of lignin) catabolism. 3-Br-4-HB inhibited 4-HB catabolism by competitively binding PHBH3 and was toxic to strain H8 cells especially at high concentrations. The existence of phbh1/phbh2 not only enabled strain H8 to utilize 3-Br-4-HB but also ensured the catabolic safety of 4-HB. Molecular docking and site-directed mutagenesis analyses revealed that Val199 and Phe384 of PHBH1/PHBH2 were required for the hydroxylation activity towards 3-Br-4-HB. Phylogenetic analysis indicated that phbh1 and phbh2 originated from a common ancestor and evolved specifically in strain H8 to adapt to 3-Br-4-HB-contaminated habitats, whereas phbh3 evolved independently. This study deepens our understanding of selective advantages of genetic redundancy in prokaryote's metabolic robustness and reveals the factors driving the divergent evolution of redundant genes in adaptation to environmental perturbation.

Understanding the Molecular Mechanism Underlying the High Catalytic Activity of p-Hydroxybenzoate Hydroxylase Mutants for Producing Gallic Acid.

p-Hydroxybenzoate hydroxylase (PHBH) is a flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (p-OHB) to 3,4-dihydroxybenzoate (3,4-DOHB). PHBH can bind to other benzoate derivatives in addition to p-OHB; however, hydroxylation does not occur on 3,4-DOHB. Replacement of Tyr385 with Phe forms a mutant, which enables the production of 3,4,5-trihydroxybenzonate (gallic acid) from 3,4-DOHB, although the catalytic activity of the mutant is quite low. In this study, we report how the L199V/Y385F double mutant exhibits activity for producing gallic acid 4.3-fold higher than that of the Y385F single mutant. This improvement in catalytic activity is primarily due to the suppression of a shunt reaction that wastes reduced nicotinamide adenine dinucleotide phosphate by producing H2O2. To further elucidate the molecular mechanism underlying this higher catalytic activity, we performed molecular dynamics simulations and quantum mechanics/molecular mechanics calculations, in addition to determining the crystal structure of the Y385F·3,4-DOHB complex. The simulations showed that the Y385F mutation facilitates the deprotonation of the 4-hydroxy group of 3,4-DOHB, which is necessary for initiating hydroxylation. Moreover, the L199V mutation in addition to the Y385F mutation allows the OH moiety in the peroxide group of C-(4a)-flavin hydroperoxide to come into the proximity of the C5 atom of 3,4-DOHB. Overall, this study provides a consistent explanation for the change in the catalytic activity of PHBH caused by mutations, which will enable us to better design an enzyme with different activities.

Hydroxyl Radical-Coupled Electron-Transfer Mechanism of Flavin-Dependent Hydroxylases.

Class A flavin-dependent hydroxylases (FdHs) catalyze the hydroxylation of organic compounds in a site- and stereoselective manner. In stark contrast, conventional synthetic routes require environmentally hazardous reagents and give modest yields. Thus, understanding the detailed mechanism of this class of enzymes is essential to their rational manipulation for applications in green chemistry and pharmaceutical production. Both electrophilic substitution and radical intermediate mechanisms have been proposed as interpretations of FdH hydroxylation rates and optical spectra. While radical mechanistic steps are often difficult to examine directly, modern quantum chemistry calculations combined with statistical mechanical approaches can yield detailed mechanistic models providing insights that can be used to differentiate reaction pathways. In the current work, we report quantum mechanical/molecular mechanical (QM/MM) calculations on the fungal TropB enzyme that shows an alternative reaction pathway in which hydroxylation through a hydroxyl radical-coupled electron-transfer mechanism is significantly favored over electrophilic substitution. Furthermore, QM/MM calculations on several modified flavins provide a more consistent interpretation of the experimental trends in the reaction rates seen experimentally for a related enzyme, para-hydroxybenzoate hydroxylase. These calculations should guide future enzyme and substrate design strategies and broaden the scope of biological spin chemistry.

Structural comparison of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida with PobA from other Pseudomonas spp. and other monooxygenases.

The crystal structure is reported of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida, a possible drug target to combat tetracycline resistance, in complex with flavin adenine dinucleotide (FAD). The structure was refined at 2.2 Šresolution with four polypeptide chains in the asymmetric unit. Based on the results of pairwise structure alignments, PobA from P. putida is structurally very similar to PobA from P. fluorescens and from P. aeruginosa. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. Additionally, the structure was compared with two enzymes from the broader class of oxygenases: 2-hydroxybiphenyl 3-monooxygenase (HbpA) from P. nitroreducens and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) from Mesorhizobium japonicum. Despite having only 14% similarity in their primary sequences, pairwise structure alignments of PobA from P. putida with HbpA from P. nitroreducens and MHPCO from M. japonicum revealed local similarities between these structures. Key secondary-structure elements important for catalysis, such as the ßαß fold, ß-sheet wall and α12 helix, are conserved across this expanded class of oxygenases.

PqsL uses reduced flavin to produce 2-hydroxylaminobenzoylacetate, a preferred PqsBC substrate in alkyl quinolone biosynthesis in Pseudomonas aeruginosa.

Alkyl hydroxyquinoline N-oxides (AQNOs) are antibiotic compounds produced by the opportunistic bacterial pathogen Pseudomonas aeruginosa They are products of the alkyl quinolone (AQ) biosynthetic pathway, which also generates the quorum-sensing molecules 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS). Although the enzymatic synthesis of HHQ and PQS had been elucidated, the route by which AQNOs are synthesized remained elusive. Here, we report on PqsL, the key enzyme for AQNO production, which structurally resembles class A flavoprotein monooxygenases such as p-hydroxybenzoate 3-hydroxylase (pHBH) and 3-hydroxybenzoate 6-hydroxylase. However, we found that unlike related enzymes, PqsL hydroxylates a primary aromatic amine group, and it does not use NAD(P)H as cosubstrate, but unexpectedly required reduced flavin as electron donor. We also observed that PqsL is active toward 2-aminobenzoylacetate (2-ABA), the central intermediate of the AQ pathway, and forms the unstable compound 2-hydroxylaminobenzoylacetate, which was preferred over 2-ABA as substrate of the downstream enzyme PqsBC. In vitro reconstitution of the PqsL/PqsBC reaction was feasible by using the FAD reductase HpaC, and we noted that the AQ:AQNO ratio is increased in an hpaC-deletion mutant of P. aeruginosa PAO1 compared with the ratio in the WT strain. A structural comparison with pHBH, the model enzyme of class A flavoprotein monooxygenases, revealed that structural features associated with NAD(P)H binding are missing in PqsL. Our study completes the AQNO biosynthetic pathway in P. aeruginosa, indicating that PqsL produces the unstable product 2-hydroxylaminobenzoylacetate from 2-ABA and depends on free reduced flavin as electron donor instead of NAD(P)H.

A peculiar IclR family transcription factor regulates para-hydroxybenzoate catabolism in Streptomyces coelicolor.

In Streptomyces coelicolor, we identified a para-hydroxybenzoate (PHB) hydroxylase, encoded by gene pobA (SCO3084), which is responsible for conversion of PHB into PCA (protocatechuic acid), a substrate of the ß-ketoadipate pathway which yields intermediates of the Krebs cycle. We also found that the transcription of pobA is induced by PHB and is negatively regulated by the product of SCO3209, which we named PobR. The product of this gene is highly unusual in that it is the apparent fusion of two IclR family transcription factors. Bioinformatic analyses, in vivo transcriptional assays, electrophoretic mobility shift assays (EMSAs), DNase I footprinting, and isothermal calorimetry (ITC) were used to elucidate the regulatory mechanism of PobR. We found that PobR loses its high affinity for DNA (i.e., the pobA operator) in the presence of PHB, the inducer of pobA transcription. PHB binds to PobR with a KD of 5.8 µM. Size-exclusion chromatography revealed that PobR is a dimer in the absence of PHB and a monomer in the presence of PHB. The crystal structure of PobR in complex with PHB showed that only one of the two IclR ligand binding domains was occupied, and defined how the N-terminal ligand binding domain engages the effector ligand.

Hybrid Quantum Mechanics/Molecular Mechanics/Coarse Grained Modeling: A Triple-Resolution Approach for Biomolecular Systems.

We present a hybrid quantum mechanics/molecular mechanics/coarse-grained (QM/MM/CG) multiresolution approach for solvated biomolecular systems. The chemically important active-site region is treated at the QM level. The biomolecular environment is described by an atomistic MM force field, and the solvent is modeled with the CG Martini force field using standard or polarizable (pol-CG) water. Interactions within the QM, MM, and CG regions, and between the QM and MM regions, are treated in the usual manner, whereas the CG-MM and CG-QM interactions are evaluated using the virtual sites approach. The accuracy and efficiency of our implementation is tested for two enzymes, chorismate mutase (CM) and p-hydroxybenzoate hydroxylase (PHBH). In CM, the QM/MM/CG potential energy scans along the reaction coordinate yield reaction energies that are too large, both for the standard and polarizable Martini CG water models, which can be attributed to adverse effects of using large CG water beads. The inclusion of an atomistic MM water layer (10 Å for uncharged CG water and 5 Å for polarizable CG water) around the QM region improves the energy profiles compared to the reference QM/MM calculations. In analogous QM/MM/CG calculations on PHBH, the use of the pol-CG description for the outer water does not affect the stabilization of the highly charged FADHOOH-pOHB transition state compared to the fully atomistic QM/MM calculations. Detailed performance analysis in a glycine-water model system indicates that computation times for QM energy and gradient evaluations at the density functional level are typically reduced by 40-70% for QM/MM/CG relative to fully atomistic QM/MM calculations.

Amperometric bienzyme screen-printed biosensor for the determination of leucine.

Leucine plays an important role in protein synthesis, brain functions, building muscle mass, and helping the body when it undergoes stress. Here, we report a new amperometric bienzyme screen-printed biosensor for the determination of leucine, by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and leucine dehydrogenase (LDH) on a screen-printed electrode with NADP(+) and p-hydroxybenzoate as the cofactors. The detection principle of the sensor is that LDH catalyzes the specific dehydrogenation of leucine by using NADP(+) as a cofactor. The product, NADPH, triggers the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a change in electron concentration at the working carbon electrode, which is detected by the potentiostat. The sensor shows a linear detection range between 10 and 600 µM with a detection limit of 2 µM. The response is reproducible and has a fast measuring time of 5-10 s after the addition of a given concentration of leucine.

Conserved and non-conserved residues and their role in the structure and function of p-hydroxybenzoate hydroxylase.

In order to elucidate the molecular mechanism of the catalytic reaction and enzyme conformation, we substituted 53 conserved residues identified by aligning 92 p-hydroxybenzoate hydroxylase sequences and 19 non-conserved residues selected from crystallographic studies of Pseudomonas fluorescens NBRC14160 p-hydroxybenzoate hydroxylase with 19 other naturally occurring amino acids, yielding a database of 619 active single mutants. The database contained 365 and 254 active single mutants for 44/53 conserved residues and 19 non-conserved residues, respectively; the data included main activity, sub-activity for NADPH and NADPH reaction specificity. Active mutations were not observed for the G14, Q102, G160, E198, R220, R246, N300, F342 and G387 conserved residues, and only one active mutant was obtained at the G9, G11, G187, D286, Y201, R214 and G295 conserved residues and the S13, E32 and R42 non-conserved residues. Only seven active mutants with higher activity than the wild-type enzyme were observed at conserved residues, and only two were observed at non-conserved residues. The 365 mutants at conserved residues included 64 active mutants with higher NADPH reaction specificity than the wild-type enzyme, and some Y181X single mutants exhibited considerable changes in NADPH reaction specificity. A Y181X/L268G double-mutant database was constructed to computationally analyze the effects of these substitutions on structural conformation and function. These results indicated that some conserved or non-conserved residues are important for structural stability or enzyme function.

Umbrella integration with higher-order correction terms.

Umbrella integration is a method to analyze umbrella sampling simulations. It calculates free-energy changes from distributions obtained from molecular dynamics. While it can be formulated on the full sampled distributions, they are generally approximated by normal distributions. This is equivalent to the truncation of a power series of the free energy with respect to the reaction coordinate after the quadratic term or by a truncation of a cumulant expansion. Here, expressions for additional terms in the power series are derived. They can be calculated from the central moments of the distributions. This extension allows to test the approximations in applications.

Structure and ligand binding properties of the epoxidase component of styrene monooxygenase .

Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudomonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 A resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains. The overall architecture is most similar to that of p-hydroxybenzoate hydroxylase (PHBH), although there are some significant differences in secondary structure. Structural comparisons suggest that a large cavity open to the surface forms the FAD binding site. At the base of this pocket is another cavity that likely represents the styrene binding site. Flavin binding and redox equilibria are tightly coupled such that reduced FAD binds apo NSMOA approximately 8000 times more tightly than the oxidized coenzyme. Equilibrium fluorescence and isothermal titration calorimetry data using benzene as a substrate analogue indicate that the oxidized flavin and substrate analogue binding equilibria of NSMOA are linked such that the binding affinity of each is increased by 60-fold when the enzyme is saturated with the other. A much weaker approximately 2-fold positive cooperative interaction is observed for the linked binding equilibria of benzene and reduced FAD. The low affinity of the substrate analogue for the reduced FAD complex of NSMOA is consistent with a preferred reaction order in which flavin reduction and reaction with oxygen precede the binding of styrene, identifying the apoenzyme structure as the key catalytic resting state of NSMOA poised to bind reduced FAD and initiate the oxygen reaction.

Monomer formation and function of p-hydroxybenzoate hydroxylase in reverse micelles and in dimethylsulfoxide/water mixtures.

It has previously been postulated that the dimeric form of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH) is important for catalysis. Here it is demonstrated that the monomeric form of PHBH is active. In a water/AOT/isooctane reverse micellar system, the function of the monomeric and dimeric forms of PHBH could be observed separately by varying the size of the micelles. A considerable decrease in the K(M) value for p-hydroxybenzoate (POHB) was found for monomeric PHBH, accompanied by a 1.5-fold decrease in enzymatic activity. The same tendency was observed when monomers of PHBH were formed by adding DMSO to the buffer. The FAD in PHBH and PHBH labeled with the fluorescence dye Alexa488 was investigated by time-resolved fluorescence anisotropy to observe monomer formation in water/DMSO mixtures. Monomer formation of PHBH occurred gradually with increasing DMSO content in the mixture. Pure PHBH monomers were detected at DMSO concentrations of 30 % (v/v) and higher.

Expression, purification, crystallization and initial crystallographic characterization of the p-hydroxybenzoate hydroxylase from Corynebacterium glutamicum.

p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction and plays an important role in the biodegradation of aromatic compounds. PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH(2)PO(4) and K(2)HPO(4) as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 A on a synchrotron beamline. The crystal belongs to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 94.72, c = 359.68 A, gamma = 120 degrees . The asymmetric unit contains two molecules, corresponding to a packing density of 2.65 A(3) Da(-1). The structure was solved by molecular replacement. Structure refinement is in progress.

Crystal structures of two aromatic hydroxylases involved in the early tailoring steps of angucycline biosynthesis.

Angucyclines are aromatic polyketides produced in Streptomycetes via complex enzymatic biosynthetic pathways. PgaE and CabE from S. sp PGA64 and S. sp. H021 are two related homo-dimeric FAD and NADPH dependent aromatic hydroxylases involved in the early steps of the angucycline core modification. Here we report the three-dimensional structures of these two enzymes determined by X-ray crystallography using multiple anomalous diffraction and molecular replacement, respectively, to resolutions of 1.8 A and 2.7 A. The enzyme subunits are built up of three domains, a FAD binding domain, a domain involved in substrate binding and a C-terminal thioredoxin-like domain of unknown function. The structure analysis identifies PgaE and CabE as members of the para-hydroxybenzoate hydroxylase (pHBH) fold family of aromatic hydroxylases. In contrast to phenol hydroxylase and 3-hydroxybenzoate hydroxylase that utilize the C-terminal domain for dimer formation, this domain is not part of the subunit-subunit interface in PgaE and CabE. Instead, dimer assembly occurs through interactions of their FAD binding domains. FAD is bound non-covalently in the "in"-conformation. The active sites in the two enzymes differ significantly from those of other aromatic hydroxylases. The volumes of the active site are significantly larger, as expected in view of the voluminous tetracyclic angucycline substrates. The structures further suggest that substrate binding and catalysis may involve dynamic rearrangements of the middle domain relative to the other two domains. Site-directed mutagenesis studies of putative catalytic groups in the active site of PgaE argue against enzyme-catalyzed substrate deprotonation as a step in catalysis. This is in contrast to pHBH, where deprotonation/protonation of the substrate has been suggested as an essential part of the enzymatic mechanism.

A systematic and comprehensive combinatorial approach to simultaneously improve the activity, reaction specificity, and thermal stability of p-hydroxybenzoate hydroxylase.

We have simultaneously improved the activity, reaction specificity, and thermal stability of p-hydroxybenzoate hydroxylase by means of systematic and comprehensive combinatorial mutagenesis starting from available single mutations. Introduction of random mutations at the positions of four cysteine and eight methionine residues provided 216 single mutants as stably expressed forms in Escherichia coli host cells. Four characteristics, hydroxylase activity toward p-hydroxybenzoate (main activity), protocatechuate-dependent NADPH oxidase activity (sub-activity), ratio of sub-activity to main activity (reaction specificity), and thermal stability, of the purified mutants were determined. To improve the above characteristics for diagnostic use of the enzyme, 11 single mutations (C152V, C211I, C332A, M52V, M52Q, M110L, M110I, M213G, M213L, M276Q, and M349A) were selected for further combinatorial mutagenesis. All possible combinations of the mutations provided 18 variants with double mutations and further combinatorial mutagenesis provided 6 variants with triple mutations and 9 variants with quadruple mutations with the simultaneously improved four properties.

Development of a glucose-6-phosphate biosensor based on coimmobilized p-hydroxybenzoate hydroxylase and glucose-6-phosphate dehydrogenase.

This work reports the development of an amperometric glucose-6-phosphate biosensor by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and glucose-6-phosphate dehydrogenase (G6PDH) on a screen-printed electrode. The principle of the determination scheme is as follows: G6PDH catalyzes the specific dehydrogenation of glucose-6-phosphate by consuming NADP(+). The product, NADPH, initiates the irreversible the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a detectable signal due to its oxidation at the working electrode. The sensor shows a broad linear detection range between 2 microM and 1000 microM with a low detection limit of 1.2 microM. Also, it has a fast measuring time which can achieve 95% of the maximum current response in 20s after the addition of a given concentration of glucose-6-phosphate with a short recovery time (2 min).

Raman crystallography and other biochemical applications of Raman microscopy.

Recent studies using a Raman microscope have shown that single protein crystals provide an ideal platform to undertake Raman difference spectroscopic analyses under nonresonance conditions. This approach, termed Raman crystallography, provides a means of characterizing chemical events within the crystal such as ligand binding and enzyme reactions. In many cases Raman crystallography goes hand in hand with X-ray crystallographic studies because the Raman results can inform the X-ray crystallographer about the status of chemical events in the crystal prior to flash freezing and X-ray analysis. In turn, the combined data from the Raman and X-ray analyses are highly synergistic and offer novel perspectives on structure and dynamics in enzyme active sites. In a related area, protein misfolding, Raman microscopy can provide detailed insights into the chemistry of the amyloid plaques associated with Alzheimer's disease and into the intermediates on the alpha-synuclein protein misfolding pathway implicated in Parkinson's disease.

Real-time enzyme dynamics illustrated with fluorescence spectroscopy of p-hydroxybenzoate hydroxylase.

We have used the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) to illustrate that a strongly fluorescent donor label can communicate with the flavin via single-pair Förster resonance energy transfer (spFRET). The accessible Cys-116 of PHBH was labeled with two different fluorescent maleimides with full preservation of enzymatic activity. One of these labels shows overlap between its fluorescence spectrum and the absorption spectrum of the FAD prosthetic group in the oxidized state, while the other fluorescent probe does not have this spectral overlap. The spectral overlap strongly diminished when the flavin becomes reduced during catalysis. The donor fluorescence properties can then be used as a sensitive antenna for the flavin redox state. Time-resolved fluorescence experiments on ensembles of labeled PHBH molecules were carried out in the absence and presence of enzymatic turnover. Distinct changes in fluorescence decays of spFRET-active PHBH can be observed when the enzyme is performing catalysis using both substrates p-hydroxybenzoate and NADPH. Single-molecule fluorescence correlation spectroscopy on spFRET-active PHBH showed the presence of a relaxation process (relaxation time of 23 micros) that is related to catalysis. In addition, in both labeled PHBH preparations the number of enzyme molecules reversibly increased during enzymatic turnover indicating that the dimer-monomer equilibrium is affected.

Conformational dynamics of the isoalloxazine in substrate-free p-hydroxybenzoate hydroxylase: single-molecule studies.

p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric enzyme in which each subunit noncovalently binds one molecule of FAD in the active site. PHBH is a model system for how flavoenzymes regulate reactions with oxygen. We report single-molecule fluorescence studies of PHBH in the absence of substrate that provide data consistent with the hypothesis that a critical step in substrate binding is the movement of the isoalloxazine between an "in" conformation and a more exposed or "open" conformation. The isoalloxazine is observed to move between these conformations in the absence of substrate. Studies with the Y222A mutant form of PHBH suggest that the exposed conformation is fluorescent while the in-conformation is quenched. Finally, we note that many of the single-molecule-fluorescence trajectories reveal a conformational heterogeneity, with populations of the enzyme characterized by either fast or slow switching between the in- and open-conformations. Our data also allow us to hypothesize a model in which one flavin in the dimer inhibits the motion of the other.