Báo cáo khoa học: The effect of pH on the initial rate kinetics of the dimeric biliverdin-IXa reductase from the cyanobacterium Synechocystis PCC6803 pptx

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The effect of pH on the initial rate kinetics of the dimericbiliverdin-IXa reductase from the cyanobacteriumSynechocystis PCC6803Jerrard M. Hayes and Timothy J. MantleSchool of Biochemistry and Immunology, Trinity College, Dublin, IrelandIntroductionCyanobacteria utilize linear tetrapyrroles as light-har-vesting pigments that are found covalently attached tophycobiliproteins in the ‘light pipes’ known as phyco-bilisomes [1]. Two genera, Synechococcus and Prochlo-rococcus, are suggested to be responsible for 25% ofglobal photosynthesis [2] and, although some strains ofProchlorococcus do not express phycobiliproteins (e.g.MED4), others (e.g. SS120) express a phycourobilin-containing type-III phycoerythrin [3]. Linear tetrapyr-role metabolism in cyanobacteria is therefore a majorphysiological pathway. Cyanobacteria express ferre-doxin-dependent bilin reductases (PcyA, PebA andPebB) that synthesize phycocyanobilin and phycoery-throbilin from biliverdin-IXa [4]. These linear tetrapyr-Keywordsbiliverdin reductase; compulsory orderedmechanism; dimer; pH; SynechocystisCorrespondenceJ. M. Hayes, School of Biochemistry andImmunology, Trinity College, Dublin 2,IrelandFax: +353 677 2400Tel: +353 895 1612E-mail: jehayes@tcd.ie(Received 23 April 2009, revised 9 June2009, accepted 11 June 2009)doi:10.1111/j.1742-4658.2009.07149.xBiliverdin-IXa reductase from Synechocystis PCC6803 (sBVR-A) is a stabledimer and this behaviour is observed under a range of conditions. This is incontrast to all other forms of BVR-A, which have been reported to behaveas monomers, and places sBVR-A in the dihydrodiol dehydrogenase ⁄ N-ter-minally truncated glucose–fructose oxidoreductase structural family ofdimers. The cyanobacterial enzyme obeys an ordered steady-state kineticmechanism at pH 5, with NADPH being the first to bind and NADP+thelast to dissociate. An analysis of the effect of pH on kcatwith NADPH ascofactor reveals a pK of 5.4 that must be protonated for effective catalysis.Analysis of the effect of pH on kcat⁄ KmNADPHidentifies pK values of 5.1and 6.1 in the free enzyme. Similar pK values are identified for biliverdinbinding to the enzyme–NADPH complex. The lower pK values in the freeenzyme (pK 5.1) and enzyme–NADPH complex (pK 4.9) are not evidentwhen NADH is the cofactor, suggesting that this ionizable group may inter-act with the 2¢-phosphate of NADPH. His84 is implicated as a crucial resi-due for sBVR-A activity because the H84A mutant has less than 1% of theactivity of the wild-type and exhibits small but significant changes in theprotein CD spectrum. Binding of biliverdin to sBVR-A is convenientlymonitored by following the induced CD spectrum for biliverdin. Binding ofbiliverdin to wild-type sBVR-A induces a P-type spectrum. The H84Amutant shows evidence for weak binding of biliverdin and appears to bind avariant of the P-configuration. Intriguingly, the Y102A mutant, which iscatalytically active, binds biliverdin in the M-configuration.AbbreviationshBVR-B, human biliverdin-IXb reductase; HSA, human serum albumin; sBVR-A, biliverdin-IXa reductase from Synechocystis PCC6803; GST,glutathione S-transferase.4414 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBSroles are then incorporated into the phycobilisomecomplex. Intriguingly, some strains of cyanobacteriaexpress biliverdin-IXa reductase (BVR-A), which catal-yses the pyridine nucleotide-dependent reduction ofbiliverdin-IXa to bilirubin-IXa. The first report of acyanobacterial BVR-A was from SynechocystisPCC6803 (sBVR-A) [5] and BVR-A-like sequences arealso clearly identifiable in Gleobacter, Anabena, Nostocand Trichodesmium (E. Franklin & T. J. Mantle,unpublished results). Schluchter and Glazer [5]reported on the unusual acidic pH optimum for sBVR-A. They also describe features of a SynechocystisPCC6803 strain lacking sBVR-A, which they interpretas indicating that the reaction product, bilirubin-Ixa,plays a role in phycobiliprotein biosynthesis [5]. Wehave been intrigued that sBVR-A can potentially divertflux from phycobilin biosynthesis and also potentiallyreduce the phycobilins to the corresponding rubin, areaction clearly catalysed in vitro [5], albeit with a rela-tively high Kmfor phycocyanobilin [5].Questions on the possible function of BVR-A incyanobacteria parallel a major re-evaluation of thefunction of BVR-A in mammals. Once considered toplay a role solely in the elimination of excess haem, itis now implicated in the maintenance of a major anti-oxidant, bilirubin-IXa [6]. At high doses, biliverdinappears to tolerize the immune system of recipientsundergoing organ transplantation in animal studies[7,8], although it is presently unclear whether thiseffect is caused by biliverdin-IXa or bilirubin-IXa.Because BVR-A is reponsible for the production of bil-irubin-IXa in neonates at birth, it is also a pharmaco-logical target for treating neonatal jaundice [9]. Therat [10,11] and human enzymes [12] have been crystal-lized; however, although there are complexes withNAD+[11] and NADP+[12], little is known aboutthe biliverdin binding site. Although the mammalianenzymes have received the most attention, comparativestudies on the salmon and Xenopus tropicalis enzymesare available [13,14]. In this respect, the enzyme fromSynechocystis is of considerable interest because itexhibits a narrow acidic pH optimum compared to thebroad range of pH that can support activity for themammalian enzymes [5,14]. The cyanobacterial enzymeis also refractory to activation by inorganic phosphatewhen NADH is the cofactor [14]. In preliminary exper-iments, we observed that sBVR-A is not subject to thepotent substrate inhibition observed with the mamma-lian enzymes and is therefore the first candidate,among all BVR-A forms studied to date, where a com-plete initial rate study can be completed in the absenceof a biliverdin-binding protein as well as at the opti-mum, presumably physiological, pH. In preliminarygel filtration experiments, we have also shown that theSynechocystis enzyme behaves as a dimer and suchstudies are extended to include the light-scattering andanalytical ultracentrifugation studies described here.We report a complete initial rate study, including theeffect of pH on the kinetic parameters and site-directedmutagenesis studies, to gain an understanding of thefunction of sBVR-A in cyanobacteria and also toincrease our knowledge of the mechanism of anenzyme closely related to a pharmacological target forneonatal jaundice.ResultsThe expression vector pETBVR-A allowed us to rou-tinely prepare 20 mg of electrophoretically homoge-nous sBVR-A from 4 L of culture using Escherichiacoli BL21 (DE3) cells. Using this approach, theenzyme has two additional residues at the N-terminus(Ser-Gly) but lacks the His-tag in the preparationreported earlier [5]. The enzyme was colourless; how-ever, the UV spectrum revealed significant absorbanceat 260 nm. Analysis of the protein sample by HPLCrevealed that, in addition to the protein, there was onemajor and two minor peaks that absorbed at 254 nm.The major peak, which eluted at 38 min, was identifiedas NADPH by its retention time, fluorescence emissionspectrum and UV absorbance spectrum. We have notpursued the identity of the two minor peaks. All threecompounds were released from the enzyme when itwas bound to 2¢,5¢-ADP-sepharose and, under theseconditions, the enzyme was eluted without contamina-tion. In preliminary experiments, we observed thatsBVR-A eluted just before BSA on gel filtration in25 mm sodium citrate pH 5 (the optimum pH foractivity; see below) and, by comparison with the elu-tion volume of standard proteins, this is consistentwith a molecular mass of 69 kDa at 20 ° C and 74 kDaat 4 °C (Table 1). Although the enzyme is less activeat pH 7.5, gel filtration was carried out at this pH andat 20 °C as well as 4 °C and, under all these condi-tions, the molecular mass of the enzyme correspondsto that of the dimer (Table 1). This result is novelbecause all BVR-As described to date have beenreported to behave as monomers [15–17]. To confirmthat sBVR-A is a dimer, we examined the nativemolecular mass using light-scattering and analyticalultracentrifugation (both sedimentation velocity andsedimentation equilibrium) and the results obtainedare provided in Table 1. These confirm the results ofthe gel filtration and are consistent with the dimericnature of sBVR-A because several purified prepara-tions have been shown to run with a subunit molecularJ. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-AFEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4415mass of 34 kDa on SDS ⁄ PAGE. Prior to a detailedkinetic analysis, the stability of sBVR-A at a range ofpH values was determined. Over the pH range 5–7, theenzyme did not lose any activity when pre-incubatedfor 180 min. The enzyme was unstable outside thisrange, being particularly unstable below pH 4.5. AtpH 4, the half life was 30 s. At pH 8, the enzymestarted to lose activity after 60 min and, at pH 9,retained 75% of the activity after 60 min of incuba-tion. The initial rate demonstrates a linear dependenceon enzyme concentration (from 0.5–5 lg ⁄ mL) whenassayed at pH 5 with NADPH or NADH as cofactor.All initial rate experiments were conducted within thisrange of enzyme concentration. In a preliminary set ofexperiments, there was no substrate inhibition up to abiliverdin concentration of 50 lm, in clear distinctionto the mammalian enzymes.Initial rate experiments with NADPH or NADH asthe variable substrate were carried out by working atvarious fixed concentrations of biliverdin-IXa andvarying the concentration of NADPH from 5–100 lmor NADH from 50–1000 lm. The ‘fixed’ concentra-tions of biliverdin were also varied from 0.5–10 lm toyield the plot for NADPH as the variable substrateshown in Fig. 1A. The apparent Vmaxvalues forNADPH as the variable substrate were then replottedagainst the biliverdin concentration (Fig. 1B) to yieldthe true Vmaxand true Kmvalues for biliverdin withNADPH as cofactor (Table 2). The initial rate mea-surements also yielded linear double-reciprocal plots(not shown) that intersected to the left of the recipro-cal initial rate axis, suggesting that the mechanism wassequential. However, these experiments could not iden-tify which of the substrates bound first or whetherthere was any particular order in their binding. A simi-lar pattern was obtained with NADH as the variablesubstrate (data not shown).Initial rate experiments with biliverdin-IXa as thevariable substrate were carried out similarly to thosedescribed for NADPH but using various ‘fixed’ con-centrations of NADPH and varying the biliverdin-IXaconcentration in the range 0.5–10 lm. The data wereTable 1. Relative molecular mass of native sBVR-A. AUC, areaunder the curve.pHTemperature(°C)MW(kDa)Gel filtration 5 20 6954 747.5 20 667.5 4 64Light scattering 5 20 73.27.5 20 66.2AUC velocity 5 11 715 21 80.17.5 11 80.47.5 21 80AUC equilibrium11 612 g 5 4 69.218 144 g 5 4 63.926 127 g 54 5511 612 g 7 4 73.818 144 g 7 4 77.726 127 g 74 72ABFig. 1. Initial rate kinetics of sBVR-A with NADPH as the variablesubstrate. (A) The reaction was conducted in 100 mM sodium cit-rate buffer (pH 5) and the reaction was initiated by the addition ofsBVR-A (5 lg). The concentrations of NADPH are indicated and theconcentrations of biliverdin-IXa were 0.5 lM ( ), 1 lM ( ), 2 lM(.), 5 lM (r) and 10 lM (•). Each point represents the mean andthe error bars represent the standard deviation of triplicate values.The curves are least squares fits to a rectangular hyperbola. (B) Areplot of the apparent Vmaxfrom (A) against the concentrations ofbiliverdin-IXa. The curve is a least squares fit to a rectangular hyper-bola and the error bars are the standard errors from the fits in (A).Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle4416 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBSfitted to a rectangular hyperbola (Fig. 2A). The trueVmaxand Kmfor NADPH were calculated by replot-ting the apparent Vmaxvalues (obtained from the fitsin Fig. 2A) against the NADPH concentration(Fig. 2B) and the kinetic constants are shown inTable 2. The initial rate data with biliverdin-IXa asthe variable substrate also yielded linear intersectingdouble-reciprocal plots that were consistent with asequential mechanism (data not shown). Althoughthese data sets indicate that the enzyme obeys asequential mechanism, product inhibition patterns arerequired to distinguish between steady-state ordered,random sequential and Theorell–Chance mechanisms.NADP+inhibition with NADPH as the variablesubstrate was carried out at saturating (10 lm) levelsof biliverdin. The inhibitory concentrations of NADP+were in the range 0–100 lm, whereas the concentrationof NADPH varied in the range 5–100 lm. Curves wereagain fitted to the initial rate data set and used to yielddouble-reciprocal plots (Fig. 3A). The pattern of thedouble-reciprocal plots shows that NADP+exhibitscompetitive kinetics against NADPH. When the slopevalues of the double-reciprocal plots were replottedagainst the inhibitor concentration, a linear relation-ship was obtained (Fig. 3B) and was used to determinethe inhibitor constant Kisfor NADP+, which is shownin Table 3. NADP+inhibition was also carried outwith biliverdin as the variable substrate. These experi-ments were performed as described for NADPH butkeeping the NADPH concentration constant at nonsat-urating (10 lm) and saturating (1 mm) levels ofNADPH and varying the biliverdin concentration inthe range 0.5–10 lm. A concentration of 1 mm wasused for NADPH to ensure saturation. NADP+showed mixed inhibition against biliverdin at nonsatu-rating levels of NADPH. When the experiment wasrepeated at saturating levels of NADPH, no inhibitionABFig. 2. Initial rate kinetics of sBVR-A with biliverdin-IXa as the vari-able substrate. (A) The reaction was conducted in 100 mM sodiumcitrate buffer (pH 5) and the reaction was initiated by the addition ofsBVR-A (5 lg). The concentrations of biliverdin-IXa are indicated andthe concentrations of NADPH were 5 lM ( ), 10 lM ( ), 20 lM (.),50 lM (r) and 100 lM (•). Each point represents the mean and theerror bars represent the standard deviation of triplicate values. Thecurves are least squares fits to a rectangular hyperbola. (B) A replotof the apparent Vmaxfrom (A) against the concentrations of NADPH.The curve is a least squares fit to a rectangular hyperbola and theerror bars are the standard errors from the fits in (A).Table 2. Kinetic parameters for wild-type and mutant forms ofsBVR-A.sBVR-AVariablesubstrateVmax(lmolÆmin)1Æmg)1)Km(lM)kcat(s)1)Wild-type NADPH 0.78 ± 0.06 10.78 ± 3.2 0.44 ± 0.034Biliverdin 0.79 ± 0.07 2.32 ± 0.59 0.45 ± 0.02NADH 0.29 ± 0.04 207 ± 66 0.17 ± 0.023Biliverdin 0.24 ± 0.027 1.6 ± 0.55 0.15 ± 0.015Y102A NADPH 0.33 ± 0.06 3.55 ± 3.2 0.18 ± 0.034Biliverdin 0.33 ± 0.022 16.19 ± 1.62 0.18 ± 0.012R185A NADPH 0.089 ± 0.006 4.54 ± 1.4 0.05 ± 0.034Biliverdin 0.089 ± 0.01 3.22 ± 0.9 0.05 ± 0.006H129A NADPH 0.68 ± 0.01 4.53 ± 0.32 0.39 ± 0.006Biliverdin 0.68 ± 0.055 1.3 ± 0.34 0.39 ± 0.03H126A NADPH 0.62 ± 0.1 23.3 ± 10 0.35 ± 0.06Biliverdin 0.64 ± 0.05 4.67 ± 0.72 0.36 ± 0.03H97A NADPH 0.58 ± 0.05 5.81 ± 2.3 0.33 ± 0.03Biliverdin 0.58 ± 0.026 2.26 ± 0.28 0.33 ± 0.015H84A NADPHBiliverdin0.0080.008Unable tocalculateE101A NADPH 0.27 ± 0.034 8.8 ± 4 0.15 ± 0.02Biliverdin 0.25 ± 0.22 21.66 ± 24.55 0.14 ± 0.13D285A NADPH 0.1 ± 0.006 1 ± 0.73 0.057 ± 0.01Biliverdin 0.11 ± 0.013 1.60 ± 0.6 0.062 ± 0.007J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-AFEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4417was observed (data not shown). The inhibition con-stants (Kisfrom the slope replot and Kiifrom the inter-cept replot) for NADP+with biliverdin as the variablesubstrate are shown in Table 3. NAD+showed com-petitive kinetics against NADH and was a mixedinhibitor against biliverdin at nonsaturating (100 lm)levels of NADH (Table 3).Bilirubin inhibition with biliverdin as the variablesubstrate was conducted at saturating (100 lm) levelsof NADPH and revealed that bilirubin is a mixedinhibitor against biliverdin at saturating levels ofNADPH. Product inhibition experiments with bilirubinas an inhibitor were also conducted with NADPH asthe variable substrate (5–100 lm) at nonsaturating(1 lm) and saturating (10 lm) levels of biliverdin. Ini-tial rate data for nonsaturating levels of biliverdinshow that bilirubin exhibits mixed inhibition kineticsat nonsaturating levels of biliverdin and, when theexperiment was repeated at saturating levels of biliver-din, the inhibition becomes uncompetitive (Fig. 4).Bilirubin exhibits mixed inhibition against NADH atnonsaturating levels of biliverdin and mixed inhibitionagainst biliverdin at saturating levels of NADH (datanot shown). Inhibition constants are shown in Table 3.These product inhibition patterns are entirely consis-tent with sBVR-A obeying a steady-state orderedmechanism at pH 5, with NADPH being the first tobind and NADP+the last to dissociate.Inorganic phosphate anion has been shown to be anactivator of human BVR-A [14]. Increasing amountsof sodium phosphate (0–100 mm) were added to thesBVR-A assay using both NADH and NADPH ascofactor and at pH 5 and pH 7. The pH was moni-tored before and after the assay to ensure that it didnot change significantly when adding increasingamounts of phosphate. The effect of ionic strength wasfound to be minimal. Inorganic phosphate was foundto have no effect on sBVR-A activity with either cofac-tor at either pH. This is a major discriminating featurebetween the cyanobacterial enzyme and the vertebrateBVR-A family members.It is often the case that, when determining the effectof pH on the kinetic parameters of a two-substrateenzyme, one substrate is held at 10 · Km(91% saturat-ing) and the variation of initial rate with the concen-tration of the second substrate is then used to estimatekcatand the Kmfor the variable substrate. However,the assumption that a concentration that saturates atone pH will saturate at all the pH values under investi-gation is not without risk. All the initial rate parame-ters reported in the present study were determined inaccordance with the classic analysis of Florini and Ves-tling [18] to calculate Kmand Vmax. The effect of pHon kcatwas investigated over the pH range 4.25–7.0with both NADPH and NADH. The values measuredfor kcatare shown when the data set is described withNADPH or biliverdin as the variable substrate. Thesame kcatprofile should be obtained (irrespective ofwhich substrate is held as the variable) and this isclearly seen in Fig. 5A. Evidently, there is a pK at 5.4for the ‘less acidic’ limb of the pH curve defining aside chain that must be protonated for catalysis tooccur. There is no co-operativity for this protonationbecause the plot of log kcatversus pH gives a slope ofapproximately –1 (Fig. 5B). On the ‘more acidic’ limbABFig. 3. Product inhibition by NADP+with NADPH as the variablesubstrate. The reaction was conducted in 100 mM sodium citratebuffer (pH 5) and the reaction was initiated by the addition ofsBVR-A (5 lg). Biliverdin-IXa was held constant (10 lM) at saturat-ing levels and the levels of NADPH are indicated. The concentra-tions of NADP+were 0 lM ( ), 10 lM ( ), 20 lM (.), 50 lM (r)and 100 lM (•). (A) The data are represented as a double-reciprocalplot and (B) a slope replot (apparent Km⁄ Vmaxfrom fits to a rectan-gular hyperbola) against the concentration of NADP+.Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle4418 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBSof this curve, there is a second pK (4.7) and proton-ation of this group reduces the kcat(but only by 50%).Great care has to be taken because the enzyme ishighly unstable at the ‘more acidic’ pH values. Initialrates were obtained at pH 4 during the first few sec-onds of the reaction under conditions when at least90% of the activity was retained; however, these areclearly not ideal conditions. With NADH as cofactor,the pK on the ‘less acidic’ limb is clearly not co-opera-tive (data not shown) and has a similar value (5.7) tothat observed with NADPH (5.4). It is intriguing thatthe kcaton the ‘more acidic’ limb shows very littledependence on pH with NADH as the cofactor.The effect of pH on the kcat⁄ Kmvalues for NADPHand NADH was also analysed. The log plot forkcat⁄ Kmis shown for the NADPH (Fig. 6A) and bili-verdin (Fig. 6B) data sets and for the NADH (Fig. 6C)and biliverdin (Fig. 6D) data sets. With NADPH asTable 3. Initial rate kinetic parameters for product inhibition studies of sBVR-A.Inhibitor Variable substrate Fixed substrate Inhibition Kis(lM) Kii(lM)NADP+NADPH Biliverdin 10 lM (saturating) Competitive 12.7 –NADP+Biliverdin NADPH 10 lM (nonsaturating) Mixed 26 51NADPH 1000 lM (saturating) No inhibition – –NAD+NADH Biliverdin 10 lM (saturating) Competitive 613NAD+Biliverdin NADH 100 lM (nonsaturating) Mixed 2615 1771NADH 1000 lM (saturating) No inhibition – –Bilirubin NADPH Biliverdin 1 lM (nonsaturating) Mixed 13 28Biliverdin 10 lM (saturating) Uncompetitive – 17.5Bilirubin NADH Biliverdin 1 lM (nonsaturating) Mixed 5.2 9.6Bilirubin Biliverdin NADPH 100 lM (saturating) Mixed 13 28Bilirubin Biliverdin NADH 1000 lM (saturating) Mixed 16 42Fig. 4. Product inhibition by bilirubin-IXa with NADPH as the vari-able substrate at saturating levels of biliverdin. (A) The reactionwas conducted in 100 mM sodium citrate buffer (pH 5) and thereaction was initiated by the addition of sBVR-A (5 lg). Biliverdin-IXa was held constant at saturating levels (10 lM) and the concen-trations of NADPH are indicated. The concentrations of bilirubin-IXawere 0 lM ( ), 1 lM ( ), 2 lM (.), 5 lM (r) and 10 lM (•). Thedata are represented as a double-reciprocal plot.ABFig. 5. Effect of pH on kcatwith NADPH as cofactor: 25 mMsodium citrate (pK values of 3.13, 4.76 and 6.4) was used as bufferover the entire pH range studied. (A) Values for kcatwere obtainedwith NADPH as the variable substrate (•) and with biliverdin-IXa asthe variable substrate (). (B) The log ⁄ log plot for (A) is shown.J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-AFEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4419cofactor, the kcat⁄ Kmdata reveal two pK values of 5.1and 6.1 with NADPH as the variable substrate and 4.9and 5.6 with biliverdin as the variable substrate. Thisis consistent with two ionizing groups in the freeenzyme with pK values of 5.1 and 6.1 that defineNADPH binding. Interestingly, with NADH (Fig. 6C)and biliverdin (Fig. 6D) as the variable substrates,there is only a single pK (i.e. 5.3 for NADH bindingto the free enzyme and 5.5 for biliverdin binding to theenzyme–NADH complex). The only difference betweenNADPH and NADH is the 2¢-phosphate on NADPHand it is tempting to suggest that there may be a disso-ciable group with a pK of 5.1 in the free enzyme (4.9in the enzyme–NADH complex) that is not involved inbinding NADH. This pK may be associated with anionizing residue that is involved in binding the 2¢-phos-phate of NADPH.The effect of pH on the initial rate kinetics is consis-tent with two ionizing groups in the enzyme active siteinvolved in binding NADPH, which may be perturbedslightly in the enzyme–NADPH complex but which areboth required for binding biliverdin. In the ternary com-plex, a group with a pK of 5.4 must be protonated forefficient catalysis with NADPH (in the case of the ter-nary complex with NADH as cofactor, this pK is 5.7).The nature of the second pK in the ternary complexwith NADPH (4.7) is unclear. There is no analogouspK in the binding of NADH and it is not readily appar-ent in the ternary complex with NADH as cofactor.To identify the ionizing residues, we have attemptedto crystallize sBVR-A, so far without success. We havetherefore built a model using the rat enzyme as a tem-plate and this is shown in Fig. 7. In this model, wehave highlighted residues from the sBVR-A model thatare candidates for the ionizing residues. These includefour His (84, 97, 126 and 129) one Glu (101), one Asp(285) and one Tyr (102) residue. All were mutated toAla residues and the sequences confirmed. The gluta-thione S-transferase (GST) fusions were purified, theGST domain cleaved and removed by affinity chroma-tography and the mutant sBVR-As analysed in termsof CD spectra, induced CD spectra for biliverdin andinitial rate kinetic parameters. The kinetic parametersof all the mutants are shown in Table 2. This clearlyrules out His97, His126 and His129, which have kcatand Kmvalues that are very close to those displayedby the wild-type enzyme. In addition. these three Histo Ala mutants show CD spectra and induced CDspectra for biliverdin that are very close to thoseexhibited by the wild-type enzyme (Fig. 8A). However,a clear candidate for a key active site residue is His84.The specific activity of the H84A mutant is 1% of thewild-type and is so low that we were unable toABCDFig. 6. log kcat⁄ Kmversus pH for NADPH and NADH as the variablesubstrates. (A) Log kcat⁄ Kmwith NADPH as the variable substrate.(B) Log kcat⁄ Kmwith biliverdin as the variable substrate and NADPHas cofactor. (C) Log kcat⁄ Kmwith NADH as cofactor. (D) Log kcat⁄ Kmwith biliverdin as the variable substrate and NADH as cofactor.Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle4420 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBSdetermine the kinetic parameters with confidence.Examination of the CD spectrum of H84A proteinreveals that it is close to, but not identical with, thatof the native enzyme (data not shown). We suggestthat H84A may be the residue responsible for proton-ating the pyrollic nitrogen prior to hydride transfer(see Discussion); however, we cannot discount a mod-est global structural change having some role in thedecreased catalytic activity. It should be noted that, inthis respect, the H84A mutant was isolated with boundnucleotide and is clearly able to bind biliverdin-IXa(Fig. 8B), suggesting that both substrates are able tobind to the H84A mutant.The binding of biliverdin to wild-type sBVR-A stabi-lizes the helical P-configuration (Fig. 8A) of the lineartetrapyrrole, also known as the ‘lock washer’ [19] and,by this criteria, biliverdin can be seen to bind weaklyto the H84A mutant (Fig. 8B), albeit with a broaden-ing of the positive ellipticity into a peak at 400 nmand a significant shoulder at 325 nm. Two of themutants (R185A and D285A) have kcatvalues that areonly 10% of wild-type (Table 2). The D285A mutanthas modest changes in the Kmvalues and the inducedCD spectrum for biliverdin is similar to wild-type. TheR185A mutant also shows similar Kmvalues to thewild-type; however, the positive ellipticity of theinduced CD for biliverdin shows a sharp peak at400 nm (the wild-type shows a broad peak centred at390 nm) and a clear minor peak at 325 nm (Fig. 8C).In the case of the E101A mutant, there is a significantincrease in the Kmfor biliverdin (nine-fold) and thekcatis reduced to a third of that of the wild-type(Table 2). The induced CD spectrum for biliverdinbound to the E101A mutant shows a considerablyreduced amplitude, with the positive ellipticity splitinto two peaks at 325 nm and 400 nm (Fig. 8D). Inthis case, the trough is centered at 580 nm (comparedto 700 nm in the wild-type). Intriguingly the Y102Amutant exhibits CD behaviour that reflects the M-con-figuration (Fig. 8E). The ability of this mutant tostabilize the opposite enantiomer is associated with amodest (50%) drop in the kcatand a seven-foldincrease in the Kmfor biliverdin.DiscussionAll mammalian forms of BVR-A are reported tobehave as monomers. These include the enzymes frompig spleen and rat liver [15], human liver [16] and oxkidney [17]. We have artificially created a dimer of ratBVR-A by using fused GST domains as sites fordimerization [20]. The Synechocystis enzyme is there-fore the first natural dimer reported for BVR-A. Wewere careful to use a range of techniques to measurethe native molecular mass of sBVR-A and to conductthese experiments under a range of conditions, includ-ing temperature, pH and the presence or absence ofphosphate, because this has such a pronounced activat-ing effect on the mammalian enzymes with NADH ascofactor [14]. Under all of these conditions, the nativeenzyme exhibits a molecular mass of 66–80 kDa and,because the molecular mass is 34 kDa as measured bySDS ⁄ PAGE, we conclude that sBVR-A is a stabledimer. In light of the recent reports on the structuresof monkey dihydrodiol dehydrogenase [21] and theN-terminally truncated dimeric form of glucose–fruc-tose oxidoreductase [22], we propose that sBVR-Ajoins this small family of pyridine nucleotide-depen-dent oxidoreductases that dimerize via the C-terminalb-sheet domain. It is intriguing that we purify sBVR-Awith bound pyridine nucleotide because this is also afeature of the glucose–fructose oxidoreductase enzyme.In addition to its unique quaternary structure,sBVR-A also exhibits a sharp pH optimum, which wereproducibly measured as pH 5. This behaviour is incontrast to that displayed by the mammalian BVR-Amonomers, which show activity over a broad range ofpH values in the range 5–9 [14]. The cyanobacterialBVR-A is not subject to the potent substrate inhibitionobserved with the mammalian forms and this hasFig. 7. A model for sBVR-A. sBVR-A model (green) superimposedon the rat BVR-A crystal structure (grey). The amino acid residuesthat mutated and their positions within the sBVR-A model areshown. The numbers indicated represent the amino acid residuesof sBVR-A.J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-AFEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4421allowed us to complete a full initial rate study on theSynechocystis enzyme and to rigorously establish thatit obeys an ordered steady-state mechanism. The effectof pH on the initial rate parameters has allowed us toidentify two ionizing groups in the free enzyme thatare required in the unprotonated (pK 5.1) and proton-ated forms (pK 6.1), respectively, for binding NADPH.The protonation state of the lower pK does not affectthe binding of NADH. This is consistent with an ioniz-ing residue, pK 5.1, in the free enzyme which, in thedeprotonated state, may promote interaction with the2¢-phosphate group of NADPH but plays no signifi-cant role in binding NADH. In the case of humanBVR-A, we have suggested that the protonation stateof Glu75, may effect the interaction with Arg44, whichis a key residue involved in binding the 2¢-phosphateof NADPH [14]. Further work is required to identifypossible analogous candidates in sBVR-A. There isclearly a pK of 5.4 in the ternary complex that isrequired to be protonated for efficient catalysis withNADPH and which, with NADH as cofactor, exhibitsapK of 5.7. Our mutagenesis studies tentatively iden-tify this residue as His84. We have recently discussedthe possibility that a His residue may be responsiblefor supplying a proton to the pyrrole nitrogen atom ofbiliverdin-IXb prior to hydride transfer in the case ofhuman biliverdin-IXb reductase (hBVR-B). Althoughstructurally distinct to BVR-A, BVR-B is a goodmodel for mechanistic studies on the reduction of thelinear tetrapyrrole ‘C10’ position by hydride. BVR-B isa ‘non-Ixa’ biliverdin reductase [23] and is unable toaccommodate biliverdin-IXa in a productive orienta-tion, although we have shown that it clearly binds,albeit rotated by 90° [24], when compared with the bil-iverdin isomers that are substrates (i.e. the IXb,IXdand IXc isomers). Mutagenesis studies on BVR-B haveindicated that a solvent hydroxonium ion may be thesource of the proton and this was found to be consis-tent with quantum mechanical ⁄ molecular mechanicalcalculations [25]. However, our studies with BVR-B asa model have demonstrated that there is a requirementfor proton transfer to the pyrrole nitrogen atom priorto hydride transfer in the hBVR-B reaction co-ordinate[25] and we suggest that His84 is a good candidate forthis function in sBVR-A. The second ‘more acidic’ pKin the kcatdata set (pK 4.7) is also prominent withNADPH but less so with NADH.We have taken advantage of the induced CD spectraof biliverdin when enantiomeric forms are stabilized bybinding to proteins, including serum albumins [26]and, as reported in the present study, sBVR-A. Insolution, these chiral forms are clearly in equilibriumso that no CD spectrum is seen. Bilirubin adopts twoenantiomeric ‘ridge tile’ configurations [19,27], whereasABCDEFig. 8. Induced CD spectra of biliverdin-IXa bound to sBVR-A and various mutants. sBVR-A and the mutants indicated (all at 29 lM) wereincubated with biliverdin-IXa (30 lM) and NADP+(100 lM). (A) Wild-type, (B) H84A, (C) D285A, (D) E101A and (E) Y102A.Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle4422 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBSbiliverdin is suggested to oscillate between two helical‘lock washer’ configurations, and one of these, the P-configuration, is clearly stabilized in a biliverdin–myo-globin complex [28]. Although it is tempting to suggestthat trapping oscillations between two helical forms isthe phenomenon responsible for the P(lus) andM(inus) spectra of biliverdin when bound to humanserum albumin (HAS) and BSA respectively [26], thisremains to be confirmed. A recent X-ray structure ofHSA with bilirubin bound [29] shows a ZZE configu-ration (not a ridge tile), whereas, in solution, HSAstabilizes a P-type induced CD spectrum so that, untilwe have CD spectra of the appropriate crystals, abso-lute assignments will not be possible. The wild-typeand most mutants that we have studied show P-behav-iour [by convention the sign of the longer wavelengthdefines (P)lus or (M)inus]. However the Y102A mutantappears to stabilize the inverted chiral M-form. Thismutant exhibits catalytic activity (kcatis approximately50% of wild-type), albeit with a seven-fold increase inthe KmBILIVERDIN. Because the KmNADPHis very simi-lar to wild-type, this suggests that hydride transferfrom the C4 of the nicotinamide ring can be accom-plished relatively efficiently, even with variable configu-rations of biliverdin bound at the active site. TheY102A mutant therefore accomodates a variant config-uration of biliverdin to the wild-type enzyme butretains the ability to catalyse the transfer of hydridefrom both pyridine nucleotides. As discussed previ-ously [30], the most likely model for the biliverdinbinding site can accommodate a number of conforma-tions of biliverdin, including the various locked iso-mers that have been shown to bind productively inhBVR-A [30] and the two helical P- and M-conformersdescribed in the present study.The description of a functional BVR-A in somecyanobacteria introduces an important issue withregard to the subcellular localization of both PcyA andsBVR-A. These two enzymes potentially compete forsubstrate and different subcellular localizations wouldprovide a way out of this hypothetical dilema. The opti-mum pH for activity for sBVR-A at acid pH values isconsistent with the hypothesis that sBVR-A may belocalized in the lumen of the thylakoid [5], which isreported to maintain a pH in the range 5.5–5 [31]. As aresult of the low abundance of this protein, we have notbeen able to confirm this using immunogold labelling(L. Weaver, J. M. Hayes & T. J. Mantle, unpublishedresults). The enzymes responsible for the synthesis ofthe light-harvesting pigments phycocyanobilin and phy-coerythrobilin (PcyA, PebA and PebB) are all ferre-doxin-dependent and their reaction product is destinedfor incorporation into the phycobilisomes that decoratethe cytosolic side of the thylakoid membrane. The bilinreductase PcyA exhibits a pH optimum of 7.5 [32],whereas PebA and PebB are assayed at pH 7.5 [4], con-sistent with a distinct subcellular localization to sBVR-A and most likely the cytosol, which has been reportedto maintain a pH in the range 6.8–7.2 [31]. Furtherwork is required to resolve this important question.Experimental proceduresThe protein coding DNA for sBVR-A was amplified fromSynechocystis PCC6803 genomic DNA using forward (5¢-CGCGGATCCCATGTCTGAAAATTTTG-3¢) and reverse(5¢-CGCCTCGAGCTAATTTTCAACTATATC-3¢) primerscontaining BamH1 and Xho1 sites, respectively, to allowdirectional cloning into a modified pET41a expression vec-tor (Novagen, Madison, WI, USA). The GST-sBVR-Afusion protein expressed from pETBVR-A in E. coli BL21(DE3) cells was purified on glutathione-sepharose (Chroma-trin Ltd, Dublin, Ireland) cleaved with thrombin (Sigma–Aldrich, St Louis, MO, USA) and the GST fragmentremoved by affinity chromatography on glutathione-sepha-rose. Prior to HPLC analysis, the purified protein wasincubated in 6 m urea at 95 °C for 2 min, centrifuged at16 000 g for 2 min and immediately loaded onto a SupelcoDiscovery C18 reversed phase HPLC column (Supelco,Bellefonte, PA, USA) (25 · 4 mm) at a flow rate of1mLÆmin)1. The HPLC column was equilibrated in100 mm potassium phosphate (pH 6) and elution wasachieved using a linear gradient of 0–40% methanol.Size-exclusion chromatography was conducted using1 · 100 cm Sephacryl 200 HR (Sigma–Aldrich) columnsequilibrated at pH 5 (25 mm sodium citrate, 100 mm NaCl)and pH 7.5 (25 mm Tris ⁄ HCl, 100 mm NaCl) at both 4 °Cand 20 °C. The calibration proteins used [b-amylase(200 kDa), alcohol dehydrogenase (150 kDa), BSA(66 kDa), carbonic anhydrase (29 kDa) and cytochrome c(12.4 kDa)] were individually applied to the column andtheir elution volumes used to construct a standard curve oflog molecular mass versus elution volume.Light scattering was performed on sBVR-A at pH 5 andpH 7.5 at 20 °C. Protein samples (0.25 mgÆmL)1) were clar-ified using a 0.22 lm filter and applied to an S-200 Super-dex HR gel-filtration column connected to an AKTAFPLC system (Amersham Biosciences, Little Chalfont,UK). The column was run at 20 °C and a flow rate of0.5 mgÆmL)1in the desired equilibration buffer (25 mmsodium citrate, pH 5, 100 m m NaCl or 25 mm Tris ⁄ HCl,pH 7.5, 100 mm NaCl). The gel-filtration column was con-nected online to a miniDawn Tristar light-scattering detec-tor (Wyatt Technology, Santa Barbara, CA, USA) and anOptilab rEX Rayleigh interference detector (Wyatt Tech-nology). The weight-average molar mass of sBVR-A wascalculated using the software astra (Wyatt Technology).J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-AFEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4423[...]... reciprocal of the apparent Vmax was plotted against the concentration of inhibitory product and the inhibition constant for the intercept effect (Kii) was determined from the intersection on the inhibitor concentration axis For pH studies, determination of pK values has been described previously [35,36] Acknowledgements We thank Tatsiana Rakovich and Kieran CrosbieStaunton for initial work on the pH kinetics. .. at 460 nm as a result of the appearance of bilirubin-IXa using a Hexios spectrophotometer (Thermo Spectronic, Cambridge, UK) with online chart recorder (Kipp & Zonen, Hilperton, UK) The typical reaction mixture contained 1–5 lg purified sBVR-A, 100 mm sodium citrate buffer (pH 5) and various concentrations of the substrate biliverdin-IXa and cofactor NAD(P)H (Calbiochem) The reaction was performed at... For initial rate and product inhibition studies, data sets were converted to double-reciprocal plots To determine inhibition constants involving slope changes, the apparent Km ⁄ Vmax was replotted against the concentration of inhibitory product The straight line intercepted the x-axis at –Kis to give the inhibition constant for the slope effect For inhibition constants involving intercept effects, the. .. initiated by the addition of enzyme or NAD(P)H The extinction coefficient for bilirubin under these conditions is 35.75 mm)1Æcm)1 For initial rate kinetics, data points (in triplicate) were fitted to the Michaelis–Menten equation using a least squares fitting routine and the computer software prism (GraphPad Software Inc., San Diego, CA, USA) or wincurve fit, 4424 version 1.3 (Kevin Raner Software, Victoria,... kinetic and physical properties of biliverdin reductase Biochem Soc Trans 9, 275–278 Florini JR & Vestling CS (1957) Graphical determination of the dissociation constants for two-substrate enzyme systems Biochim Biophys Acta 25, 575–578 Trull FR, Ibars O & Lightner DA (1992) Conformation inversion of bilirubin formed by reduction of the biliverdin-human serum albumin complex: evidence from circular... TJ (2007) Activation of biliverdinIXalpha reductase by inorganic phosphate and related anions Biochem J 405, 61–67 Noguchi M, Yoshida T & Kikuchi G (1979) Purification and properties of biliverdin reductases from pig spleen and rat liver J Biochem 86, 833–848 Maines MD & Trakshel GM (1993) Purification and characterization of human biliverdin reductase Arch Biochem Biophys 300, 320–326 Phillips O & Mantle... circular dichroism Arch Biochem Biophys 298, 710–714 Ennis O, Maytum R & Mantle TJ (1997) Cloning and overexpression of rat kidney biliverdin IX alpha reductase as a fusion protein with glutathione S-transferase: stereochemistry of NADH oxidation and evidence that the presence of the glutathione S-transferase domain does not effect BVR-A activity Biochem J 328, 33–36 Carbone V, Sumii R, Ishikura S, Asada... interpretation of analytical sedimentation data for proteins In Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding SE, Rowe AJ & Horton JC, eds), pp 90–125 Royal Society of Chemistry, Cambridge 34 Fisher CL & Pei GK (1997) Modification of a PCR-based site-directed mutagenesis method BioTechniques 23, 570–571 35 O’Fagain C, Butler BM & Mantle TJ (1983) The effect of pH on the kinetics of. .. Structure of monkey dimeric dihydrodiol dehydrogenase in complex with isoascorbic acid Acta Crystallogr D Biol Crystallogr 64, 532–542 Lott JS, Halbig D, Baker HM, Hardman MJ, Sprenger GA & Baker EN (2000) Crystal structure of a truncated mutant of glucose-fructose oxidoreductase shows that an N-terminal arm controls tetramer formation J Mol Biol 304, 575–584 Effect of pH on the dimeric Synechocystis. .. ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms Plant Cell 13, 965–978 5 Schluchter WM & Glazer AN (1997) Characterization of cyanobacterial biliverdin reductase Conversion of biliverdin to bilirubin is important for normal phycobiliprotein biosynthesis J Biol Chem 272, 13562–13569 6 Baranano DE, Rao M, Ferris CD & Snyder SH (2002) Biliverdin reductase: a major physiologic cytoprotectant . The effect of pH on the initial rate kinetics of the dimeric biliverdin-IXa reductase from the cyanobacterium Synechocystis PCC6803 Jerrard. two-substrateenzyme, one substrate is held at 10 · Km(91% saturat-ing) and the variation of initial rate with the concen-tration of the second substrate is then used
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