Crystallographic study of coenzyme, coenzyme analogue and substrate binding in 6-phosphogluconate dehydrogenase: implications for NADP specificity and the enzyme mechanism

Adams, Margaret; Ellis, Grant H; Gover, S.; Naylor, C.E.; Phillips, Christopher

doi:10.1016/s0969-2126(00)00066-6

Cited by 112 publications

(156 citation statements)

References 49 publications

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“…The development of a partial positive charge on the thioester carbonyl during the reaction could be stabilized by the oxyanion hole created by hydrogen bonds donated by the amide nitrogen atoms of Phe 68 and Gly…”

Section: Discussionmentioning

confidence: 99%

“…3A). Amino acid residues observed to hydrogen bond to the cofactor in monofunctional HACDs (67,68) and PfMFP are conserved and have similar conformations in AtMFP2-HACD (Glu 342 , Glu 401 , Lys 406 , and Asn 624 ), but no significant electron density is observed in the AtMFP2 co-factor-binding site. A similar absence of positive electron density for the co-factor is observed in the crystal structure of the truncated recombinant RnMFE-1-HACD (15), whereas NAD ϩ is present in the active site of the PfMFP ␤-oxidation complex but with an average B-factor of 98 Å 2 (13).…”

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confidence: 99%

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The Multifunctional Protein in Peroxisomal β-Oxidation

Arent¹,

Christensen²,

Pye³

et al. 2010

Journal of Biological Chemistry

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Plant fatty acids can be completely degraded within the peroxisomes. Fatty acid degradation plays a role in several plant processes including plant hormone synthesis and seed germination. Two multifunctional peroxisomal isozymes, MFP2 and AIM1, both with 2-trans-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase activities, function in mouse ear cress (Arabidopsis thaliana) peroxisomal ␤-oxidation, where fatty acids are degraded by the sequential removal of two carbon units. A deficiency in either of the two isozymes gives rise to a different phenotype; the biochemical and molecular background for these differences is not known. Structure determination of Arabidopsis MFP2 revealed that plant peroxisomal MFPs can be grouped into two families, as defined by a specific pattern of amino acid residues in the flexible loop of the acylbinding pocket of the 2-trans-enoyl-CoA hydratase domain. This could explain the differences in substrate preferences and specific biological functions of the two isozymes. The in vitro substrate preference profiles illustrate that the Arabidopsis AIM1 hydratase has a preference for short chain acyl-CoAs compared with the Arabidopsis MFP2 hydratase. Remarkably, neither of the two was able to catabolize enoyl-CoA substrates longer than 14 carbon atoms efficiently, suggesting the existence of an uncharacterized long chain enoyl-CoA hydratase in Arabidopsis peroxisomes.Fatty acids are degraded by the sequential removal of two carbon units in a process known as ␤-oxidation (Fig. 1). This process is ubiquitous and takes its name from the oxidation taking place at the carbon atom ␤ to the carboxyl group. The discovery of a peroxisomal ␤-oxidation system was made in plants (1), and whereas peroxisomal ␤-oxidation in animals appears to be merely a fatty acid chain-shortening machine feeding mitochondrial ␤-oxidation, plant and fungal ␤-oxidation takes place almost entirely in the peroxisomes. The products of the reaction are H 2 O 2 , acetyl-CoA, reducing equivalents, and a variety of chain length-shortened acyl-containing molecules like the plant hormones jasmonic acid (2) and indole-3-acetic acid (3, 4).The conversion of storage triacylglycerols by ␤-oxidation provides metabolic energy and carbon skeletons for germination and early post-germinative seedling growth in oil seed plants (5) and metabolic energy and carbon for the production of hydrolytic enzymes in cereals (6). It is a salvage pathway for fatty acids during foliar senescence (7), supplies respiratory substrates to carbohydrate-deprived tissue (8), and at a lower level, is a constitutive property of all plant tissues most likely involved with membrane lipid turnover (7).Three proteins (each present as several isozymes) that host a total of four enzyme activities constitute the core of peroxisomal ␤-oxidation. Acyl-CoA oxidase (ACX) 6 oxidizes acyl-CoA to 2-trans-enoyl-CoA using FAD as co-enzyme. A multifunctional protein (MFP) adds water over the 2-trans-enoyl-CoA double bond and oxidizes the resultant L-3-hydroxyacylCoA usin...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

The Multifunctional Protein in Peroxisomal β-Oxidation

Arent¹,

Christensen²,

Pye³

et al. 2010

Journal of Biological Chemistry

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“…This is an attractive model since significant structural differences between the HAD-NAD + and HAD-NADH models are not readily apparent. In contrast, crystallographic studies of a structurally related enzyme, 6-phosphogluconate dehydrogenase, have suggested that reduced and oxidized cofactor bind with dramatically different conformations of the nicotinamide ring, accounting for the considerable differences in binding affinities (19). Direct comparison with 6-phosphogluconate dehydrogenase, however, may not be relevant since its reduced cofactor may participate in catalysis subsequent to hydride transfer.…”

Section: Model Of the Abortive Ternary Complexmentioning

confidence: 97%

Sequestration of the active site by interdomain shifting: crystallographic and spectroscopic evidence for distinct conformations of L-3-Hydroxyacyl-CoA dehydrogenase

Barycki¹

2000

Journal of Biological Chemistry

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“…In the E⅐6PG binary complex, the 6-phosphate of 6PG is surrounded by the following five active-site residues that are within hydrogen-bond distance: Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 (6). Multiple sequence alignment of 6PGDH shows that these five residues are completely conserved (Fig.…”

mentioning

confidence: 99%

“…Interestingly, Arg-287 participates in a hydrogen-bond network through water 528 to and the 1-carboxyl of 6PG. It is suggested that this network is important in defining the bound conformation of 6PG (6).…”

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confidence: 99%

Importance in Catalysis of the 6-Phosphate-binding Site of 6-Phosphogluconate in Sheep Liver 6-Phosphogluconate Dehydrogenase

Dworkowski

Cook

2006

Journal of Biological Chemistry

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The 6-phosphate of 6-phosphogluconate (6PG) is proposed to anchor the sugar phosphate in the active site and aid in orientating the substrate for catalysis. In order to test this hypothesis, alanine mutagenesis was used to probe the contribution of residues in the vicinity of the 6-phosphate to binding of 6PG and catalysis. The crystal structure of sheep liver 6-phosphogluconate dehydrogenase shows that Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 contribute a mixture of ionic and hydrogen bonding interactions to the 6-phosphate, and these interactions are likely to provide the majority of the binding energy for 6PG. All mutant enzymes, with the exception of T262A, exhibit an increase in K 6PG that ranges from 5-to 800-fold. There is also a less pronounced increase in K NADP , ranging from 3-to 15-fold, with the exception of T262A. The R287A and R446A mutant enzymes exhibit a dramatic decrease in V/E t (600-and 300-fold, respectively) as well as in V/K 6PG E t (10 5 -and 10 4 -fold), and therefore no further characterization was carried out with these two mutant enzymes. No change in V/E t was observed for the Y191A mutant enzyme, whereas 20-and 3-fold decreases were obtained for the K260A and T262A mutant enzymes, respectively, resulting in a decrease in V/K 6PG E t range from 3-to 120-fold. All mutant enzymes also exhibit at least an order of magnitude increase in 13 C-isotope effect ؊1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 in providing the binding energy for 6PG. In addition, these residues also likely ensure proper orientation of 6PG for catalysis and aid in inducing the conformation change that precedes, and sets up the active site for, catalysis.6-Phosphogluconate dehydrogenase (6PGDH 2 ; EC 1.1.1.44) catalyzes the reversible oxidative decarboxylation of 6-phosphogluconate (6PG), producing ribulose 5-phosphate (Ru5P) and CO 2 with the concomitant reduction of NADP to NADPH. The kinetic mechanism is rapid equilibrium random on the basis of a complete kinetic characterization of the sheep liver enzyme and Candida utilis (1, 2). The pH dependence of kinetic parameters indicates a general acid-general base chemical mechanism (1, 2), and sitedirected mutagenesis (3, 4) studies suggest that Lys-183 and Glu-190 are likely the general base and the general acid, respectively. In this mechanism, the general base (Lys-183) is required to accept a proton from the 3-hydroxyl group of 6PG concomitant with hydride transfer from C-3 of 6PG to the coenzyme. Reduction of the nicotinamide ring is accompanied by rotation around the N-glycosidic bond such that the ring occupies the position formerly occupied by the 1-carboxylate of the substrate (5). The resulting 3-keto-6-phosphogluconate intermediate is decarboxylated to produce the enediol of Ru5P with the general base used to protonate the carboxyl oxygen. A general acid (Glu-190) is needed to facilitate the tautomerization of the enediol of ...

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Crystallographic study of coenzyme, coenzyme analogue and substrate binding in 6-phosphogluconate dehydrogenase: implications for NADP specificity and the enzyme mechanism

Cited by 112 publications

References 49 publications

The Multifunctional Protein in Peroxisomal β-Oxidation

The Multifunctional Protein in Peroxisomal β-Oxidation

Sequestration of the active site by interdomain shifting: crystallographic and spectroscopic evidence for distinct conformations of L-3-Hydroxyacyl-CoA dehydrogenase

Importance in Catalysis of the 6-Phosphate-binding Site of 6-Phosphogluconate in Sheep Liver 6-Phosphogluconate Dehydrogenase

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