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Structural basis for substrate specificity in human monomeric carbonyl reductases.

Pilka ES, Niesen FH, Lee WH, El-Hawari Y, Dunford JE, Kochan G, Wsol V, Martin HJ, Maser E, Oppermann U - PLoS ONE (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Oxford, Headington, United Kingdom.

ABSTRACT

Unlabelled: 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 narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further investigate 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.

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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 crystal 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.
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pone-0007113-g005: 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 crystal 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.

Mentions: A major distinguishing feature between CBR1 and CBR3 is Trp229, which is replaced by a prolyl residue in CBR3 (cf Figure 2C). 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 (Figure 5), 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 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 (Fig. 5). 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 (Figure 3). Both CBR1 Trp229 mutants showed significant decrease in activity for 1,4-naphthoquinone and a modest decrease for its ortho- isomer (Table 2). Data for isatin (Table 3) 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 2), indicating the possible occurrence 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 a significant drop of the Km value, indicating improved binding of the substrate in the active site (Table 3). These data suggest that the residues at both positions are strongly involved in substrate and product binding, indicated by data with swapped residues that either reduce (CBR1) or increase (CBR3) catalytic efficiencies. These residues 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.


Structural basis for substrate specificity in human monomeric carbonyl reductases.

Pilka ES, Niesen FH, Lee WH, El-Hawari Y, Dunford JE, Kochan G, Wsol V, Martin HJ, Maser E, Oppermann U - PLoS ONE (2009)

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 crystal 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2741203&req=5

pone-0007113-g005: 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 crystal 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.
Mentions: A major distinguishing feature between CBR1 and CBR3 is Trp229, which is replaced by a prolyl residue in CBR3 (cf Figure 2C). 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 (Figure 5), 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 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 (Fig. 5). 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 (Figure 3). Both CBR1 Trp229 mutants showed significant decrease in activity for 1,4-naphthoquinone and a modest decrease for its ortho- isomer (Table 2). Data for isatin (Table 3) 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 2), indicating the possible occurrence 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 a significant drop of the Km value, indicating improved binding of the substrate in the active site (Table 3). These data suggest that the residues at both positions are strongly involved in substrate and product binding, indicated by data with swapped residues that either reduce (CBR1) or increase (CBR3) catalytic efficiencies. These residues 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.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Oxford, Headington, United Kingdom.

ABSTRACT

Unlabelled: 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 narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further investigate 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.

Enhanced version: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Show MeSH
Related in: MedlinePlus