Crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding.

Pig aldehyde reductase containing the active site mutation tyrosine(50) to phenylalanine has been crystallized in the presence of the cofactor NADP(H) to a resolution of 2.2 A. This structure clearly shows loss of the tyrosine hydroxyl group and no other significant perturbations compared with previously determined structures. The mutant binds cofactor (both oxidized and reduced) more tightly than the wild-type enzyme but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone, as determined by fluorescence titrations. Numerous attempts at preparing a ternary complex with a range of small aldehyde substrates were unsuccessful. This result, in addition to the inability of the mutant protein to bind the inhibitor, provides strong evidence for the proposal that the tyrosine hydroxyl group is essential for substrate binding in addition to catalysis.

Expression of B-type natriuretic peptide in atrial natriuretic peptide gene disrupted mice.

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are two hormones produced and secreted by the heart to control blood pressure, body fluid homeostasis and electrolyte balance. Each peptide binds to a common family of 3 receptors (GC-A, GC-B and C-receptor) with varying degrees of affinity. The proANP gene disrupted mouse model provides an excellent opportunity to examine the regulation and expression of BNP in the absence ofANP. A new radioimmunoassay (RIA) was developed in order to measure mouse BNP peptide levels in the plasma, atrium and ventricle of the mouse. A detection limit of 3-6 pg/tube was achieved by this assay. Results show that plasma and ventricular level of BNP were unchanged among the three genotypes of mice. However, a significant decrease in the BNP level was noted in the atrium. The homozygous mutant (ANP-/-) had undetectable levels of BNP in the atrium, while the heterozygous (ANP+/-) and wild-type (ANP+/+) mice had 430 and 910 pg/mg in the atrium, respectively. Northern Blot analysis shows the ANP-/- mice has a 40% reduction of BNP mRNA level in the atrium and a 5-fold increase in the ventricle as compared with that of the ANP+/+ mouse. Our data suggest that there is a compensatory response of BNP expression to proANP gene disruption. Despite the changes in the atrial and ventricular tissue mRNA and peptide levels, the plasma BNP level remains unaltered in the ANP-/- mice. We conclude that the inability of BNP to completely compensate for the lack of ANP eventually leads to chronic hypertension in the proANP gene disrupted mice.

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding.

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid-base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR-Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.

Altered expression of natriuretic peptide receptors in proANP gene disrupted mice.

BACKGROUND: The atrial natriuretic peptide (ANP) family is a complex system consisting of at least three polypeptides and at least three types of receptor. Each peptide interacts with different types of receptor at varying degrees of affinity. To determine if natriuretic peptide levels influence natriuretic peptide receptor expression and regulation, we examined the expression of guanylyl cyclase linked GC-A, GC-B and C-receptor in the lungs of mice with a mutation that inactivates the ANP gene (Nppa). METHODS: The mRNA level of GC-A, GC-B and C-receptor in the lung were studied by ribonuclease protection assays (RPA). RESULTS: Results of RPA showed that although the mRNA level of GC-A and GC-B of heterozygous ANP+/- was not different from wild type ANP+/+ mice, they were significantly higher in the homozygous mutant ANP-/- mice. In addition, C-receptor mRNA level in ANP+/- and ANP-/- was significantly lower than ANP+/+ mice. The C-receptor results were confirmed by receptor binding assays and affinity cross-linking studies. CONCLUSIONS: Taken together these data suggest that permanent removal of ANP from the natriuretic peptide system results in an up-regulation of GC-A and GC-B, and a corresponding down-regulation of C-receptor in the lung of proANP gene disrupted mice. We postulated that changes in the natriuretic peptide receptor population may result in chronic hypertension and cardiac hypertrophy in the ANP-/- mice.

Crystal structure of CHO reductase, a member of the aldo-keto reductase superfamily.

Chinese hamster ovary (CHO) reductase is an enzyme belonging to the aldo-keto reductase (AKR) superfamily that is induced by the aldehyde-containing protease inhibitor ALLN (Inoue, Sharma, Schimke, et al., J Biol Chem 1993;268: 5894). It shows 70% sequence identity to human aldose reductase (Hyndman, Takenoshita, Vera, et al., J Biol Chem 1997;272:13286), which is a target for drug design because of its implication in diabetic complications. We have determined the crystal structure of CHO reductase complexed with nicotinamide adenine dinucleotide phosphate (NADP)+ to 2.4 A resolution. Similar to aldose reductase and other AKRs, CHO reductase is an alpha/beta TIM barrel enzyme with cofactor bound in an extended conformation. All key residues involved in cofactor binding are conserved with respect to other AKR members. CHO reductase shows a high degree of sequence identity (91%) with another AKR member, FR-1 (mouse fibroblast growth factor-regulated protein), especially around the variable C-terminal end of the protein and has a similar substrate binding pocket that is larger than that of aldose reductase. However, there are distinct differences that can account for differences in substrate specificity. Trp111, which lies horizontal to the substrate pocket in all other AKR members is perpendicular in CHO reductase and is accompanied by movement of Leu300. This coupled with movement of loops A, B, and C away from the active site region accounts for the ability of CHO reductase to bind larger substrates. The position of Trp219 is significantly altered with respect to aldose reductase and appears to release Cys298 from steric constraints. These studies show that AKRs such as CHO reductase are excellent models for examining the effects of subtle changes in amino acid sequence and alignment on binding and catalysis.

Chronic hypertension in ANP knockout mice: contribution of peripheral resistance.

Atrial Natriuretic Peptide (ANP) exerts a chronic hypotensive effect which is mediated by a reduction in total peripheral resistance (TPR). Mice with a homozygous disruption of the pro-ANP gene (-/-) fail to synthesize ANP and develop chronic hypertension in comparison to their normotensive wild-type (+/+) siblings. In order to determine whether alterations in basal hemodynamics underlie the hypertension associated with lack of endogenous ANP activity, we used anesthetized mice to measure arterial blood pressure (ABP) and heart rate (HR), as well as cardiac output (CO) by thermodilution technique. -/- (n = 7) and +/+ (n = 10) mice of comparable weight and age were used. Stroke volume (SV) and TPR were derived from CO, HR, and ABP by a standard formula. ABP (mm Hg) was significantly higher in -/- (132+/-4) (P < 0.0001) than in +/+ mice (95+/-2). CO (ml min(-1)), HR(beats min(-1))and SV (microl beat(-1)) did not differ significantly between -/- and +/+ mice (CO -/- = 7.3+/-0.5, +/+ = 8.3+/-0.6; HR -/- = 407+/-22, +/+ = 462+/-21; SV -/- = 17.6+/-1.1, +/+ = 17.6+/-1.7). However, TPR (mm Hg ml(-1) min(-1)) was significantly elevated in -/- mice (18.4+/-0.7) compared to +/+ mice (12.3+/-1) (P = 0.0003). Autonomic ganglion blockade with a mixture of hexamethonium and pentolinium was followed by comparable percent reductions in CO (-/- = 28+/-4, +/+ = 29+/-3), HR (-/- = 9+/-4, +/+ = 16+/-4) and SV(-/- = 21+/-4, +/+ = 15+/-6) in both genotypes. However, the concomitant decrease in ABP (%) in -/- (41+/-2) was significantly greater than in +/+ (23+/-4) mice (P = 0.0009) and was accompanied by a significant reduction in TPR. We conclude that the hypertension associated with lack of endogenous ANP is due to elevated TPR, which is determined by an increase in cardiovascular autonomic tone.

Sequence and expression levels in human tissues of a new member of the aldo-keto reductase family.

We have isolated a human cDNA clone from small intestine that represents a new member of the aldo-keto reductase family. This new member showed 70% identity at the protein level to human aldose reductase and around 80% identity to other Chinese hamster and mouse reductases. The expression pattern shows that this message is located primarily in the adrenal gland, thus suggesting an involvement in steroid metabolism. It is also strongly expressed in the intestinal tract and has been called human small intestine reductase.

Aldehyde reductase: the role of C-terminal residues in defining substrate and cofactor specificities.

The only major structural difference between aldehyde reductase, a primarily NADPH-dependent aldo-keto reductase, and aldose reductase, a dually coenzyme-specific (NADPH/NADH) member of the same superfamily, is an additional eight amino acid residues in the substrate/inhibitor binding site (C-terminal region) of aldehyde reductase. On the premise that this segment defines the substrate specificity of the enzyme, a mutant of aldehyde reductase lacking residues 306-313 was constructed. In contrast to wild-type enzyme, the mutant enzyme reduced a narrower range of aldehydes and the new substrate specificity was not similar to aldose reductase as might have been predicted. A major change in coenzyme specificity was observed, however, the mutant enzyme being distinctly NADH preferring(Km, NADH = 35 microM, compared to <5 mM for wild-type and Km, NADPH = 670 microM, compared to 35 microM for wild type). Upon analyzing coordinates of aldehyde and aldose reductase, we found that deletion of residues 306-313 may have created a truncated enzyme that retained the three-dimensional structural features of the enzyme's C-terminal segment. The change in substrate specificity could be explained by the new alignment of amino acids. The reversal of coenzyme specificity appeared to be due to a significant backbone shift initiated by the formation of a strong hydrogen bond between Tyr319 and Val300. A similar bond exists in aldose reductase (Tyr309-Ala299). It appears, therefore, that as far as coenzyme specificity is concerned, deletion of residues 306-313 has converted aldehyde reductase into an aldose reductase-like enzyme.

Salt-sensitive hypertension in ANP knockout mice: potential role of abnormal plasma renin activity.

Atrial natriuretic peptide (ANP), a peptide hormone produced by the heart, exerts a chronic hypotensive effect. Knockout mice with a homozygous disruption of the pro-ANP gene (-/-) are incapable of producing ANP and are hypertensive relative to their wild-type (+/+) siblings. Previous studies showed that arterial blood pressure (ABP) was further increased in conscious -/- mice kept for 2 wk on 2% salt, but not in anesthetized -/- mice after 1 wk on 8% salt. To determine whether inconsistencies in observed effects of salt on ABP of -/- mice are due to duration of increased salt intake and/or the state of consciousness of the animals, we measured ABP from an exteriorized carotid catheter during and after recovery from anesthesia with ketamine-xylazine in adult +/+ and -/- mice kept on low (LS; 0.008% NaCl)- or high (HS; 8% NaCl)-salt diets for 3-4 wk. Conscious ABP +/- SE (mmHg) of +/+ mice did not differ significantly on either diet (HS, 113 +/- 3; LS, 110 +/- 5). However, on HS diet -/- mice had significantly higher ABP (135 +/- 3; P < 0.001) than both -/- (115 +/- 2) and +/+ (110 +/- 5) mice on LS diet. Anesthesia decreased ABP in all groups, but the the genotype- and diet-related differences were preserved. Plasma renin activity (PRA, ng ANG I.ml-1.h-1) in blood collected at termination of experiment was appropriately different on the 2 diets in +/+ mice (HS, 4.9 +/- 1.9; LS, 21 +/- 2.8). However, PRA failed to decrease in -/- mice on HS diet (HS, 18 +/- 2.9; LS, 19 +/- 3.7). Independent of genotype, concentration of endothelin-1 (ET-1, pg/mg protein) and endothelial constitutive NOS (ecNOS, density/100 micrograms protein) was significantly elevated in kidneys of mice fed on HS diet (ET-1 -/-, 31 +/- 4.7 and +/+, 32 +/- 4.1; ecNOS -/-, 160 +/- 19 and +/+, 156 +/- 19) compared with mice fed on LS diet (ET-1 -/-, 19 +/- 1.9 and +/+, 21 +/- 1.8; ecNOS -/-, 109 +/- 13 and +/+, 112 +/- 18). We conclude that, regardless of the state of alertness, -/- mice develop salt-sensitive hypertension after prolonged feeding on HS, in part due to their inability to reduce PRA, whereas the specific renal upregulation of ecNOS and ET-1 in response to HS intake may be an ANP-independent adaptive adjustment aimed at improving kidney function and counteracting the pressor effect of salt.

Studies on the inhibitor-binding site of porcine aldehyde reductase: crystal structure of the holoenzyme-inhibitor ternary complex.

Aldehyde reductase is an enzyme capable of metabolizing a wide variety of aldehydes to their corresponding alcohols. The tertiary structures of aldehyde reductase and aldose reductase are similar and consist of an alpha/beta-barrel with the active site located at the carboxy terminus of the strands of the barrel. We have determined the X-ray crystal structure of porcine aldehyde reductase holoenzyme in complex with an aldose reductase inhibitor, tolrestat, at 2.4 A resolution to obtain a picture of the binding conformation of inhibitors to aldehyde reductase. Tolrestat binds in the active site pocket of aldehyde reductase and interacts through van der Waals contacts with Arg 312 and Asp 313. The carboxylate group of tolrestat is within hydrogen bonding distance with His 113 and Trp 114. Mutation of Arg 312 to alanine in porcine aldehyde reductase alters the potency of inhibition of the enzyme by aldose reductase inhibitors. Our results indicate that the structure of the inhibitor-binding site of aldehyde reductase differs from that of aldose reductase due to the participation of nonconserved residues in its formation. A major difference is the participation of Arg 312 and Asp 313 in lining the inhibitor-binding site in aldehyde reductase but not in aldose reductase.

A new nomenclature for the aldo-keto reductase superfamily.

The aldo-keto reductases (AKRs) represent a growing oxidoreductase superfamily. Forty proteins have been identified and characterized as AKRs, and an additional fourteen genes may encode proteins related to the superfamily. Found in eukaryotes and prokaryotes, the AKRs metabolize a wide range of substrates, including aliphatic aldehydes, monosaccharides, steroids, prostaglandins, and xenobiotics. This broad substrate specificity has caused problems in naming these proteins. Enzymes capable of these reactions have been referred to as aldehyde reductase (ALR1), aldose reductase (ALR2), and carbonyl reductase (ALR3); however, ALR3 is not a member of the AKR superfamily. Also, some AKRs have multiple names based upon substrate specificity. For example, human 3alpha-hydroxysteroid dehydrogenase (3apha-HSD) type I is also known as dihydrodiol dehydrogenase 4 and chlordecone reductase. To address these issues, we propose a new nomenclature system for the AKR superfamily based on amino acid sequence identities. Cluster analysis of the AKRs shows seven distinct families at the 40% amino acid identity level. The largest family (AKR1) contains the aldose reductases, aldehyde reductases, and HSDs. Other families include the prokaryotic AKRs, the plant chalcone reductases, the Shaker channels, and the ethoxyquin-inducible aflatoxin B1 aldehyde reductase. At the level of 60% amino acid identity, subfamilies are discernible. For example, the AKR1 family includes five subfamilies: (A) aldehyde reductases (mammalian); (B) aldose reductases; (C) HSDs; (D) delta4-3-ketosteroid-5beta-reductases; and (E) aldehyde reductases (plant). This cluster analysis forms the basis for our nomenclature system. Recommendations for naming an aldo-keto reductase include the root symbol "AKR," an Arabic number designating the family, a letter indicating the subfamily when multiple subfamilies exist, and an Arabic numeral representing the unique protein sequence. For example, human aldehyde reductase would be assigned as AKR1A1. Our nomenclature is both systematic and expandable, thereby allowing assignment of consistent designations for newly identified members of the superfamily.

Cloning, sequencing, and enzymatic activity of an inducible aldo-keto reductase from Chinese hamster ovary cells.

Treatment of Chinese hamster ovary (CHO) cells by the aldehyde containing calpain inhibitor I resulted in the induction of a 35-kDa protein that was partially sequenced and shown to be a member of the aldo-keto reductase superfamily (Inoue, S., Sharma, R. C., Schimke, R. T., and Simoni, R. D. (1993) J. Biol. Chem. 268, 5894-5898). Using rapid amplification of cDNA ends polymerase chain reaction, we have sequenced the cDNA for this protein (CHO reductase). This enzyme is a new member of the aldo-keto reductase superfamily and shows greatest amino acid sequence identity to mouse fibroblast growth factor-regulated protein and mouse vas deferens protein (92 and 80% sequence identity, respectively). The enzyme exhibits about 70% sequence identity with the aldose reductases (ALR2; EC 1.1.1.21) and about 47% with the aldehyde reductases (ALR1; EC 1.1.1.2). Northern analysis showed that it is induced in preference to either ALR1 or ALR2 and RNase protection assays showed gene expression in bladder, testis, jejunum, and ovary in descending order of expression. The cDNA for this inducible reductase was cloned into the pET16b vector and expressed in BL21(DE3) cells. Expressed CHO reductase showed kinetic properties distinct from either ALR1 or ALR2 including the ability to metabolize ketones. This protein joins a growing number of inducible aldo-keto reductases that may play a role in cellular regulation and protection.

A nomenclature system for the aldo-keto reductase superfamily.

As new members of the AKR superfamily are identified the need for a systematic and expandable nomenclature has risen, especially since some members of the superfamily have multiple names based on substrate specificity. We have proposed a nomenclature system for the AKR superfamily that is similar to the P450 system but based on amino acid sequence comparisons instead of nucleotide sequence comparisons. Our system uses percent amino acid identities to delineate families and subfamilies within the larger superfamily. Although there are not as many AKRs as P450s, having a flexible nomenclature system will allow for easy incorporation of new proteins into the superfamily.

Gene expression of A- and B-type natriuretic peptides in response to acute ethanol ingestion.

Given that ethanol ingestion is associated with a disruption of water and electrolyte balance in addition to being a significant risk factor for cardiovascular disease, we have investigated the gene expression of ANP and BNP in response to acute doses of ethanol. Wistar rats were administered either a 5 g/kg dose of ethanol or an equivalent volume of water, and atrial and ventricular tissue samples were removed at 30, 60, and 120 min for analyses. Although no differences in ANP mRNA were observed between ethanol and water-treated rats during the time course, BNP mRNA levels in ethanol-treated rats were 43% of those present in water-treated animals in atrial tissue at 120 min. In ventricular tissue, BNP mRNA levels were reduced similarly to 38% of control. These results suggest a possible differential regulation of A- and B-type natriuretic peptides under the influence of ethanol ingestion.

Structure of porcine aldehyde reductase holoenzyme.

Aldehyde reductase, a member of the aldo-keto reductase superfamily, catalyzes the NADPH-dependent reduction of a variety of aldehydes to their corresponding alcohols. The structure of porcine aldehyde reductase-NADPH binary complex has been determined by x-ray diffraction methods and refined to a crystallographic R-factor of 0.20 at 2.4 A resolution. The tertiary structure of aldehyde reductase is similar to that of aldose reductase and consists of an alpha/beta-barrel with the active site located at the carboxy terminus of the strands of the barrel. Unlike aldose reductase, the N epsilon 2 of the imidazole ring of His 113 in aldehyde reductase interacts, through a hydrogen bond, with the amide group of the nicotinamide ring of NADPH.

Crystallization and preliminary structure of porcine aldehyde reductase-NADPH binary complex.

Porcine aldehyde reductase-NADPH binary complex has been crystallized from a buffered ammonium sulfate solution. The crystal form is hexagonal, space group P6(5)22, with a = b = 67.2, c = 243.7 A, alpha = beta = 90.0 and gamma = 120.0 degrees. A molecular-replacement structure solution has been successfully obtained by using the refined structure of the apoenzyme as the search model. The crystallographic R-factor is currently equal to 0.24 after energy minimization using data between 8 and 3.0 A resolution. The aldehyde reductase-NADPH complex model is supported by electron density corresponding to NADPH not included in the search model. The tertiary structure of aldehyde reductase consists of a beta/alpha-barrel with the coenzyme-binding site located at the carboxy-terminal end of the strands of the barrel. The structure of aldehyde reductase-NADPH binary complex will help clarify the mechanism of action for this enzyme and will lead to the development of pharmacologic agents to delay or prevent diabetic complications.

Studies on human aldose reductase. Probing the role of arginine 268 by site-directed mutagenesis.

Aldose reductase (ALR2) shows a strong specificity for its nucleotide coenzyme, binding NADPH much more tightly than NADH (KD of < 1 microM versus 1.2 mM respectively). Interactions responsible for this specificity include salt linkages between the highly conserved residues Lys-262 and Arg-268, and the 2'-phosphate of NADP(H). Previous studies show that mutation of Lys-262 results in an increase in the Km for both coenzyme and aldehyde substrate, as well as in the kcat of reduction. The present study shows that mutation of Arg-268 to methionine results in a 36-fold increase in Km and 205-fold increase in KD for NADPH, but little change in Km for DL-glyceraldehyde or in the kcat of the reaction. Calculation of free energy changes show that the 2'-phosphate of NADPH contributes 4.7 kcal/mol of binding energy to its interaction with WT-hALR2. For the R268M mutant, the interaction of NADPH was destabilized by 3.2 kcal/mol, indicating that the mutation decreases the binding energy of NADPH by 65%. The effect of removing Arg-268 in the absence of the 2'-phosphate of NADPH was virtually identical to the destabilization of the activation energy in the absence of the 2'-phosphate itself (1.9 versus 2.0 kcal/mol, respectively). Therefore, while the 2'-phosphate of the coenzyme plays a role in both coenzyme binding and transition state stabilization during catalysis, the role of Arg-268 lies strictly in tighter coenzyme binding.