Pimchai Chaiyen | Mahidol University (original) (raw)

Papers by Pimchai Chaiyen

… et Biophysica Acta (BBA …, 2004

The reaction of flavoenzymes with oxygen remains a fascinating area of research because of its re... more 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.

Journal of Physical Chemistry B, 2009

Ultrafast fluorescence dynamics of flavin adenine dinucleotide (FAD) in wild type pyranose 2-oxid... more Ultrafast fluorescence dynamics of flavin adenine dinucleotide (FAD) in wild type pyranose 2-oxidase (P2O) has been investigated in solution by means of fluorescence up-conversion method. Fluorescence decays were well described by two-exponential model function. Fluorescence lifetimes were 1 ∼ 110 fs and 2 ∼ 360 ps, respectively. The ( 2 / 1 ) ratio (∼3200) was extraordinary high compared to other flavoproteins without subunit structure. The heterogeneous distribution of emission lifetimes were elucidated in terms of two different conformers of P2O; conformer 1 with 1 and conformer 2 with 2 . Emission peaks of conformer 1 and conformer 2 were determined to be at ∼540 nm and 510 nm, respectively, using transient spectral reconstruction procedure. Using dynamics analysis by Kakitani and Mataga (KM) theory, both quenching processes were ascribed to photoinduced electron transfer (ET) reactions mainly from Trp168 to the excited isoalloxazine (Iso*) in different protein tetramers having different static dielectric constants (ε 1 ∼ 3.25 for conformer 1 and ε 2 ∼ 5.93 for conformer 2). The quaternary structure seems to be responsible for the observed conformational heterogeneity.

Biochemistry, 2012

Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monoo... more Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB), the ring-fission fuel of the gentisate pathway. In this study, the kinetics of reduction of the enzyme-bound flavin by NADH was investigated at pH 8.0 using a stopped-flow spectrophotometer, and the data were analyzed comprehensively according to kinetic derivations and simulations. Observed rate constants for reduction of the free enzyme by NADH under anaerobic conditions were linearly dependent on NADH concentrations, consistent with a one-step irreversible reduction model with a bimolecular rate constant of 43 ± 2 M −1 s −1 . In the presence of 3-HB, observed rate constants for flavin reduction were hyperbolically dependent on NADH concentrations and approached a limiting value of 48 ± 2 s −1 . At saturating concentrations of NADH (10 mM) and 3-HB (10 mM), the reduction rate constant is ∼51 s −1 , whereas without 3-HB, the rate constant is 0.43 s −1 at a similar NADH concentration. A similar stimulation of flavin reduction was found for the enzyme− product (2,5-DHB) complex, with a rate constant of 45 ± 2 s −1 . The rate enhancement induced by aromatic ligands is not due to a thermodynamic driving force because E m 0 for the enzyme−substrate complex is −179 ± 1 mV compared to an E m 0 of −175 ± 2 mV for the free enzyme. It is proposed that the reduction mechanism of 3HB6H involves an isomerization of the initial enzyme− ligand complex to a fully activated form before flavin reduction takes place.

Journal of Photochemistry and Photobiology A-chemistry, 2010

Reported time-resolved Stokes shifts (TRSS) of free tryptophan (Trp) and free p-coumaric acid (CA... more Reported time-resolved Stokes shifts (TRSS) of free tryptophan (Trp) and free p-coumaric acid (CA) in water, and Trp in monellin, apomyoglobin, and isoalloxazine (Iso) of flavin mononucleotide (FMN) in the reductase component (C1 protein) of p-hydroxyphenylacetate hydroxylase were analyzed with continuum model. All unknown parameters of these systems in the theoretical equations were determined to obtain the best fit between the observed and calculated TRSS, according to a non-linear least square method. TRSS of free Trp at 295 K was also analyzed with four sets of reported dielectric constants and solvent relaxation times of water. Agreement between the observed and calculated TRSS of the free Trp was excellent. In CA the calculated TRSS could satisfactorily reproduce the observed one. Frequency-dependent dielectric constants of Trp in the proteins and Iso in C1 protein were expressed with 2- and 3-relaxation times. Static dielectric constant, ɛ0, intermediate permittivity, ɛ1, dielectric constant of Iso, ɛc, 2-relaxation times, τ1 and τ2, μe and D0 in the 2-relaxation time analyses were determined by the best-fit procedures. Agreements between the observed and calculated TRSS of Trp in native, denatured monellins, apomyoglobin, and Iso in C1 protein were excellent. No further improvements were obtained with 3-relaxation time analyses. Origin of the slow decaying component of TRSS in apomyoglobin was interpreted with continuum model and compared with molecular dynamics (MD) simulation model and a continuum model by Halle and Nilsson [J. Phys. Chem. B 113 (2009) 8210]. Frozen states revealed with MD model were reproduced with the 3-relaxation time analysis.

Journal of Chemical Education, 2010

The dissociation constant, K d , of the binding of riboflavin-binding protein (RP) with neutral r... more The dissociation constant, K d , of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the ...

Archives of Biochemistry and Biophysics, 2010

2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (MHPCO) and 5-pyridoxic acid oxygen... more 2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (MHPCO) and 5-pyridoxic acid oxygenase are flavoenzymes catalyzing an aromatic hydroxylation and a ring-cleavage reaction. Both enzymes are involved in biodegradation of vitamin B6 in bacteria. Oxygen-tracer experiments have shown that the enzymes are monooxygnases since only one atom of molecular oxygen is incorporated into the products. Kinetics of MHPCO has shown that the enzyme is similar to single-component flavoprotein hydroxylases in that the binding of MHPC is required prior to the flavin reduction by NADH, and C4a-hydroperoxy-FAD and C4a-hydroxy-FAD are found as intermediates. Investigation on the protonation status of the substrate upon binding to the enzyme has shown that only the tri-ionic form of MHPC is bound at the MHPCO active site. Using a series of FAD analogues with substituents at the 8-position of the isoalloxazine ring, the oxygenation of MHPC by the C4a-hydroperoxy-FAD was shown to occur via an electrophilic aromatic substitution mechanism. Recently, the X-ray structures of MHPCO and a complex of MHPC–MHPCO at 2.1 Å have been reported and show the presence of nine water molecules in the enzyme active site. Based on structural data, a few residues, Tyr82, Tyr223, Arg181, were suggested to be important for catalysis of MHPCO.

Biochemistry, 1997

Titrations of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with the substrate MH... more Titrations of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with the substrate MHPC identified the MHPC species bound to the enzyme as the tripolar ionic species. This result was supported by studies of the binding to the enzyme of N-methyl-5-hydroxynicotinic acid (NMHN), an MHPC analog existing only in the tripolar ionic form. The Kd is 55 microM compared to a Kd of 9.2 microM for MHPC and 5.2 microM for 5-hydroxynicotinic acid. Kinetics studies of the binding of NMHN to MHPC oxygenase show that its binding, like that for MHPC and for 5HN, is also a two-step process. Since NMHN never exists as an anionic form, neither of the observed steps is due to the binding of an anionic species as an intermediate step. Investigations of the reduction and oxygenation half reactions demonstrate that the mechanism of catalysis with NMHN is basically the same as with MHPC or with 5-hydroxynicotinic acid. Product analysis from reactions using NMHN, a compound that possesses positive charge on the nitrogen atom, indicates that the product of NMHN is an aliphatic compound, similar to the products derived from MHPC and from another substrate analog, 5-hydroxynicotinic acid. These results indicate that the nitrogen atom of the substrate is invariably protonated during the catalytic reaction.

Journal of Molecular Biology, 2010

Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes ca... more Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. The enzyme–flavin bond helps to increase the flavin's redox potential to facilitate substrate oxidation in several oxidases. The formation of the enzyme–flavin covalent bond—the flavinylation reaction—has been studied for the past 40 years. For the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of developing negative charges at the flavin N(1) and N(5) loci is crucial. Whereas the structural basis for stabilization at N(1) is relatively well studied, the structural requisites for charge stabilization at N(5) remain less clear. Here, we show that flavinylation of histidine 167 of pyranose 2-oxidase from Trametes multicolor requires hydrogen bonding at the flavin N(5)/O(4) locus, which is offered by the side chain of Thr169 when the enzyme is in its closed, but not open, state. Moreover, our data show that additional stabilization at N(5) by histidine 548 is required to ensure high occupancy of the histidyl–flavin bond. The combination of structural and spectral data on pyranose 2-oxidase mutants supports the quinone methide mechanism. Our results demonstrate an elaborate structural fine-tuning of the active site to complete its own formation that couples efficient holoenzyme synthesis to conformational substates of the substrate-recognition loop and concerted movements of side chains near the flavinylation ligand.

Febs Journal, 2009

The putative gene of Plasmodium vivax serine hydroxymethyltransferase (PvSHMT; EC 2.1.2.1) was cl... more The putative gene of Plasmodium vivax serine hydroxymethyltransferase (PvSHMT; EC 2.1.2.1) was cloned and expressed in Escherichia coli. The purified enzyme was shown to be a dimeric protein with a monomeric molecular mass of 49 kDa. PvSHMT has a maximum absorption peak at 422 nm with a molar absorption coefficient of 6370 m−1·cm−1. The Kd for binding of the enzyme and pyridoxal-5-phosphate was 0.14 ± 0.01 μm. An alternative assay for measuring the tetrahydrofolate-dependent SHMT activity based on the coupled reaction with 5,10-methylenetetrahydrofolate reductase (EC 1.5.1.20) from E. coli was developed. PvSHMT uses a ternary-complex mechanism with a kcat value of 0.98 ± 0.06 s−1 and Km values of 0.18 ± 0.03 and 0.14 ± 0.02 mm for l-serine and tetrahydrofolate, respectively. The optimum pH of the SHMT reaction was 8.0 and an Arrhenius’s plot showed a transition temperature of 19 °C. Besides l-serine, PvSHMT forms an external aldimine complex with d-serine, l-alanine, l-threonine and glycine. PvSHMT also catalyzes the tetrahydrofolate-independent retro-aldol cleavage of 3-hydroxy amino acids. Although l-serine is a physiological substrate for SHMT in the tetrahydrofolate-dependent reaction, PvSHMT can also use d-serine. In the absence of tetrahydrofolate at high pH, PvSHMT forms an enzyme–quinonoid complex with d-serine, but not with l-serine, whereas SHMT from rabbit liver was reported to form an enzyme–quinonoid complex with l-serine. The substrate specificity difference between PvSHMT and the mammalian enzyme indicates the dissimilarity between their active sites, which could be exploited for the development of specific inhibitors against PvSHMT.

Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2005

The Toll signalling pathway, which is crucial for innate immunity, is transduced in insect haemol... more The Toll signalling pathway, which is crucial for innate immunity, is transduced in insect haemolymph via a proteolytic cascade consisting of three serine proteases. The proteolytic cascade is downregulated by a specific serine protease inhibitor (serpin). Recently, the serpin SPN48 was found to show an unusual specific reactivity towards the terminal serine protease, Spä tzle-processing enzyme, in the beetle Tenebrio molitor. In this study, the mature form of SPN48 was overexpressed in Escherichia coli and purified. The purified SPN48 protein was crystallized using 14% polyethylene glycol 8000 and 0.1 M 2-(N-morpholino)ethanesulfonic acid pH 6.0 as the precipitant. The crystals diffracted X-rays to 2.1 Å resolution and were suitable for structure determination. The crystals belonged to space group P2 1 . The crystal structure will provide information regarding how SPN48 achieves its unusual specificity for its target protease.

Biochemistry, 2010

Pyranose 2-oxidase (P2O) from Trametes multicolor contains a flavin adenine dinucleotide (FAD) co... more Pyranose 2-oxidase (P2O) from Trametes multicolor contains a flavin adenine dinucleotide (FAD) cofactor covalently linked to the N 3 atom of His167. The enzyme catalyzes the oxidation of aldopyranoses by molecular oxygen to generate 2-keto-aldoses and H 2 O 2 as products. In this study, the transient kinetics and primary and solvent kinetic isotope effects of the mutant in which His167 has been replaced with Ala (H167A) were investigated, to elucidate the functional role of the 8a-N 3 -histidyl FAD linkage and to gain insights into the reaction mechanism of P2O. The results indicate that the covalent linkage is mainly important for a reductive half-reaction in which the FAD cofactor is reduced by D-glucose, while it is not important for an oxidative half-reaction in which oxygen reacts with the reduced FAD to generate H 2 O 2 . D-Glucose binds to H167A via multiple binding modes before the formation of the active Michaelis complex, and the rate constant of flavin reduction decreases ∼22-fold compared to that of the wild-type enzyme. The reduction of H167A using D-glucose isotopes (2-d-D-glucose, 3-d-D-glucose, and 1,2,3,4,5,6,6-d 7 -D-glucose) as substrates indicates that the primary isotope effect results only from substitution at the C2 position, implying that H167A catalyzes the oxidation of D-glucose regiospecifically at this position. No solvent kinetic isotope effect was detected during the reductive half-reaction of the wild-type or H167A enzyme, implying that the deprotonation of the D-glucose C2-OH group may occur readily upon the binding to P2O and is not synchronized with the cleavage of the D-glucose C2-H bond. The mutation has no drastic effect on the oxidative half-reaction of P2O, as H167A is very similar to the wild-type enzyme with respect to the kinetic constants and the formation of the C4a-hydroperoxyflavin intermediate. Kinetic mechanisms for both halfreactions of H167A were proposed on the basis of transient kinetic data and were verified by kinetic simulations and steady-state kinetic parameters.

Biochemistry, 2009

Pyranose 2-oxidase (P2O) from Trametes multicolor is a flavoprotein oxidase that catalyzes the ox... more Pyranose 2-oxidase (P2O) from Trametes multicolor is a flavoprotein oxidase that catalyzes the oxidation of aldopyranoses by molecular oxygen to yield the corresponding 2-keto-aldoses and hydrogen peroxide. P2O is the first enzyme in the class of flavoprotein oxidases, for which a C4ahydroperoxy-flavin adenine dinucleotide (FAD) intermediate has been detected during the oxidative half-reaction. In this study, the reduction kinetics of P2O by D-glucose and 2-d-D-glucose at pH 7.0 was investigated using stopped-flow techniques. The results indicate that D-glucose binds to the enzyme with a two-step binding process; the first step is the initial complex formation, while the second step is the isomerization to form an active Michaelis complex (E-Fl ox :G). Interestingly, the complex (E-Fl ox : G) showed greater absorbance at 395 nm than the oxidized enzyme, and the isomerization process showed a significant inverse isotope effect, implying that the C2-H bond of D-glucose is more rigid in the E-Fl ox :G complex than in the free form. A large normal primary isotope effect (k H /k D = 8.84) was detected in the flavin reduction step. Steady-state kinetics at pH 7.0 shows a series of parallel lines. Kinetics of formation and decay of C-4a-hydroperoxy-FAD is the same in absence and presence of 2-keto-D-glucose, implying that the sugar does not bind to P2O during the oxidative half-reaction. This suggests that the kinetic mechanism of P2O is likely to be the ping-pong-type where the sugar product leaves prior to the oxygen reaction. The movement of the active site loop when oxygen is present is proposed to facilitate the release of the sugar product. Correlation between data from presteady-state and steady-state kinetics has shown that the overall turnover of the reaction is limited by the steps of flavin reduction and decay of C4a-hydroperoxy-FAD. † Abbreviations: P2O, pyranose oxidase; FAD, flavin adenine dinucleotide; k obs , observed rate constant; 2FG, 2-fluoro-deoxy-Dglucose; GMC, glucose-methanol-choline family; ABTS, 2,2azino-bis(3-ethylbenzthiazoline-6-sulfonic acid diammonium salt); E-Fl ox , P2O in the oxidized form; E-Fl red , P2O in the reduced form; E-Fl ox /G, a complex of P2O and D-glucose; E-Fl-OOH, a C4ahydroperoxy-FAD intermediate.

Molecular and Biochemical Parasitology, 2009

Serine hydroxymethyltransferase (SHMT) is a ubiquitous enzyme required for folate recycling and d... more Serine hydroxymethyltransferase (SHMT) is a ubiquitous enzyme required for folate recycling and dTMP synthesis. A cDNA encoding Plasmodium falciparum (Pf) SHMT was expressed as a hexa-histidine tagged protein in Escherichia coli BL21-CodonPlus® (DE3)-RIL. The protein was purified and the process yielded 3.6 mg protein/l cell culture. Recombinant His6-tagged PfSHMT exhibits a visible spectrum characteristic of pyridoxal-5′-phosphate enzyme and catalyzes the reversible conversion of l-serine and tetrahydrofolate (H4folate) to glycine and 5,10-methylenetetrahydrofolate (CH2-H4folate). Steady-state kinetics study indicates that His6-tagged PfSHMT catalyzes the reaction by a ternary-complex mechanism. The sequence of substrate binding to the enzyme was also examined by glycine product inhibition. A striking property that is unique for His6-tagged PfSHMT is the ability to use d-serine as a substrate in the folate-dependent serine–glycine conversion. Kinetic data in combination with expression result support the proposal of SHMT reaction being a regulatory step for dTMP cycle. This finding suggests that PfSHMT can be a potential target for antimalarial chemotherapy.

Archives of Biochemistry and Biophysics, 2005

β-Glucosidases from cassava and Thai rosewood can synthesize a variety of alkyl glucosides using ... more β-Glucosidases from cassava and Thai rosewood can synthesize a variety of alkyl glucosides using various alcohols as glucosyl acceptors for transglucosylation. Both enzymes were inactivated by 2-deoxy-2-fluoro-sugar analogues to form the covalent glycosyl-enzyme intermediates, indicating that the reaction mechanism was of the double-replacement type. The trapped enzyme intermediates were used for investigating transglucosylation specificity, by measuring the rate of reactivation by various alcohols. The glucosyl-enzyme intermediate from the cassava enzyme showed a 20- to 120-fold higher rate of glucose transfer to alcohols than the glucosyl-enzyme intermediate from the Thai rosewood enzyme. Kinetic analysis indicated that the aglycone binding site of the cassava enzyme was hydrophobic, since the enzyme bound better to more hydrophobic alcohols and showed poor transfer of glucose to hydrophilic sugars. With butanol, transglucosylation was faster with the primary alcohols than with the secondary or tertiary alcohol. Studies with ethanol and chloro-substituted ethanols indicated that the rate of transglucosylation was significantly faster with alcohols with lower pKa values, where the reactive alkoxide was more readily generated, indicating that the formation of the alkoxide species was a major step governing the formation of the transition state in the cassava enzyme.

Journal of Biological Chemistry, 2010

Pyranose 2-oxidase (P2O) catalyzes the oxidation by O 2 of D-glucose and several aldopyranoses to... more Pyranose 2-oxidase (P2O) catalyzes the oxidation by O 2 of D-glucose and several aldopyranoses to yield the 2-ketoaldoses and H 2 O 2 . Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr 169 forms H-bonds to the flavin-N5/O4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the P2O⅐sugar complex. Transient kinetics of wild-type (WT) and Thr 169 3 S/N/ G/A replacement variants show that D-Glc binds to T169S, T169N, and WT with the same K d (45-47 mM), and the hydride transfer rate constants (k red ) are similar (15.3-9.7 s ؊1 at 4°C ). k red of T169G with D-glucose (0.7 s ؊1 , 4°C) is significantly less than that of WT but not as severely affected as in T169A (k red of 0.03 s ؊1 at 25°C). Transient kinetics of WT and mutants using D-galactose show that P2O binds D-galactose with a one-step binding process, different from binding of D-glucose. In T169S, T169N, and T169G, the overall turnover with D-Gal is faster than that of WT due to an increase of k red . In the crystal structure of T169S, Ser 169 O␥ assumes a position identical to that of O␥1 in Thr 169 ; in T169G, solvent molecules may be able to rescue H-bonding. Our data suggest that a competent reductive half-reaction requires a side chain at position 169 that is able to form an H-bond within the ES complex. During the oxidative half-reaction, all mutants failed to stabilize a C4a-hydroperoxyflavin intermediate, thus suggesting that the precise position and geometry of the Thr 169 side chain are required for intermediate stabilization.

Journal of Biochemistry, 2007

A new luciferase from V. campbellii (Lux_Vc) was cloned and expressed in Escherichia coli and pur... more A new luciferase from V. campbellii (Lux_Vc) was cloned and expressed in Escherichia coli and purified to homogeneity. Although the amino acid sequences and the catalytic reactions of Lux_Vc are highly similar to those of the luciferase from V. harveyi (Lux_Vh), the two enzymes have different affinities toward reduced FMN (FMNH À ). The catalytic reactions of Lux_Vc and Lux Vh were monitored by stopped-flow absorbance and luminescence spectroscopy at 48C and pH 8. The measured K d at 48C for the binding of FMNH À to Lux_Vc was 1.8 kM whereas to Lux_Vh, it was 11 kM. Another difference between the two enzymes is that Lux_Vc is more stable than Lux_Vh over a range of temperatures; Lux_Vc has t 1/2 of 1020 min while Lux_Vh has t 1/2 of 201 min at 378C. The superior thermostability and tighter binding of FMNH À make Lux_Vc a more tractable luciferase than Lux_Vh for further structural and functional studies, as well as a more suitable enzyme for some applications. The kinetics results reported here reveal transient states in the reaction of luciferase that have not been documented before.

Journal of Bacteriology, 2008

The luxG gene is part of the lux operon of marine luminous bacteria. luxG has been proposed to be... more The luxG gene is part of the lux operon of marine luminous bacteria. luxG has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study, luxG from Photobacterium leiognathi TH1 was cloned and expressed in Escherichia coli in both native and C-terminal His 6 -tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of LuxG were purified to homogeneity, and their biochemical properties were characterized. Purified LuxG is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the LuxG reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4°C pH 8.0, K m for NADH, K m for FMN, and k cat were calculated to be 15.1 M, 2.7 M, and 1.7 s ؊1 , respectively. Coupled assays between LuxG and luciferases from P. leiognathi TH1 and Vibrio campbellii also showed that LuxG could supply FMNH ؊ for light emission in vitro. A luxG gene knockout mutant of P. leiognathi TH1 exhibited a much dimmer luminescent phenotype compared to the native P. leiognathi TH1, implying that LuxG is the most significant source of FMNH ؊ for the luminescence reaction in vivo.

Biochemistry, 2007

p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylatio... more p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). HPAH is composed of two proteins: a flavin mononucleotide (FMN) reductase (C 1 ) and an oxygenase (C 2 ). C 1 catalyzes the reduction of FMN by NADH to generate reduced FMN (FMNH -) for use by C 2 in the hydroxylation reaction. C 1 is unique among the flavin reductases in that the substrate HPA stimulates the rates of both the reduction of FMN and release of FMNHfrom the enzyme. This study quantitatively shows the kinetics of how the C 1 -bound FMN can be reduced and released to be used efficiently as the substrate for the C 2 reaction; additional FMN is not necessary. Reactions in which O 2 is rapidly mixed with solutions containing C 1 -FMNHand C 2 are very similar to those in which solutions containing O 2 are mixed with one containing the C 2 -FMNHcomplex. This suggests that in a mixture of the two proteins FMNHbinds more tightly to C 2 and has already been completely transferred to C 2 before it reacts with oxygen. Rate constants for the transfer of FMNHfrom C 1 to C 2 were found to be 0.35 and g74 s -1 in the absence and presence of HPA, respectively. The reduction of cytochrome c by FMNHwas also used to measure the dissociation rate of FMNHfrom C 1 . In the absence of HPA, FMNHdissociates from C 1 at 0.35 s -1 , while with HPA present it dissociates at 80 s -1 ; these are the same rates as those for the transfer from C 1 to C 2 . Therefore, the dissociation of FMNHfrom C 1 is rate-limiting in the intermolecular transfer of FMNHfrom C 1 to C 2 , and this process is regulated by the presence of HPA. This regulation avoids the production of H 2 O 2 in the absence of HPA. Our findings indicate that no protein-protein interactions between C 1 and C 2 are necessary for efficient transfer of FMNHbetween the proteins; transfer can occur by a rapid-diffusion process, with the rate-limiting step being the release of FMNHfrom C 1 . 1 Abbreviations: HPA, p-hydroxyphenylacetate; HPAH, p-hydroxyphenylacetate hydroxylase; DHPA, 3,4-dihydroxy-phenylacetate; FMNH -, reduced flavin mononucleotide; C1, reductase component of HPAH from A. baumannii; C2, oxygenase component of HPAH from A. baumannii; C2-FMN, complex of C2 and oxidized FMN; C2-FMNH -, complex of C2 and reduced FMN; C2-FMNH -HPA, complex of C2-FMNHand HPA; cyt c, cytochrome c.

Journal of Biotechnology, 2009

Pyranose 2-oxidase (P2Ox) has several proposed biotechnological applications such as a bio-compon... more Pyranose 2-oxidase (P2Ox) has several proposed biotechnological applications such as a bio-component in biofuel cells or for carbohydrate transformations. To improve some of the catalytic properties of P2Ox from Trametes multicolor, we selected a semi-rational approach of enzyme engineering, saturation mutagenesis of active-site residues and subsequent screening of mutant libraries for improved activity. One of the active-site mutants with improved catalytic characteristics identified was V546C, which showed catalytic constants increased by up to 5.7-fold for both the sugar substrates (d-glucose and d-galactose) and alternative electron acceptors (1,4-benzoquinone, BQ and ferricenium ion, Fc+], albeit at the expense of increased Michaelis constants. By combining V546C with other amino acid replacements, we obtained P2Ox variants that are of interest for biofuel cell applications due to their increased kcat for both BQ and Fc+, e.g., V546C/E542K showed 4.4- and 17-fold increased kcat for BQ compared to the wild-type enzyme when d-glucose and d-galactose, respectively, were the saturating substrates, while V546C/T169G showed approx. 40- and 50-fold higher kcat for BQ and Fc+, respectively, with d-galactose in excess. This latter variant also shows significantly modulated sugar substrate selectivity, due to an increase in kcat/KM for d-galactose and a decrease in kcat/KM for d-glucose when oxygen is the electron acceptor, as well as improved catalytic efficiencies for d-galactose, regardless of the electron acceptor used. While the wild-type enzyme strongly prefers d-glucose over d-galactose as its substrate, V546C/T169G converts both sugars equally well as was shown by the kinetic constants determined as well as by biotransformation experiments.