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This is an enhanced and peer-reviewed version of an original article published by PLoS ONE. The primary difference in this version is the direct interactivity with the 3D representations, with animated transitions triggered by the links in the main text. As such this is not the version of record or the version that should be cited. The original citation for this article is Pilka ES et al. 2009. PLoS ONE 4(5): e5613. doi:10.1371/journal.pone.0005613.
Structural Basis For Substrate Specificity In Human Monomeric Carbonyl Reductases
Ewa S Pilka 1* , Frank H Niesen 1* , Wen Hwa Lee 1* , Yasser El-Hawari 2 , James E Dunford 3 , Grazyna Kochan 1 , Vladimir Wsol 4 , Hans-Joerg Martin 2 , Edmund Maser 2 , and Udo Oppermann 1,3,#
1 Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Headington, OX3 7DQ, UK.
2 University of Kiel, Kiel, Germany.
3 Nuffield Department of Orthopedic Surgery, Rheumatology and Musculoskeletal Sciences, Botnar Research Center, University of Oxford, Oxford, OX3 7LD, UK.
4 Faculty of Pharmacy, Charles University, Hradec Kralove, Czech Republic.
* These authors contributed equally to this work.
#Corresponding author e-mail: udo.oppermann@sgc.ox.ac.uk
Funding: The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Abstract
Carbonyl reduction constitutes a phase I reaction for many xenobiotics and is carried out in mammals mainly by members of two protein families, namely aldo-keto reductases and short-chain dehydrogenases/reductases. In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids. One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum. A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date. Screening of a focused xenobiotic compound library revealed that CBR3 has a narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further structure-activity relationships between these enzymes we crystallized CBR3, performed substrate docking, site-directed mutagenesis and compared its kinetic features to CBR1. Despite high sequence similarities, the active sites differ in shape and surface properties. The data reveal that the differences in substrate specificity are largely due to a short segment of a substrate binding loop comprising critical residues Trp229/Pro230, Ala235/Asp236 as well as part of the active site formed by Met141/Gln142 in CBR1 and CBR3, respectively. The data suggest a minor role in xenobiotic metabolism for CBR3.
Introduction
Reduction of carbonyl groups to the corresponding alcohols constitutes a significant metabolic step both for endogenous and xenobiotic compounds [1-3]. These reactions are carried out by distinct NAD(P)(H) dependent oxidoreductases mainly belonging to three protein superfamilies, namely the short-chain dehydrogenases/reductases (SDR), aldo-keto-reductases (AKR), or medium-chain dehydrogenases/reductases (MDR) [1-3].
A unifying feature of carbonyl reductases appears to be their broad and often overlapping substrate specificity. Endogenous substrates comprise steroids, eicosanoids, cofactors, neurotransmitters and polyols. In addition, a large set of xenobiotics has been identified as substrates for carbonyl reducing enzymes including drugs such as warfarin, daunorubicin or ketoprofen, environmental pollutants (PAH quinones derived from diesel exhaust such as phenanthrenequinone) or tobacco derived carcinogens like NNK [2,3].
In humans, several enzymes contribute significantly to the metabolic reductive transformation, mainly found in tissues such as liver, kidney, placenta or the central nervous system. The major cytosolic enzymes identified are carbonyl reductase (CBR1, according to the official nomenclature system SDR21C1)[4,5], belonging to the SDR family, and members of the AKR family such as aldehyde reductase (AKR1A1), aldose reductase (AKR1B1), several dihydrodiol/hydroxysteroid dehydrogenases (of the AKR1C subfamily) or aflatoxin aldehyde reductase (AKR7A2) [1-3,6].
CBR1 fulfills an important role in the phase I metabolism of xenobiotics. Substrates include o-quinones derived from polycyclic aromatic hydrocarbons (PAH) or p-quinones such as menadione, besides an extraordinarily wide spectrum of xenobiotic carbonyls such as anthracyclines, metyrapone or the carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone. The endogenous compound spectrum comprises steroids, eicosanoids and lipid derived aldehydes [7-9].
Recent studies indicate a role for CBR1 in apoptosis, tumor metastasis and oxygen induced stress [10-12]). At present, no clear evidence is available which specific endogenous substrate is responsible for these effects, however a recent study showed that CBR1 effectively inactivates in vitro the lipid aldehyde 4-oxononenal [13], indicating that CBR1 is involved in the stress response and elimination of metabolites produced by reactive oxygen species.
A human paralog, CBR3 (SDR21C2)[4,5], which is 71% identical to CBR1 [6] is located in the vicinity of the CBR1 gene on chromosome 21 at 21q22.12. However, thus far limited information on enzymatic properties of CBR3 is available [14]; in this and another study [15] only CBR3 activity towards the model substrate menadione, 4-nitrobenzaldehyde or 4-benzoylpyridine was reported. Given the importance of CBR1 and other carbonyl reductases in endogenous and xenobiotic carbonyl metabolism, we performed a study to establish a substrate specificity profile of human CBR3. To understand structure-function relationships between the two related human CBR enzymes, we analyzed substrate specificity features of CBR1 and CBR3, and furthermore establish a structural basis for the activity differences through mutational, kinetic and crystallographic studies.
Results
Substrate screening of CBR3 and comparison to CBR1
Human CBR3 was expressed as N-terminally His6-tagged protein in
E. coli
and purified to apparent homogeneity by consecutive chromatographic steps comprising immobilized metal affinity and size-exclusion chromatography. The enzyme was subjected to a substrate screening against a focused library of 111 different carbonyl substrates, using spectrophotometric and HPLC-based assays. The library consisted of a variety of endogenous carbonyl containing ligands such as polyols, eicosanoids or steroids, as well as a diverse set of xenobiotic carbonyl compounds, shown to be substrates for distinct types of carbonyl reductases for review see [6].
The screening was carried out side-by-side with human CBR1 under identical conditions and revealed that CBR3 has a much narrower substrate spectrum compared to CBR1. Our results confirm the previously recognized broad substrate specificity of CBR1 [1,3,6,9] which is able to metabolize a wide range of substrates including endogenous compounds such as prostaglandins or lipid-derived aldehydes, a wide spectrum of xenobiotics such as ortho- and paraquinones and anthracyclins (Supplementary Information Table 1). In total, we found significant activity (see below, Table 1) for 43 out of 111 substrates, with a large fraction of quinones. In contrast, a limited set of substrates were reduced by CBR3 in an NADPH-dependent manner (31 substrates), usually with significantly less activity than CBR1. Among the best substrates was 1,2-naphthoquinone, for which an activity of 2.5 μmol/(min mg) was observed. Compared to the CBR3 activity for 1,2-naphthoquinone a significant (> 10 activity was observed for 12 out of the 22 tested quinones (Table 1). Interestingly, a preference of CBR3 for ortho-quinones is apparent; no activity was found towards menadione, one of the standard substrates used in activity screens for carbonyl reductases. This is in line with the lack of activity against any other quinone in para configuration. Among the non-quinone compounds that were identified as substrates for CBR3 were isatin and oracin, coniferyl aldehyde and acetohexamide.
We investigated in more detail the difference in activity between CBR1 and CBR3 towards important xenobiotics such as isatin and naphthoquinones. The comparison of activites towards isatin and 1,2-naphthoquinone is in line with the observation from the screen: CBR1 showed higher catalytic efficiency for both substrates.
In summary, there are a number of differences between the activity profiles of the two carbonyl reductases. The most striking of them is the strong difference in the activity towards the two naphthoquinones whose only structural difference is the position of the two carbonyl groups (i.e. para vs. ortho). Furthermore, in contrast to CBR1 [9,11], CBR3 shows no activity towards eicosanoids or aliphatic carbonyls like 4-oxononenal.. At this point in time the activity observed against coniferyl aldehyde cannot be assigned to a specific chemical group, i.e. double bond or carbonyl group. Further experiments are required to verify product formation for several of the hits identified.
Active site architecture of CBR enzymes
To understand the substrate specificity differences between CBR1 and CBR3 we determined the structure of human CBR3 by X-ray crystallography and compared it to recently determined human and porcine structures [12,19] of CBR1. Based on these structures, residues of potential mechanistic importance were selected for site-directed mutagenetic replacement, and activity of resulting mutants was tested.
The 3D structures of CBR1 and CBR3 are similar , as expected with a canonical Rossmann-fold for nucleotide cofactor binding enzymes of the SDR family [20]). CBRs represent prototypes of monomeric SDRs with a two-helical insertion stabilizing an interface that in other SDRs constitutes the main oligomerization surface (Fig. 1C).
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Figure 1: Structure of human CBR3. Panel A: The substrate binding loop in CBR3 is engaged in contacts to a symmetry related copy (red circle), resulting in an open conformation of the active site. Panel B: Comparison of active site configurations of human CBR enzymes. The overlay of the complex structure of human CBR1 (1wma, in grey) with cofactor (magenta) and inhibitor (ball and stick model) with the binary complex of human CBR3 with NADP (2hrb, in red) shows the open and closed active site loop conformations. Panel C: Sequence alignment of human carbonyl reductases CBR1, CBR3 and dicarbonyl reductase DCXR. The 2-helical insertion found in CBR enzymes is highlighted by green boxing, the active site loop region discussed in this paper is highlighted by a red box. Secondary structure elements are shown for CBR1 and DCXR below the alignment. Click here for full size figure. |
Inspection of the active sites of the CBR structures reveals an arrangement consistent with the postulated reaction mechanism [20,21]. Accordingly, Tyr193/194 functions as the catalytic acid/base, Ser139/140 stabilizes the substrate by forming interactions to the substrate carbonyl, and Lys197/198 forms hydrogen bonds with the nicotinamide ribose moiety, thereby lowering the pKa of the Tyr-OH to promote proton transfer. Hydride transfer is from the S-side of C4 of the nicotinamide to the substrate. The role of Asn113/114 is to stabilize the position of Lys197/198 via a conserved water molecule, and furthermore, to establish a proton relay at the active site, including coenzyme, substrate, Tyr193/194, ribose 2'OH, Lys197/198, water, and Asn113/114.
The main distinguishing feature of the crystal structures of human/porcine CBR1 (ternary cofactor inhibitor complex PDB 1wma [12]; binary cofactor complex PDB 1n5d [19])) and human CBR3 (binary cofactor complex, PDB 2hrb) is the conformation of the substrate binding loop : whereas the CBR1 structures show a conformation with a more closed active site, in CBR3 the loop is engaged in crystal contacts with a symmetry related molecule (Fig. 1A and 1B). Despite extensive crystal screening and attempts to obtain ternary complexes, we were unsuccessful in finding different crystal forms. Inspection of the “open” structure reveals that substrate docking in this conformation is not useful to produce models explaining the observed substrate features. We therefore decided to model the CBR3 sequence using the CBR1 structure as template (Fig. 2), assuming a similar loop arrangement . The loop modelling results in a conformation with all residues in acceptable regions of a Ramachandran plot, moreover docking analysis with different substrates allowed us to successfully identify critical residues for substrate recognition and catalysis. A comparison of the two CBR structures in the loop-closed conformation shows a wide opening to a gorge-like active site. In the CBR1-inhibitor complex structure (1wma), the inhibitor molecule occupies large parts of the entrance and is also covered by a PEG molecule derived from crystallization. CBR1 has a slightly narrower substrate binding cleft (Fig. 4) than CBR3, mainly as a result of the terminal sulf-methyl group of Met141. This residue is replaced in CBR3 by Gln142 (Fig. 4F), which has a similar but not identical conformation, as observed in structures 1wma and 2hrb.
 |
Figure 2: Modelled loop in CBR3 (orange) and comparison to CBR1 (green). Positions and residues used for mutagenesis are highlighted by inclusion of their side-chains. Inhibitor and cofactor from CBR1 (PDB id 1wma) are included. Click here for full size figure. |
Critical residues for quinone specificity in CBR enzymes
Comparison of the active sites of CBR1 and CBR3 suggests that three residue positions are
critical for substrate recognition and catalysis
. In particular, we identified position 229/230 (Trp229 in CBR1, Pro230 in CBR3), position 235/236 (Ala235 in CBR1, Asp236 in CBR3) and position 141/142 (Met141 in CBR1, Gln142 in CBR3) as the most likely candidates for determination of substrate specificity. To analyze the effect of site-directed mutagenetic replacements on activity differences between the two enzymes, we selected isatin and two structural quinone isomers, 1,2- and 1,4-naphthoquinone, as model ortho- and para-quinone substrates. In case of naphthoquinones, often no saturation could be reached within the tested range of concentrations or there was very little activity observed and thus, calculation of kinetic constants was not possible.
 |
Figure 3:Active site of human CBR1 with 1,4-naphthoquinone docked into a catalytically competent orientation (the water molecules WAT1 and WAT2 were present in the crystl structure of CBR1, but were not used in the docking). The catalytic residues Ser139 and Tyr194 orient the substrate carbonyl, whereas residue Trp229 makes aromatic-stacking interactions and coordination of a water molecule (WAT1) through the indole nitrogen. As a result, the water WAT1 is positioned to form hydrogen bond with the carbonyl group in position to the substrate carbonyl. Note the crystallographic water molecule WAT2 found in the same position as the carbonyl oxygen from the docked substrate. Distances are shown in Å. Click here for full size figure. |
A major distinguishing feature between CBR1 and CBR3 is Trp229, which is replaced by a prolyl residue in CBR3 (Fig. 1C). The ternary complex of CBR1 with NADP and OH-PP[12], a high affinity inhibitor, as well as docking studies with different CBR1 substrates suggest a critical role of this residue for substrate selectivity. As deduced from the CBR1-NADP-1,4-naphthoquinone complex model (Fig. 3), Trp229 serves two possible functions, namely to provide a chemical moiety for aromatic stacking interactions with the substrate, and also to coordinate a water molecule through the indole nitrogen. This water molecule (Wat1), observed in the structure of human CBR1 (pdb code 1WMA) is putatively responsible for the CBR1 specificity towards para-quinones. This seems to be further reinforced by the position of another water molecule (Wat2) seen in the structure of CBR1, which matches with the C4-carbonyl group of the pose adopted by 1,4-Naphthoquinone docked into the active site of CBR1. We tested this hypothesis, by replacing Trp229 by Pro or Phe, as well as by creating a double mutant Trp229Pro/Ala235Asp. In CBR1 this second position is located close to Trp229 as well as to the nicotinamide and pyrophosphate portions of the cofactor (Fig. 2). Both CBR1 Trp229 mutants showed significant decrease in activity for 1,4- naphthoquinone and a modest decrease for its ortho- isomer (Table 3). Data for isatin (Table 4) showed drastic increase in Km for Trp229Pro mutant while both Trp229 substitutions led to faster substrate turnover highlighting the importance of aromatic stacking interactions for substrate recognition and binding. Destabilisation of the active site was much more significant in the Trp229Pro/Ala235Asp double mutant, where CBR1 residues were exchanged with the corresponding CBR3 residues. It resulted in a 1000-fold increase in Km and a 50-fold increase in Vmax towards isatin in comparison to WT CBR1 and in a complete loss of activity towards 1,4- naphthoquinone. Both CBR3 Pro230 mutants showed some activity towards the para-naphthoquinone but decreased activity for the ortho-naphthoquinone and isatin, as compared with the WT. However, the behaviour of the double Pro230Trp/ Asp326Ala mutant towards naphthoquinones was very similar to the WT CBR3 (Table 3), indicating the possible occurence of a steric clash between introduced aromatic residue and Asp236. In case of isatin, a 2-fold decrease in Vmax was observed for the Pro230Phe mutant, while replacing Asp236 with Ala resulted in significant drop of Km value, indicating improved binding of the substrate in the active site (Table 4). These data suggest that the residues at both positions are strongly involved in substrate and product binding, and that they are part of a more complex set of factors that combine to determine the activity. A major role within this proposed network of interactions falls to Trp229 in CBR1.
 |
Figure 4: Comparison of active site properties of human CBR1 and CBR3. A-C: CBR1 (green), D-E: CBR3, with modelled loop (orange). First Column (A and D): solvent accessible surface representation of the active site pockets coloured according to electrostatic potentials, with the cofactor represented as sticks. Yellow line marks the plane cutting through the active site. The plane divides the pocket into two halves that are depicted in the following two columns. Second column (B and E): 'left' half of the pocket. Third column (C and F): 'right' half of the pocket. Cofactor is shown for orientation purpose. Residues that were mutated in this study are marked with asterisks. Catalytic residue labels are underlined. Click here for full size figure. |
Residues Met141 and Gln142 , found at homologous positions in CBR1 and CBR3, respectively (Fig. 1C), and displaying similar side-chain conformations are located on helix aF and form the wall opposite of Trp229/Pro230 (Fig. 4). Replacing this position had a dramatic effect on CBR1: while Met141Gln and Met141Ala mutants showed modest decrease in activity towards naphthoquinones (Table 3), combining these mutations with Trp229Pro led to destabilisation of the protein (very low solubility, no activity). The opposite effect was observed for CBR3, with the Gln142Met mutant showing a modest increase in activity,and with the double mutants still active towards 1,2- naphthoquinone.
Taken together, these data highlight the significance of the side-chain chemistry at position 142 in determination of CBR activity.
Discussion
The objective of this study was to establish a substrate profile for human CBR3, to compare its enzymatic properties to its paralog CBR1, and to establish determinants for activity and substrate specificity. First, CBR3 catalyzes the carbonyl reduction of a much narrower spectrum of xenobiotic substrates in contrast to the exceptionally broad substrate profile of CBR1 [1,3,6,9] Furthermore, no endogenous substrate could be unequivocally detected to this end for CBR3. Of the known CBR1 substrates, only quinone compounds with ortho substitution or compounds like isatin and the cytostatic oracin could be identified as substrates for human CBR3.
Second, this work establishes the structural basis for narrower substrate specificity in CBR3, and highlights the active site loop found in CBRs as flexible entity that is one critical factor for substrate specificity. Exchange of non-conserved residues between CBR1 and CBR3 in this loop results in position-specific effects that control catalysis. In particular, the data reveal critical roles for Trp229 and Pro230 in CBR1 and CBR3, respectively, for activity towards para-quinones. These data suggest that hydrophobic interactions as well as possible contacts made through a water molecule coordinated by the indole nitrogen of Trp229 could contribute to substrate orientation and possibly product release in the active site. This is supported by the fact, that in the experimental structures, the main-chain of the loop starts to deviate at position 229. This clearly indicates that a major difference between CBR1 and CBR3 is a large, hydrophobic wall built by Trp229 in CBR1, and a more open substrate site in CBR3, irrespective if the loop modelling, as performed in this work, is correct or not. Other variable residues found on the loop also contribute to a varying extent to substrate specificity, such as residue Asp236 in CBR3, introducing an additional charge into the active site in comparison to CBR1. Other determinants for activity are residues found at position 141/142, namely Met141 in CBR1 and Gln142 in CBR3. Although of similar size, these residues have significant differential effects on catalytic properties of the active site. A preliminary study [22] showed the whole region encompassing residues 235/236 – 243/244 as crucial in determination of activities of CBR1 and CBR3. Replacing this region in CBR3 with residues from CBR1, combined with Pro230Trp mutation, was sufficient for a 1000-fold increase in activity to ~40% of activity of CBR1. To summarise, the substrate pockets of CBR1 and CBR3 show fundamental differences in size, as manifested through residues found at position 229, as well as in surface properties [pocket electrostatics CBR1 ; CBR3 ], as seen with the more polar residues lining the active site in CBR3 (Fig. 4) [active site residues: CBR1 ; CBR3 ].
Although not specifically tested in this study, it is conceivable that some of the activities observed in this study are related to “propinquity” effects [23]). It has been previously shown that orthoquinones can be reduced to the corresponding hydroquinones in a manner not involving a protein derived catalytic base, as seen with mutant studies performed on members of the aldo-keto reductase [23] or medium-chain dehydrogenase/reductase (MDR) families [29]. Instead, the enzyme is used as a “scaffold” to bring the reduced nicotinamide cofactor and the orthoquinone into close proximity, to allow hydride and direct solvent proton transfer to the adjacent carbonyl groups [23]. This possibility underlines the importance of correct cofactor and substrate positioning in the active site.
As observed from in silico screening ( http://www.genecards.org ) and other experimental data [15], CBR1 is an ubiquitously expressed enzyme with highest levels found in liver and the central nervous system, with a significantly lower transcription level but overlapping expression pattern found for CBR3. This suggests some redundancy in substrate specificity, but could also point to different substrates classes and hence different roles for these paralogous enzymes. CBR1 plays without doubt a major role in the phase I metabolism of xenobiotic compounds including xenobiotic quinones [1,3,6,24], a function which we at present cannot wholeheartedly postulate for CBR3, in light of the observed narrow substrate spectrum. In addition, several more recently conducted studies also suggest a critical role for CBR1 in the metabolism of endogenous lipid mediators such as prostaglandins or lipid oxidation products such as the highly reactive and genotoxic 4-oxonon-2-enal, which is produced under oxidative stress [25-27]. This could relate the observations that CBR1 is involved in metastasis, neurodegeneration and apoptosis to its properties to catalyze prostaglandin and lipid aldehyde inactivation [9,11,25], however, final experimental proof for this hypothesis is necessary. In light of these data, the structural differences determined, and the apparent lack of activity of CBR3 towards lipid mediators like prostaglandins or oxononenal we suggest that CBR3 is likely involved in the metabolism of structurally and chemically different substrates. The precise identity of these compounds needs to be established in further metabolomic and molecular genetic studies.
Acknowledgements
We thank Frank von Delft for help with data collection at the Swiss Light Source (SLS). We thank Julián Atienza-Herrero for help with preparation of the web-based activeiSee version of this article.
Abbreviations: SDR: short-chain dehydrogenase/reductase; AKR: aldo-keto reductase; CBR1: carbonyl reductase 1; CBR3: carbonyl reductase 3.
Keywords: carbonyl reductase, phase I drug metabolism, carbonyl reduction; quinone reduction; short-chain dehydrogenase/reductase.
Footnotes: The atomic coordinates and structure factors (code 2HRB ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ ( http://www.rcsb.org ).
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