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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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Structure of human CBR3.Panel A: The substrate binding loop in CBR3 is engaged in contacts (red oval) to a symmetry related copy (grey), resulting in an open conformation of the active site. The CBR-specific helical insertion involved in dimerization is highlighted in green. 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.
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pone-0007113-g002: Structure of human CBR3.Panel A: The substrate binding loop in CBR3 is engaged in contacts (red oval) to a symmetry related copy (grey), resulting in an open conformation of the active site. The CBR-specific helical insertion involved in dimerization is highlighted in green. 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.

Mentions: 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 [21]. CBRs represent prototypes of monomeric SDRs with a two-helical insertion stabilizing an interface that in other SDRs constitutes the main oligomerization surface (Figure 2).


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)

Structure of human CBR3.Panel A: The substrate binding loop in CBR3 is engaged in contacts (red oval) to a symmetry related copy (grey), resulting in an open conformation of the active site. The CBR-specific helical insertion involved in dimerization is highlighted in green. 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2741203&req=5

pone-0007113-g002: Structure of human CBR3.Panel A: The substrate binding loop in CBR3 is engaged in contacts (red oval) to a symmetry related copy (grey), resulting in an open conformation of the active site. The CBR-specific helical insertion involved in dimerization is highlighted in green. 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.
Mentions: 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 [21]. CBRs represent prototypes of monomeric SDRs with a two-helical insertion stabilizing an interface that in other SDRs constitutes the main oligomerization surface (Figure 2).

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