Biliverdin-IXa reductase BVR-A is most active with its physiological substrate biliverdin-IXa, but can also reduce the three other biliverdin isomers IXb, IXd and IXc.. We now show that
Trang 1verdin sites of human biliverdin-IXa reductase and human biliverdin-IXb reductase
Edward M Franklin1, Seamus Browne1, Anne M Horan1, Katsuhiko Inomata2, Mostafa A S
Hammam3, Hideki Kinoshita2, Tilman Lamparter4, Georgia Golfis1 and Timothy J Mantle1
1 School of Biochemistry and Immunology, Trinity College, Dublin, Ireland
2 Division of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Ishikawa, Japan
3 Department of Chemistry, School of Science, Nagoya University, Aichi, Japan
4 Institut fu¨r Botanik I, Universita¨t Karlsruhe (TH), Germany
Introduction
The reduction of biliverdin-IXa to bilirubin-IXa
catal-ysed by biliverdin-IXa reductase (BVR-A) comprises
an ancient reaction that has been conserved
through-out evolution from cyanobacteria to man [1,2] Until
recently, the pathway in mammals has been considered
functionally as the catabolic elimination of excess
haem This view has been challenged by recent observations that haem oxygenase plays a major cytoprotective role [3], and there is clear evidence that bilirubin-IXa is a physiologically significant antioxi-dant [4] Central to this model is the enzyme biliverdin reductase, which is responsible for the maintenance of
Keywords
Biliverdin, dimethyl ester, ditaurate, inhibitor,
jaundice, tolerance
Correspondence
E M Franklin, School of Biochemistry
and Immunology, Trinity College Dublin,
Dublin 2, Ireland
Fax: +353 1677 2400
Tel: +353 1896 1612
E-mail: efrankli@tcd.ie
(Received 23 April 2009, revised 9 June
2009, accepted 11 June 2009)
doi:10.1111/j.1742-4658.2009.07148.x
Many vertebrate species express two enzymes that are capable of catalysing the reduction of various isomers of biliverdin Biliverdin-IXa reductase (BVR-A) is most active with its physiological substrate biliverdin-IXa, but can also reduce the three other biliverdin isomers IXb, IXd and IXc Bili-verdin-IXb reductase (BVR-B) catalyses the reduction of only the IXb, IXd and IXc isomers of biliverdin Therefore, the activity of BVR-A can be measured using biliverdin-IXa as a specific substrate We now show that the dimethyl esters of biliverdin-IXb and biliverdin-IXd are substrates for BVR-B, but not for BVR-A This provides a useful method for specifically assaying the activity of both BVR-A and BVR-B in crude mixtures, using biliverdin-IXa for BVR-A and the dimethyl ester of either biliverdin-IXb
or biliverdin-IXd for BVR-B Human BVR-A has been suggested as a pharmacological target for neonatal jaundice Because of the absence of a crystal structure with biliverdin bound to BVR-A, we have investigated indirect ways of examining tetrapyrrole binding In the present study, we report that a number of sterically locked conformers of 18-ethylbiliverdin-IXa are substrates for human BVR-A, and discuss the implications for the biliverdin binding site The oxidation of bilirubin-IXa ditaurate to biliver-din-IXa ditaurate is also described We show that biliverbiliver-din-IXa ditaurate
is a substrate for human BVR-A and discuss the possibility of using a com-peting substrate, which is reduced to a water soluble and excretable rubin,
as a prototypic inhibitor of BVR-A
Abbreviations
18EtBV, 18-ethylbiliverdin-IXa; BVR-A, biliverdin-IXa reductase; BVR-B, biliverdin-IXb reductase; DDQ, 2,3-dichloro-5,6-dicyanobenzoquinone; hBVR-A, human biliverdin-IXa reductase; hBVR-B, human biliverdin-IXb reductase.
Trang 2cytoprotective levels of bilirubin-IXa [5] This
antioxi-dant mechanism has been suggested to involve cycling
between biliverdin-IXa and bilirubin-IXa [5]
Intrigu-ing evidence has been obtained from animal
transplan-tation studies [6–8] showing that the administration of
biliverdin-IXa is cytoprotective for the heart, colon
and liver In a notable study, Yamashita et al [8]
demonstrated that short-term treatment (3 weeks) with
biliverdin-IXa is sufficient to induce tolerance in a
recipient to the donor heart for 120 days The
intro-duction of an allogeneic third heart at 120 days was
rejected, whereas the introduction of a third syngeneic
heart was accepted, clearly indicating tolerance [8]
The exact mechanism for achieving tolerance remains
obscure, although it is clear that components of the
haem catabolic pathway play a major role in successful
organ tranplants as well as in physiological
cytopro-tection [4,8]
BVR-A has also been identified as a
pharmacologi-cal target for treating neonatal jaundice [9] Mammals
maintain relatively high levels of circulating
bilirubin-IXa This is excreted in the bile, predominantly as a
conjugate with glucuronic acid At birth, the delayed
expression of glucuronyltransferase UGT1A1 results in
increased levels of unconjugated bilirubin in the liver,
which reflux into plasma This results in transiently
elevated levels of bilirubin-IXa, which manifests as
neonatal jaundice If the albumin binding capacity is
exceeded, bilirubin-IXa can partition into the brain
and cause irreversible damage or death
The design of pharmacological inhibitors for
BVR-A would not only combat the hyperbilirubinemia
asso-ciated with neonatal jaundice, but also might prove
significant during organ transplantation by elevating
the concentration of cytosolic biliverdin-IXa The
pau-city of information on the biliverdin binding site has
hampered the development of inhibitors of BVR-A
With this in mind, and in the absence of a crystal
structure of the ternary complex of BVR-A with
pyri-dine nucleotide and biliverdin, we set out to further
probe the nature of the biliverdin binding pocket in a
significant extension to our earlier studies [2] In the
present study, sterically-locked conformers of
18-ethyl-biliverdin-IXa (18EtBV) are shown to be substrates for
human BVR-A (hBVR-A), and the implications for
the nature of the binding of biliverdin-IXa to BVR-A
are discussed
We also show that biliverdin ditaurate is a substrate
for BVR-A and discuss the possibility of using a
com-peting substrate such as this to reduce the levels of the
lipophilic and potentially toxic bilirubin-IXa by
pro-ducing bilirubin ditaurate, which is water soluble and
readily excreted
Additionally, by studying various biliverdin-IX iso-mers, we present a method that specifically allows the assay of both BVR-A and biliverdin-IXb reductase (BVR-B) enzymes in crude preparations Catalytically, the major difference identified to date between the two human enzymes is that BVR-B catalyses the reduction
of biliverdin-IXb, biliverdin-IXd and biliverdin-IXc, but not IXa [2,10] BVR-A prefers biliverdin-IXa as substrate, but can reduce all three other isomers [10] Thus, although it is possible to specifically measure the activity of BVR-A with biliverdin-IXa, it is not pos-sible to specifically measure the activity of BVR-B with any of the free acids of biliverdin (IXa, IXb, IXc or IXd) We now show that BVR-B can reduce biliverdin-IXb, biliverdin-IXd and biliverdin-IXc as their dimethyl esters in clear distinction to BVR-A This permits the specific assay of the activity of BVR-A and BVR-B in crude cytosols Increasingly, straightforward assays are required that are specific for measuring the activity of BVR-A and BVR-B Protein levels of BVR-A are signi-ficantly elevated in chronic hypoxia in the rat lung [11] and after focal cerebral ischaemia in mice [12] In addi-tion, lipopolysaccharide has been reported to increase rat kidney BVR-A by a post-transcriptional mechanism [13] and thymidine induces BVR-A in human K562 ery-throleukemic cells [14] Furthermore, both BVR-A and BVR-B have recently been reported to form adducts with environmental toxins [15] and endogenous ligands [16] BVR-B is adducted at equimolar stoichiometry by electrophilic metabolites of 1-nitronapthalene [15] and BVR-A by 15-deoxy-D12,14-prostaglandin J2 [16] It is unclear whether the adducted proteins retain activity Measurement of both the RNA and protein level of both BVR-A and BVR-B is straightforward; however,
to date, there has been no method of specifically assay-ing the activity of both enzymes in crude preparations with biliverdin substrates
Results
Non-IXa biliverdin dimethyl esters as substrates for human BVR-B (hBVR-B)
All three ‘non-IXa’ biliverdin dimethyl esters were reduced to the corresponding bilirubins by BVR-B The spectra of the dimethyl ester of biliverdin-IXb in the presence and absence of 37 lm BSA revealed no significant change in the pigment spectrum (Fig 1A) The addition of hBVR-B (18.5 lg) revealed a time-dependent decrease at 660 nm and an increase at
460 nm (Fig 1B) Similar results were obtained with the dimethyl ester of biliverdin-IXd (data not shown)
We did not have sufficient material for detailed studies
Trang 3with the dimethyl ester of biliverdin-IXc Initial rate
kinetics show that the dimethyl esters of the IXb and
IXd isomers all demonstrate substrate inhibition with
apparent substrate inhibitory Kivalues in the lm range
(data not shown)
Probing the biliverdin binding pocket of BVR-A
using synthetic biliverdin isomers
The substrate specificity of hBVR-A was examined
using a range of synthetic biliverdins locked in
various conformations These biliverdin derivatives have their C and D rings sterically locked by cycliz-ing with an additional two- or three-carbon chain [22] (and are termed 15Z-syn 18EtBV, 15Z-anti 18EtBV, 15E-syn 18EtBV and 15E-anti 18EtBV; Fig 2) All of the fixed isomers were substrates for human BVR-A (Fig 4) The spectra of all of the fixed isomers exhibited the characteristic immediate
A
B
Fig 1 Spectra of the hBVR-B catalysed reaction of the dimethyl
ester of biliverdin-IXb (A) Biliverdin-IXb dimethyl ester (7 l M ) in
100 m M potassium phosphate (pH 7.5) containing 1% (v ⁄ v)
metha-nol from the stock solution of ester (blue); addition of 37 l M BSA
from a stock solution of 50 mgÆmL)1produced the red trace and
subsequent addition of 50 l M NADPH produced the green trace.
After addition of enzyme, the reaction was allowed to go to
completion (orange trace) (B) The BVR-B reaction was initiated by
the addition of hBVR-B (18.5 lg) and the spectra recorded over a
period of 20 min.
Fig 2 The structures of the open chain and locked tetrapyrroles The structures shown are 15Z-syn 18EtBV, 15Z-anti 18EtBV, 15E-syn 18EtBV and 15E-anti 18EtBV.
Trang 4red shift (for the ‘390’ and ‘660’ peaks) on the
addi-tion of BSA and this was particularly marked for
the 15E-syn 18EtBV isomer (Fig 3C) On addition
of human BVR-A (1.4 lg), all the locked isomers
showed an increase in absorbance in the 460 nm
region coupled with a decrease at 650 nm, which
was consistent with reduction to the corresponding
bilirubins (Fig 4A–D) When the initial rate was
measured (DA460) at a range of concentrations of the
various locked isomers, the characteristic substrate
inhibition profile was evident (Fig 5A–C) Although
we have not isolated or characterized the putative
bilirubin products from the locked isomer reactions,
the spectral changes that we observed are entirely
consistent with enzyme-catalysed formation of the
corresponding bilirubin The possibility of a C-10
adduct (other than hydride from NADPH) is highly
unlikely Thiol compounds will adduct to biliverdin
to give a yellow product; however, because neither
NADPH nor BVR-A alone were capable of initiating
an increase in A460, adduct formation of this type
can be ruled out The fact that the linear increase
with time at A460 required NADPH, BVR-A and the
locked biliverdin isomer is entirely consistent with
enzyme-catalysed hydride transfer from NADPH to
the various locked isomers The demonstration that
locked isomers of biliverdin can be substrates for
BVR-A has been reported previously for the rat liver
enzyme [26], although the physiologically less
rele-vant IXc isomer and its locked variants were used in
that study
Competitive substrates as inhibitors
As an alternative strategy to biliverdin reductase
inhibitors for human BVR-A, we were interested in
the possibility that competing substrates may provide
a means of inhibiting BVR-A in vivo In the case of
the natural isomer, biliverdin-IXa, the reduction
product bilirubin-IXa is lipophilic and requires
sub-sequent conjugation with glucuronic acid for efficient
elimination However, replacing the propionate side
chains of biliverdin-IXa by sulfonate analogues
should produce a water soluble bilirubin that would
not need conjugation prior to excretion Figure 6
shows the initial rate kinetics for human BVR-A
with biliverdin-IXa ditaurate The substrate
inhibi-tion with this sulfonate is less potent than that seen
with biliverdin-IXa Biliverdin-IXa sulfonate is also a
good substrate (data not shown) and, similar to
bili-verdin-IXa ditaurate, the substrate inhibition is not
as potent as that seen with biliverdin-IXa
A
B
C
D
Fig 3 Spectra of the locked conformers of 18EtBV The spectra of the fixed isomers are shown in (A) 15Z-syn 18EtBV, (B) 15Z-anti 18EtBV, (C) 15E-syn 18EtBV and (D) 15E-anti 18EtBV in 100 m M Tris ⁄ HCl (pH 8) The immediate effect of the addition of 37 l M BSA
is also shown.
Trang 5The induction of haem oxygenase has been implicated
in a number of models for cytoprotection, and subse-quently, there has been an increasing interest in the enzymes of tetrapyrrole metabolism The two down-stream products of haem oxygenase activity, the linear tetrapyrroles biliverdin-IXa and bilirubin-IXa, have been identified as agents that improve the efficacy of organ transplantation [8,27] BVR-A has been identi-fied as a pharmacological target for treating neonatal jaundice [9] Increasingly, there is a requirement for straightforward assays that are specific for measuring the activity of BVR-A and BVR-B in crude prepara-tions Until now, no method of specifically assaying the activity of both enzymes in crude preparations with biliverdin substrates has been proposed
As described previously, the free acid of biliverdin-IXa is not a substrate for hBVR-B [2], and the dimethyl ester behaves similarly The bridging propio-nate side chains of biliverdin-IXa preclude access to the substrate pocket of hBVR-B in a productive mode and the tetrapyrrole rotates 90 compared to meso-biliverdin-IVa [28] The rotated configuration observed with biliverdin-IXa bound to BVR-B is not consistent with hydride transfer [28] It is likely that a similar binding orientation is adopted when the dimethyl ester
of biliverdin-IXa binds to hBVR-B Biliverdin-IXa dimethyl ester clearly does bind because it inhibits hBVR-B activity with an apparent Ki in the micro-molar range The dimethyl ester of biliverdin-IXa is not a substrate for BVR-A (data not shown), which is
in agreement with previous findings [29] Clearly, the activity of BVR-A can be measured specifically using biliverdin-IXa as a substrate [2] and, in the present study, we report that it is now possible to specifically measure the activity of BVR-B using the dimethyl ester
of biliverdin-IXb or biliverdin-IXd It is not possible
to use the free acids of the IXb, IXd and IXc biliver-din isomers as specific substrates because they are reduced by BVR-A as well as BVR-B [10]
The substrate specificity of hBVR-A was also analy-sed using a range of synthetic biliverdins locked in various conformations to gain further insight on the biliverdin binding site of human BVR-A It is highly
A
B
C
D
Fig 4 Spectra of the BVR-A catalysed reactions of the locked con-formers of biliverdin-IXa The spectra of the fixed isomers are shown in (A) 15Z-syn 18EtBV, (B) 15Z-anti 18EtBV, (C) 15E-syn 18EtBV and (D) 15E-anti 18EtBV in 100 m M Tris ⁄ HCl (pH 8) Zero time has 120 l M NADPH added and the subsequent scans, over 10 and 12 min (as indicated), show the hBVR-A dependent reduction
of the pigment and the associated oxidation of NADPH.
Trang 6likely that BVR-A and BVR-B follow the same reac-tion mechanism with hydride transfer to a carbocareac-tion intermediate [30] The angle of attack is likely to be the same such that the reaction centre for both BVR-B and BVR-A is likely to involve the B-face hydrogen atom attached to C4 of the nicotinamide ring and the two bridging pyrroles (rings B and C) with the linking C10 methene bridge as the electrophilic centre Over-lapping conformationally stable forms of the sterically-locked conformers of 18EtBV, where the two C-10 bridging pyrroles (B and C) are ‘fixed’, reveals that the outer pyrrole rings (A and D) can adopt a variety of conformations in the verdin binding site of BVR-A There are two models that require consideration to define biliverdin binding in light of the present obser-vations The first model assumes that all four pyrroles are bound in the active site, which must be able to accommodate various conformations of the tetrapyr-role If we assume a helical ‘lock washer’ conformation for the ‘nonlocked’ physiological substrate biliverdin-IXa, then, although the B and C rings may deviate slightly from planarity, the deviation for the outer A and D rings is sufficient to suggest that they may inter-act with the ‘roof’ and ‘floor’ of a hypothetical ‘verdin binding site’, with the nicotinamide ring forming part
of the floor If the bound conformation is helical, there would be a significant distance between the ‘roof’ and the ‘floor’, allowing a variety of conformational states
to be accommodated, as appears to be the case for the locked isomers shown to be substrates in the present study Such a model would also allow the binding of both P- and M-helical configurations described by Hayes & Mantle [31] for the wild-type and Y102A
A
B
C
Fig 5 Initial rate kinetics of the BVR-A catalysed reactions of the
locked conformers of biliverdin-IXa The initial rate kinetics of the
fixed isomers are shown in (A) 15Z-syn 18EtBV, (B) 15Z-anti
18EtBV, (C) 15E-anti 18EtBV in 100 m M Tris ⁄ HCl (pH 8) with
120 l M NADPH The initial rates were measured by recording
DA 460 at the range of concentrations indicated for the various
locked isomers The data are fitted to partial substrate inhibition
[25] The limited availability of the locked isomers curtailed the
range of substrate concentrations studied.
Fig 6 Initial rate kinetics of BVR-A with biliverdin-IXa ditaurate The reaction was carried out at 30 C and was initiated by the addi-tion of enzyme.
Trang 7mutant forms, respectively, of the cyanobacterial
enzyme BVR-A from Synechocystis PCC6803 The
second model requires that only the central B and C
pyrroles are bound with the two outer pyrroles in
solvent and therefore not constrained This would
allow the various locked conformers to be substrates
but would not be consistent with the CD spectra
dem-onstrating that helical conformations bind to human
(E Franklin and J M Hayes, unpublished work) and
cyanobacterial [31] forms of BVR-A The second
model is clearly applicable for BVR-B, where the inner
pyrroles are in good density [28], although the outer
pyrroles show low occupancy and are modelled in the
solvent The second model does not allow protein
binding to stabilize either P- or M-helical forms, which
we clearly see in the case of both human and
cyano-bacterial forms of BVR-A
Clearly, we require the crystal structures of ternary
complexes with biliverdin-IXa to define the biliverdin
binding site It is likely that conformational changes
may be needed to facilitate biliverdin binding once the
enzyme-pyridine nucleotide complex has formed This
information will allow the development of potential
BVR-A inhibitors In this regard, we have shown that
biliverdin-IXa sulfonate is a substrate for human
BVR-A, although its synthesis is not straightforward
The rubin product is water soluble and readily
excreted, whereas bilirubin-IXa requires conjugation
with glucuronic acid prior to excretion The concept of
a competing substrate as a BVR-A inhibitor, where
the product is readily excretable, is of some interest In
the present study, we have also shown that the
synthe-sis of biliverdin ditaurate is relatively straighforward
and that it is a substrate for human BVR-A Our
preli-minary investigations indicate that an in vivo
evalua-tion of this compound is warranted to determine
whether it can competitively reduce the levels of the
potentially toxic bilirubin-Ixa, whereas the bilirubin
ditaurate is predicted to retain the ability to be
elimi-nated efficiently from circulation The supplementation
of competitive substrates that are reduced to a soluble
bilirubin product might also boost the degree of
physi-ological cytoprotection afforded by the cycling of
bilirubin described by Baranano et al [5]
Experimental procedures
Preparation of biliverdin-IXd dimethyl ester and
biliverdin-IXb dimethyl ester
The dimethyl esters of biliverdin-IXb, biliverdin-IXc and
biliverdin-IXd were synthesized by coupled oxidation of
haem, producing the linear free acids Esterification of the
resulting free acids with BF3⁄ MeOH and separation of all three biliverdin dimethyl esters by TLC was performed as described [17] The band corresponding to the various dimethyl esters was scraped off, and the pigment extracted into acetone Isomers with the greatest degree of separation
on TLC were used in the present study The IXd dimethyl ester runs as a distinctly separate band, whereas the upper-most fraction of the IXb dimethyl ester band was isolatable
in a homogenous form The NMR spectra of the dimethyl ester of the IXd and IXb isomers were in accordance with a previous study [17], and the absorption spectra in methanol were as reported previously [18] The isomers also showed the red shift and doubling of extinction coefficient on the addition of HCl [18] The dimethyl ester of biliverdin-IXa was synthesized by oxidation of the free acid of bilirubin-IXa to biliverdin-bilirubin-IXa using 2,3-dichloro-5,6-dicyanobenzo-quinone (DDQ) [19] and subsequent esterification with
BF3⁄ MeOH [17] The synthesis of the sterically locked bilins has been described previously [20,21] and the recorded absorbance spectra are reported [22] Biliverdin ditaurate was synthesized by oxidation of bilirubin-IXa ditaurate using DDQ Briefly, bilirubin-IXa ditaurate (53 mg) was dissolved in 20 mL of sterile distilled water in
a round bottomed flask, to which 30 mg of DDQ was added and mixed The solution was allowed to react for
5 min and then silica gel (3 g) was added to the flask The biliverdin ditaurate was dried onto silica gel by rotary evap-oration A silica gel 60G column of 20 mL bed volume was equilibrated in ethyl acetate (100 mL) The dried biliverdin ditaurate⁄ silica gel material was loaded onto the column The column was washed with 300 mL of ethyl acetate to remove the reduced quinone The column was then washed with 150 mL of ethyl acetate⁄ methanol (4 : 1, v ⁄ v) to elute the unreacted quinone and bilirubin ditaurate Finally, the biliverdin ditaurate was eluted with 100% methanol and dried to a powder by rotary evaporation This material was homogenous by TLC and the NMR spectrum of the final product was consistent with oxidation at C10 The extinc-tion coefficient at 660 nm at pH 6.8 is 11.3 mm)1Æcm)1 Biliverdin-IXa sulfonate was a generous gift from Professor David Lightner (University of Reno, NV, USA)
Enzyme assays Recombinant hBVR-A and hBVR-B were purified and assayed as described previously [2] The biliverdin dimethyl esters were solubilized in methanol and additions from this stock solution were made to the assay mix The final con-centration of methanol in the assay mix never exceeded 1%, which was shown not to affect enzyme activity The assay mix also contained 37 lm BSA to aid solubilization
of the dimethyl ester The addition of BSA lowers the free concentration of the various verdins and their dimethyl esters For this reason, we have not reported the Kior Km
values, although we show the data plotted against the total
Trang 8verdin concentration for comparative purposes with the
lit-erature This issue has been discussed previously [23], along
with kinetic studies on BVR-A conducted in the absence of
biliverdin-binding proteins [24] and the difficulties in
cor-recting for biliverdin binding when it is known to occur
[25] When sterically-locked conformers of 18EtBV were
used as substrates, the assay mix contained 120 lm
NADPH in 100 mm Tris⁄ HCl (pH 8) Spectra of the
vari-ous locked bilins were measured in 100 mm Tris⁄ HCl (pH
8) using a Cary 300 UV-Vis spectrophotometer (Varian
Inc., Palo Alto, CA, USA) The effect of 37 lm BSA and
also the subsequent addition of 120 lm NADPH on the
spectra were recorded BVR-A (1.4 lg) was then added,
and spectra were recorded at intervals over a period of
20 min
Acknowledgement
This work was supported by a grant from Science
Foundation Ireland
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