Kinetics of carbonyl reductase from human brain

Bohren, Kurt M.; Wartburg, J. P. von; Wermuth, Bendicht

doi:10.1042/bj2440165

Cited by 35 publications

(21 citation statements)

References 28 publications

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“…Yields from one placenta were -0.25 mg (the more acidic form) and 0.75 mg (the more basic form). They were largely indistinguishable enzymatically, in agreement with previous reports (13,16,17,29,30), both giving similar activity with prostaglandin B1 (=1 unit/mg of protein). Their total compositions were also largely indistinguishable.…”

Section: Resultssupporting

confidence: 92%

See 1 more Smart Citation

Carboxyethyllysine in a protein: native carbonyl reductase/NADP(+)-dependent prostaglandin dehydrogenase.

Krook

Ghosh

Strömberg

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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Two different forms of the monomeric NADP+-ilnked prostaglandin dehydrogenase/carbonyl reductase were purified from human placenta and shown to differ by the m ation of a lysine residue. The modified and the unmodified proteins were reproducibly recovered In a ratio of -1:3, and both were chemically stable. The modifed form was more acidic (pI 7.4 versus pI 7.7) but I i habl from the unmoded form in specificity and activity. Amino acid analysis, sequence analysis, mass spectrometry, and chemical synthesis identified the modified residue as N6-(lcarboxyethyl)lysine with C-2 of propionic acid attached to the side-chain N of Lys-238. This compound can be formed from the lysine residue and pyruvate via a Schiff base and subsequent reduction. The enzyme and Its NAD+-dependent counterpart are distantly related (23% residue identity) and have the same family assiment to short-chain dehydrogenases. Alignments and model-building into the tertiary structure of 3a/20j3-hydroxysteroid dehydrogenase show that carbonyl reductase has an extra loop (positions 149-189) that forms a separate extension and replaces a backbone C-terminal ,3-strand. This change affects the substrate pocket, explaining the different substrate specificities but conserves residues of known functional importance. Carboxyethyllysine at position 238 corresponds to a proteolysis-sensitive position in several short-chain dehydrogenases, less well-defined in the model but close to a surface, and is compatible with the accessibility and enzyme properties observed.The biological inactivation of prostaglandins is mediated by 15-hydroxyprostaglandin dehydrogenase. Two different types of this cytosolic activity, the most active one using NAD+ and the other using NADP+, were detected early in a number of tissues (1-3). We have characterized the NAD+-linked enzyme as a short-chain dehydrogenase (4), with properties typical of that enzyme family (5). A crystallographic structure of the protein 3a/20,B-hydroxysteroid dehydrogenase, which is within this family, has recently become available (6), as has that of another, distantly related member of the family, dihydropteridine reductase, which is strikingly similar, except for some loop and turn regions (7). These and other short-chain dehydrogenases contain a functionally important tyrosine residue (4, 8-10) and a segment sensitive to proteolysis (10).The NADP+ enzyme type also acts on the 9-keto group of prostaglandins in the reverse direction (3, 11, 12), thus constituting a prostaglandin 9-keto reductase and can then convert prostaglandin E2 to prostaglandin F2

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Section: Resultssupporting

confidence: 92%

“…Notably, the carboxyethylation identifies a protein modification that appears not to be regulatory because the multiple forms are enzymatically quite similar (29). It is also not just a coenzyme adduct, like subforms in this (13) and several other dehydrogenases.…”

Section: Discussionmentioning

confidence: 99%

Carboxyethyllysine in a protein: native carbonyl reductase/NADP(+)-dependent prostaglandin dehydrogenase.

Krook

Ghosh

Strömberg

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…) (Wermuth, 1981;Bohren et al, 1987;Lakhman et al, 2005). Experiments were performed in a Cary-Varian Bio 300 UV-visible spectrophotometer equipped with thermal control and proprietary software for enzyme kinetics analysis (Varian, Walnut Creek, CA).…”

Section: Methodsmentioning

confidence: 99%

A Functional Genetic Polymorphism on Human Carbonyl Reductase 1 (CBR1V88I) Impacts on Catalytic Activity and NADPH Binding Affinity

González-Covarrubias

Ghosh²,

Lakhman³

et al. 2007

Drug Metab Dispos

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ABSTRACT:Human carbonyl reductase 1 (CBR1) metabolizes endogenous and xenobiotic substrates such as the fever mediator, prostaglandin E2 (PGE 2 ), and the anticancer anthracycline drug, daunorubicin. We screened 33 CBR1 full-length cDNA samples from white and black liver donors and performed database analyses to identify genetic determinants of CBR1 activity. We pinpointed a single nucleotide polymorphism on CBR1 (CBR1 V88I) that encodes for a valine-toisoleucine substitution for further characterization. Human carbonyl reductase 1 (CBR1) is a monomeric cytosolic enzyme that catalyzes the two-electron reduction of biologically and pharmacologically active substrates by using NADPH as cofactor . For example, CBR1 converts the potent fever mediator prostaglandin E 2 (PGE 2 ) into the less active metabolite PGF 2␣ . Recent studies have shown that the transcription of CBR1 in lung and liver is down-regulated to suppress the catabolism of PGE 2 during the course of the febrile response (Ivanov et al., 2003;Ivanov and Romanovsky, 2004). CBR1 also plays a predominant role in the metabolism of the anticancer anthracyclines doxorubicin and daunorubicin by catalyzing the formation of their corresponding C-13 alcohol metabolites (doxorubicinol and daunorubicinol). Anthracycline C-13 alcohol metabolites circulate in plasma, have half-lives similar to those of the parent compounds, and are devoid of significant tumor cell-killing activity. The use of anthracyclines for cancer treatment is hampered by the development of clinical cardiotoxicity in some patients (Wouters et al., 2005). The pathogenesis of anthracycline-related cardiotoxicity is complex and appears to be mediated by a combination of oxidative stress and intracardiac metabolic perturbations induced by C-13 anthracycline alcohol metabolites (Minotti et al., 2004). In human myocardium, cytosolic carbonyl reductases (CBRs), and aldo/keto reductases catalyze the two-electron reduction of the anthracycline side chain C-13 carbonyl group to form cardiotoxic alcohol metabolites (e.g., doxorubicinol, daunorubicinol). CBR activity is the main source of quinone detoxification in humans, and biochemical studies showed that aldo/keto reductases have 7-to 18-fold lower catalytic efficiencies (V max /K m ) for the reduction of anthracycline substrates than CBRs (Wermuth et al., 1986;Ohara et al., 1995;Rosemond and Walsh, 2004;Covarrubias et al., 2006). The key role of CBR1 during the pathogenesis of anthracycline-related cardiotoxicity has been established in biochemical studies and in experiments with murine models (Mordente et al., 2001). Mice overexpressing human CBR1 in heart showed high intracardiac levels of doxorubicinol and increased signs of myocardial damage after doxorubicin administration . Conversely, mice with a

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“…Two independent CBR1 V88 and CBR1 I88 protein preparations were used for this study. CBR1 enzymatic activities were measured with a validated kinetic method that records the rate of oxidation of the NADPH cofactor at 340 nm (NADPH molar absorption coefficient, 6,220 M -1 cm -1 ) (17,18). Under conditions of initial velocity (V 0 ), the method was linear (r 2 > 0.90) and reproducible (CV % range: 94 -111%) for all CBR1 substrates.…”

Section: Kinetic Studiesmentioning

confidence: 99%

Inhibition of Polymorphic Human Carbonyl Reductase 1 (CBR1) by the Cardioprotectant Flavonoid 7-monohydroxyethyl Rutoside (monoHER)

2008

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Purpose-Carbonyl reductase 1 (CBR1) reduces the anticancer anthracyclines doxorubicin and daunorubicin into the cardiotoxic metabolites doxorubicinol and daunorubicinol. We evaluated whether the cardioprotectant monoHER inhibits the activity of polymorphic CBR1.Methods-We performed enzyme kinetic studies with monoHER, CBR1 (CBR1 V88 and CBR1 I88) and anthracycline substrates. We also characterized CBR1 inhibition by the related flavonoids triHER and quercetin.Results-MonoHER inhibited the activity of CBR1 V88 and CBR1 I88 in a concentrationdependent manner. The IC 50 values of monoHER were lower for CBR1 I88 compared to CBR1 V88 for the substrates daunorubicin and doxorubicin (daunorubicin, IC 50-CBR1 I88: 164 μM vs. IC 50 -CBR1 V88: 219 μM; doxorubicin, IC 50 -CBR1 I88: 37 μM vs. IC 50 -CBR1 V88: 59 μM; p < 0.001). Similarly, the flavonoids triHER and quercetin exhibited lower IC 50 values for CBR1 I88 compared to CBR1 V88 (p < 0.001). MonoHER acted as a competitive CBR1 inhibitor when using daunorubicin as a substrate (Ki = 45 ± 18 μM). MonoHER acted as an uncompetitive CBR1 inhibitor for the small quinone substrate menadione (Ki = 33 ± 17 μM). Conclusions-The cardioprotectant monoHER inhibits CBR1 activity. CBR1 V88I genotype status and the type of anthracycline substrate dictate the inhibition of CBR1 activity.

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Kinetics of carbonyl reductase from human brain

Cited by 35 publications

References 28 publications

Carboxyethyllysine in a protein: native carbonyl reductase/NADP(+)-dependent prostaglandin dehydrogenase.

Carboxyethyllysine in a protein: native carbonyl reductase/NADP(+)-dependent prostaglandin dehydrogenase.

A Functional Genetic Polymorphism on Human Carbonyl Reductase 1 (CBR1V88I) Impacts on Catalytic Activity and NADPH Binding Affinity

Inhibition of Polymorphic Human Carbonyl Reductase 1 (CBR1) by the Cardioprotectant Flavonoid 7-monohydroxyethyl Rutoside (monoHER)

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