Purification and Characterization ofζ-Crystallin from the Camel Lens

Duhaiman, Ali S.; Rabbani, Nayyar; Al‐Jafari, Abdulaziz A.; Alhomida, Abdullah S.

doi:10.1006/bbrc.1995.2511

Cited by 33 publications

(18 citation statements)

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“…The protein has a nucleotide-binding fingerprint motif, AXX GXXG, and is expected to bind NADH or NADPH. The medium-chain dehydrogenase/reductase superfamily has such enzymes as -crystallin (5,11,(25)(26)(27)(28)32), the quinone oxidoreductase from Escherichia coli (QOR Ec ) (6,7,18,31), and horse liver alcohol dehydrogenase (LADH) (3,4,8,9), all of which catalyze NADH-or NADPH-dependent redox reactions of various substrates. This superfamily consists of two subfamilies: dehydrogenases and reductases.…”

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confidence: 99%

“…-Crystallin reduces such naturally occurring quinones as 1,2-naphthoquinone and phenanthraquinone but is inactive with menadione, ubiquinone, and vitamins K 1 and K 2 (25,26,28). The physiological function of -crystallin is speculated to be detoxification or the metabolism of a quinone (5,11,27,32). Genes encoding -crystallin homologs are widely distributed from bacteria to higher plants and animals, but the only well characterized one is the P1 -crystallin from Arabidopsis thaliana (19,20).…”

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confidence: 99%

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Crystal Structures of the Quinone Oxidoreductase from Thermus thermophilus HB8 and Its Complex with NADPH: Implication for NADPH and Substrate Recognition

2003

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The crystal structures of the -crystalline-like soluble quinone oxidoreductase from Thermus thermophilus HB8 (QOR Tt ) and of its complex with NADPH have been determined at 2.3-and 2.8-Å resolutions, respectively. QOR Tt is composed of two domains, and its overall fold is similar to the folds of Escherichia coli quinone oxidoreductase (QOR Ec ) and horse liver alcohol dehydrogenase. QOR Tt forms a homodimer in the crystal by interaction of the ␤F-strands in domain II, forming a large ␤-sheet that crosses the dimer interface. High thermostability of QOR Tt was evidenced by circular dichroic measurement. NADPH is located between the two domains in the QOR Tt -NADPH complex. The disordered segment involved in the coenzyme binding of apo-QOR Tt becomes ordered upon NADPH binding. The segment covers an NADPH-binding cleft and may serve as a lid. The 2-phosphate group of the adenine of NADPH is surrounded by polar and positively charged residues in QOR Tt , suggesting that QOR Tt binds NADPH more readily than NADH. The putative substratebinding site of QOR Tt , unlike that of QOR Ec , is largely blocked by nearby residues, permitting access only to small substrates. This may explain why QOR Tt has weak p-benzoquinone reduction activity and is inactive with such large substrates of QOR Ec as 5-hydroxy-1,4-naphthoquinone and phenanthraquinone.

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confidence: 99%

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confidence: 99%

Crystal Structures of the Quinone Oxidoreductase from Thermus thermophilus HB8 and Its Complex with NADPH: Implication for NADPH and Substrate Recognition

2003

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“…As has been reported by Fernández et al [8], human f-crystallin is a homotetramer, like camel lens f-crystallin [24]. However, this quaternary structure changed with the ionic strength of the chromatography buffer.…”

Section: Expression Purification and Molecular Weight Determination mentioning

confidence: 57%

Kinetic and structural evidence of the alkenal/one reductase specificity of human ζ-crystallin

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Shafqat

et al. 2010

Cell. Mol. Life Sci.

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Human ζ-crystallin is a Zn(2+)-lacking medium-chain dehydrogenase/reductase (MDR) included in the quinone oxidoreductase (QOR) family because of its activity with quinones. In the present work a novel enzymatic activity was characterized: the double bond α,β-hydrogenation of medium-chain 2-alkenals and 3-alkenones. The enzyme is especially active with lipid peroxidation products such as 4-hydroxyhexenal, and a role in their detoxification is discussed. This specificity is novel in the QOR family, and it is similar to that described in the distantly related alkenal/one reductase family. Moreover, we report the X-ray structure of ζ-crystallin, which represents the first structure solved for a tetrameric Zn(2+)-lacking MDR, and which allowed the identification of the active-site lining residues. Docking simulations suggest a role for Tyr53 and Tyr59 in catalysis. The kinetics of Tyr53Phe and Tyr59Phe mutants support the implication of Tyr53 in binding/catalysis of alkenal/one substrates, while Tyr59 is involved in the recognition of 4-OH-alkenals.

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“…~-Crystallin was purified from the cortex of the camel (Camelus drornedarius) lens to homogeneity according to a method as previously reported (Duhaiman et al, 1995). The enzyme purity was estimated to be >95% and the specific activity of this preparation was 25/zmol NADPH oxidized/min/mg protein in the presence of 25 #M 9,10-phenanthrenequinone.…”

Section: Enzyme Preparationmentioning

confidence: 99%

“…In a previous study we reported the isolation of a novel homotetramer enzyme which has NADPH:quinone oxidoreductase activity, the "(-crystallin" from the camel lens, which constitutes about 10% of the total protein of the camel (Garland et al, 1991;Duhaiman et al, 1995) and guinea pig (Huang et al, 1987) lenses. It has been shown that a mutation in the ~'-crystallin gene is associated with autosomal dominant congenital cataract (Huang et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of ζ-crystallin by coumarins: A structure-activity study

Duhaiman

1996

J Protein Chem

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A structure-activity study was carried out to determine the important groups of coumarin derivatives in inhibiting the oxidoreductase activity of the camel lens zeta-crystallin. Coumarin, 4-hydroxycoumarin, 7-hydroxy-4-methylcoumarin, dicoumarol, and warfarin were screened for their inhibitory effect on zeta-crystallin activity. The sequence of potency for the inhibitors was dicoumarol > 4-hydroxycoumarin > warfarin > > coumarin. 7-Hydroxy-4-methylcoumarin was ineffective as an inhibitor. Only dicoumarol, 4-hydroxycoumarin, and warfarin were found to inhibit the oxidoreductase activity in micromolar ranges. All tested inhibitors seem to act in reversible and time-independent manner. Concentration causing 50% inhibition of the enzyme activity (IC50 value) was 34 microM for dicoumarol, 76 microM for 4-hydroxycoumarin, and approximately 515 microM for warfarin, while 1 mM coumarin showed less than 10% inhibition. Kinetic analysis revealed inhibition of camel lens zeta-crystallin by coumarin derivatives to occur in a competitive manner with respect to dichlorophenolindophenol (DCIP) as an electron acceptor and uncompetitive manner with respect to NADPH as an electron donor. The Ki values were found to be 16 microM for dicoumarol, 40 microM for 4-hydroxycoumarin, and 220 microM for warfarin. The structure-activity relationship of coumarin derivatives indicates that the phenolic hydroxyl group at the C-4 position in the coumarin skeleton is important for the maximal inhibition.

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Purification and Characterization ofζ-Crystallin from the Camel Lens

Cited by 33 publications

References 0 publications

Crystal Structures of the Quinone Oxidoreductase from Thermus thermophilus HB8 and Its Complex with NADPH: Implication for NADPH and Substrate Recognition

Crystal Structures of the Quinone Oxidoreductase from Thermus thermophilus HB8 and Its Complex with NADPH: Implication for NADPH and Substrate Recognition

Kinetic and structural evidence of the alkenal/one reductase specificity of human ζ-crystallin

Inhibition of ζ-crystallin by coumarins: A structure-activity study

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