Stereochemical inversion at C-15 accompanies the enzymatic isomerization of all-trans- to 11-cis-retinoids

all-trans-Retinol (vitamin A) is processed by membranes from the pigment epithelium of the amphibian or bovine eye to form 11-cis-retinoids. When the isomerization reaction is performed with either [15(S)-3H,14C]-all-trans-retinol or [15(R)-3H,14C]-all-trans-retinol as substrate, the resultant 11-cis-retinals, formed by the in vitro enzymatic oxidation of the retinols, retain their 3H in the former case and lose it in the latter. The ocular all-trans- (pro-R specific) and 11-cis-retinol (pro-S specific) dehydrogenases operate with different stereochemistries with respect to the prochiral methylene hydroxyl centers of their substrates. Inversion of stereochemistry at the prochiral retinol centers was shown to accompany the isomerization process in both the amphibian and bovine systems. The 11-cis-retinol formed from [15(S)-3H,14C]-all-trans-retinol was chemically isomerized with I2 to produce [15(R)-3H,14C]-all-trans-retinol. The 11-cis-retinol formed from [15(R)-3H,14C]-all-trans-retinol was chemically isomerized with I2 to produce [15(S)-3H,14C]-all-trans-retinol. The stereochemistry at the prochiral center of retinol is not affected by the I2-catalyzed double-bond isomerization process and, hence, inversion of stereochemistry at C-15 must accompany isomerization. The same inverted stereochemistry was found with the associated retinyl palmitates. Possible mechanistic reasons for the observed inversion of stereochemistry during isomerization are discussed.     

Substrate specificities and mechanism in the enzymatic processing of vitamin A into 11-cis-retinol

The biosynthesis of 11-cis-retinol in the retinal pigment epithelium requires two consecutive enzymatic reactions. The first involves the esterification of all-trans-retinol by lecithin retinol acyltransferase (LRAT). The second reaction involves the direct conversion of an all-trans-retinyl ester into 11-cis-retinol by an isomerase-like enzyme. This latter reaction couples the free energy of hydrolysis of an ester to the thermodynamically uphill trans to cis conversion, thus providing the energy to drive the latter process. In this paper both enzymes are studied with respect to their substrate specificities to provide information on mechanism. The isomerase is shown to be highly specific with respect to the ionylidene ring system and substitution at C15, whereas sterically bulkier substituents at C9 and C11 are permitted. C5 and C13 demethyl retinoids are isomerized, removing from consideration isomerization mechanisms involving C-H abstraction at the C5 or C13 methyl groups of the retinoid. On the other hand, C9 demethyl retinoids are not isomerized. A C-H abstraction mechanism is unlikely at the C9 methyl group as well, because no kinetic deuterium isotope effect is found with all-trans-19,19,19-trideuterioretinoids and isomerization of unlabeled retinoids occurs without the incorporation of deuterium when the isomerization is performed in D2O. LRAT proved to be broadly specific for retinols but was relatively inert with other hydrophobic alcohols including cholesterol. The enzyme is also highly specific for phosphatidylcholine analogues versus other potential membranous acyl donors such as phosphatidylethanolamine and phosphatidylserine.     

Isomerization of 11-cis-retinoids to all-trans-retinoids in vitro and in vivo

The regeneration of 11-cis-retinal, the universal chromophore of the vertebrate retina, is a complex process involving photoreceptors and adjacent retinal pigment epithelial cells (RPE). 11-cis-Retinal is coupled to opsins in both rod and cone photoreceptor cells and is photoisomerized to all-trans-retinal by light. Here, we show that RPE microsomes can catalyze the reverse isomerization of 11-cis-retinol to all-trans-retinol (and 13-cis-retinol), and membrane exposure to UV light further enhances the rate of this reaction. This conversion is inhibited when 11-cis-retinol is in a complex with cellular retinaldehyde-binding protein (CRALBP), providing a clear demonstration of the protective effect of retinoid-binding proteins in retinoid processes in the eye, a function that has been long suspected but never proven. The reverse isomerization is nonenzymatic and specific to alcohol forms of retinoids, and it displays stereospecific preference for 11-cis-retinol and 13-cis-retinol but is much less efficient for 9-cis-retinol. The mechanism of reverse isomerization was investigated using stable isotope-labeled retinoids and radioactive tracers to show that this reaction occurs with the retention of configuration of the C-15 carbon of retinol through a mechanism that does not eliminate the hydroxyl group, in contrast to the enzymatic all-trans-retinol to 11-cis-retinol reaction. The activation energy for the conversion of 11-cis-retinol to all-trans-retinol is 19.5 kcal/mol, and 20.1 kcal/mol for isomerization of 13-cis-retinol to all-trans-retinol. We also demonstrate that the reverse isomerization occurs in vivo using exogenous 11-cis-retinol injected into the intravitreal space of wild type and Rpe65-/- mice, which have defective forward isomerization. This study demonstrates an uncharacterized activity of RPE microsomes that could be important in the normal flow of retinoids in the eye in vivo during dark adaptation.     

Inhibitors of retinyl ester formation also prevent the biosynthesis of 11-cis-retinol

Lecithin retinol acyl transferase (LRAT) from the retinyl pigment epithelium is potently inhibited by all-trans-retinyl alpha-bromoacetate in the micromolar range. The inhibition is competitive and reversible. The retinyl pigment epithelium also contains an enzymatic activity capable of converting added all-trans-retinol into 11-cis-retinol. This isomerization is likely to require the intermediate formation of all-trans-retinyl esters, which are themselves produced by LRAT action. Here this possibility is directly tested by studying the effect of all-trans-retinyl alpha-bromoacetate on the isomerization reaction. When pigment epithelium membranes are preincubated with all-trans-retinyl alpha-bromoacetate, they form neither retinyl esters nor 11-cis-retinol from added all-trans-retinol. However, if the pigment epithelium membranes are first allowed to form all-trans-retinyl esters from all-trans-retinol before the addition of all-trans-retinyl alpha-bromoacetate, then 11-cis-retinol formation proceeds at close to the rate found in the absence of inhibitor. In addition, 11-cis-retinyl esters are not formed under these conditions, eliminating the possibility of a direct ester-ester isomerization route. Therefore, all-trans-retinyl esters are obligate intermediates in the biosynthesis of 11-cis-retinol.     

RPE65, Visual Cycle Retinol Isomerase, Is Not Inherently 11-cis-specific: SUPPORT FOR A CARBOCATION MECHANISM OF RETINOL ISOMERIZATION*

The mechanism of retinol isomerization in the vertebrate retina visual cycle remains controversial. Does the isomerase enzyme RPE65 operate via nucleophilic addition at C₁₁ of the all-trans substrate, or via a carbocation mechanism? To determine this, we modeled the RPE65 substrate cleft to identify residues interacting with substrate and/or intermediate. We find that wild-type RPE65 in vitro produces 13-cis and 11-cis isomers equally robustly. All Tyr-239 mutations abolish activity. Trp-331 mutations reduce activity (W331Y to ∼75% of wild type, W331F to ∼50%, and W331L and W331Q to 0%) establishing a requirement for aromaticity, consistent with cation-π carbocation stabilization. Two cleft residues modulate isomerization specificity: Thr-147 is important, because replacement by Ser increases 11-cis relative to 13-cis by 40% compared with wild type. Phe-103 mutations are opposite in action: F103L and F103I dramatically reduce 11-cis synthesis relative to 13-cis synthesis compared with wild type. Thr-147 and Phe-103 thus may be pivotal in controlling RPE65 specificity. Also, mutations affecting RPE65 activity coordinately depress 11-cis and 13-cis isomer production but diverge as 11-cis decreases to zero, whereas 13-cis reaches a plateau consistent with thermal isomerization. Lastly, experiments using labeled retinol showed exchange at 13-cis-retinol C₁₅ oxygen, thus confirming enzymatic isomerization for both isomers. Thus, RPE65 is not inherently 11-cis-specific and can produce both 11- and 13-cis isomers, supporting a carbocation (or radical cation) mechanism for isomerization. Specific visual cycle selectivity for 11-cis isomers instead resides downstream, attributable to mass action by CRALBP, retinol dehydrogenase 5, and high affinity of opsin apoproteins for 11-cis-retinal.     

Retinyl esters are the substrate for isomerohydrolase

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.     

Isomerization of all-trans-9- and 13-desmethylretinol by retinal pigment epithelial cells.

Photoisomerization of 11-cis-retinal to all-trans-retinal triggers phototransduction in the retinal photoreceptor cells and causes ultimately the sensation of vision. 11-cis-Retinal is enzymatically regenerated through a complex set of reactions in adjacent retinal pigment epithelial cells (RPE). In this study using all-trans-9-desmethylretinol (lacking the C(19) methyl group) and all-trans-13-desmethylretinol (lacking the C(20) methyl group), we explored the effects of C(19) and C(20) methyl group removals on isomerization of these retinols in RPE microsomes. The C(19) methyl group may be involved in the substrate activation, whereas the C(20) methyl group causes steric hindrance with a proton in position C(10) of 11-cis-retinol; thus, removal of this group could accelerate isomerization. We found that all-trans-9-desmethylretinol and all-trans-13-desmethylretinol are isomerized to their corresponding 11-cis-alcohols, although with lower efficiencies than isomerization of all-trans-retinol to 11-cis-retinol. These findings make the mechanism of isomerization through the C(19) methyl group unlikely, because in the case of 9-desmethylretinol, the isomerization would have to progress by proton abstraction from electron-rich olefinic C(9). The differences between all-trans-retinol, all-trans-9-desmethylretinol, and all-trans-13-desmethylretinol appear to be a consequence of the enzymatic properties, and binding affinities of the isomerization system, rather than differences in the chemical or thermodynamic properties of these compounds. This observation is also supported by quantum chemical calculations. It appears that both methyl groups are not essential for the isomerization reaction and are not likely involved in formation of a transition stage during the isomerization process.     

Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers

The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well.     

Mechanism of isomerization of 11-cis-retinal in lipid dispersions by aromatic amines

It has previously been shown that retinotoxic, primary aromatic amines catalyze the isomerization of 11-cis-retinal to its all-trans congener after Schiff base formation [Bernstein, P.S., Fulton, B.S., & Rando, R.R. (1986) Biochemistry 25, 3370-3377]. This process led to the short-circuiting of the visual cycle and the observed retinotoxicity when it occurred in vivo. The catalysis was also observed to occur in vitro in phosphatidylcholine-based vesicles but not in hydrocarbon solutions. The rate of isomerization of an aromatic amine Schiff base of 11-cis-retinal in the phospholipid vesicles was typically 10(3)-fold more rapid than in hydrocarbon solutions. In this article, the mechanistic basis of this apparently membrane-specific catalysis is described. It was found that the rate enhancement effect observed was independent of the lipid used. Moreover, a bilayer structure was not important because rate enhancements were also observed in micelles. The rapid isomerization rates observed in lipid dispersions appear not be free radical initiated because free radical quenching agents, such as alpha-tocopherol and beta-carotene, had little effect on the isomerization rates. It was further found that aliphatic amines, such as n-dodecylamine, could be substituted for the aromatic amines in phospholipid. Finally, and most importantly, it was found that the isomerization of the aromatic amine retinal Schiff bases in phospholipid vesicles was acid-catalyzed. It is concluded that the rate enhancements observed for the isomerization of 11-cis-retinal-aromatic amine Schiff bases in lipid dispersions over that in hydrocarbon solvents are due to the occurrence of acid-base catalysis in the former.     

Preferential release of 11-cis-retinol from retinal pigment epithelial cells in the presence of cellular retinaldehyde-binding protein

In photoreceptor cells of the retina, photoisomerization of 11-cis-retinal to all-trans-retinal triggers phototransduction. Regeneration of 11-cis-retinal proceeds via a complex set of reactions in photoreceptors and in adjacent retinal pigment epithelial cells where all-trans-retinol is isomerized to 11-cis-retinol. Our results show that isomerization in vitro only occurs in the presence of apo-cellular retinaldehyde-binding protein. This retinoid-binding protein may drive the reaction by mass action, overcoming the thermodynamically unfavorable isomerization. Furthermore, this 11-cis-retinol/11-cis-retinal-specific binding protein potently stimulates hydrolysis of endogenous 11-cis-retinyl esters but has no effect on hydrolysis of all-trans-retinyl esters. Apo-cellular retinaldehyde-binding protein probably exerts its effect by trapping the 11-cis-retinol product. When retinoid-depleted retinal pigment epithelial microsomes were preincubated with different amounts of all-trans-retinol to form all-trans-retinyl esters and then [3H]all-trans-retinol was added, as predicted, the specific radioactivity of [3H]all-trans-retinyl esters increased during subsequent reaction. However, the specific radioactivity of newly formed 11-cis-retinol stayed constant during the course of the reaction, and it was largely unaffected by expansion of the all-trans-retinyl ester pool during the preincubation. The absence of dilution establishes that most of the ester pool does not participate in isomerization, which in turn suggests that a retinoid intermediate other than all-trans-retinyl ester is on the isomerization reaction pathway.     

The effect of amino-acid substitutions I112P, D147E and K152N in CYP11B2 on the catalytic activities of the enzyme.

By replacing specific amino acids at positions 112, 147 and 152 of the human aldosterone synthase (CYP11B2) with the corresponding residues from human, mouse or rat 11beta-hydroxylase (CYP11B1), we have been able to investigate whether these residues belong to structural determinants of individual enzymatic activities. When incubated with 11-deoxycorticosterone (DOC), the 11beta-hydroxylation activity of the mutants was most effectively increased by combining D147E and I112P (sixfold increase). The two substitutions displayed an additive effect. The same tendency can be observed when using 11-deoxycortisol as a substrate, although the effect is less pronounced. The second step of the CYP11B2-dependent DOC conversion, the 18-hydroxylation activity, was not as strongly increased as the 11beta-hydroxylation potential. Activity was unaffected by D147E, whereas the single mutant I112P displayed the most pronounced activation (70% enhancement), thus causing different increasing effects on the first two enzymatic reaction steps. A slightly enhanced aldosterone synthesis from DOC could be measured due to increased levels of the intermediates. However, the 18-oxidation activity of all the mutants, except for I112S and D147E, was slightly reduced. The strongly enhanced 18-hydroxycorticosterone and aldosterone formation observed in the mutants provides important information on a possible role of such amino-acid replacements in the development of essential hypertension. Furthermore, the results indicate the possibility of a differential as well as independent modification of CYP11B2 reaction steps. The combination of functional data and computer modelling of CYP11B2 suggests an indirect involvement of residue 147 in the regulation of CYP11B isoform specific substrate conversion due to its location on the protein surface. In addition, the results indicate the functional significance of amino-acid 112 in the putative substrate access channel of human CYP11B2. Thus, we present the first example of substrate recognition and conversion being attributed to the N-terminal part of human CYP11B2.     

Monoterpene cyclases: use of the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate to uncouple the isomerization step of the coupled isomerization-cyclization reaction

Enzymes from Salvia officinalis capable of catalyzing the isomerization and subsequent cyclization of geranyl pyrophosphate to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate were examined with the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate in an attempt to dissect the cryptic isomerization step from the normally coupled reaction sequence. The analog inhibited the cyclization of geranyl pyrophosphate and was itself catalytically active, affording acyclic terpene olefins and alcohols as products. The enzymatic products generated from 6,7-dihydrogeranyl pyrophosphate qualitatively resembled the solvolysis products of 6,7-dihydrolinalyl pyrophosphate, yet they constituted a far higher proportion of olefins, suggesting that enzymatic product formation occurs in an environment relatively inaccessible to water. Since the normal cyclization of geranyl pyrophosphate is considered to proceed via preliminary isomerization to the bound tertiary intermediate (3R)-linalyl pyrophosphate, the results suggest that the analog undergoes the normal pyrophosphate ionization-migration step, giving rise in this case to (3R)-6,7-dihydrolinalyl pyrophosphate which is reionized, and because the subsequent cyclizations are precluded, the resulting cation is either deprotonated or captured by water. In divalent metal ion requirement, pH optimum, and other characteristics, the enzymatic transformation of the analog resembles the normal monoterpene cyclase reaction.     

Nonstereospecific biosynthesis of 11-cis-retinal in the eye

[3H]-all-trans-Retinol injected intraocularly into rats is processed to [3H]-11-cis-retinal, the visually active retinoid that binds to opsin. After 18 h, virtually all (93%) of the radioactive retinals recovered were in the form of 11-cis-retinal. At earlier times, however, both all-trans- and 13-cis-retinals, the latter being a nonphysiological isomer, were formed. Both of these isomers disappeared concomitant with the formation of 11-cis-retinal. The rise and fall of 13-cis-retinal suggest that this isomer can be converted into 11-cis-retinal either directly or indirectly in vivo and, hence, that the biosynthesis of the latter is nonstereospecific. This hypothesis was verified by showing that in double-labeling experiments [14C]-13-cis-retinol was converted into 11-cis-retinal nearly as well (approximately 70%) as [3H]-all-trans-retinol. These studies show that the biosynthesis of 11-cis-retinal can be nonstereospecific and, hence, that the process may be chemically rather than enzymatically mediated in vivo. In contrast, double-labeling studies with [14C]-9-cis-retinol and [3H]-all-trans-retinol showed that very little, if any, of the 9-cis isomer was processed to 11-cis-retinal in vivo although it did form isorhodopsin. This is consistent with what is known about the relative chemical stabilities of 9-cis-retinoids from model studies. The isomerization of 9-cis-retinoids is much slower than that of their all-trans, 13-cis, or 11-cis congeners. These results are discussed in terms of a possible mechanism for the biosynthesis of 11-cis-retinal in vivo and suggest that the isomerization event need not necessarily be enzyme mediated.     

Interaction of 11-cis-retinol dehydrogenase with the chromophore of retinal g protein-coupled receptor opsin

Vertebrate opsins in both photoreceptors and the retinal pigment epithelium (RPE) have fundamental roles in the visual process. The visual pigments in photoreceptors are bound to 11-cis-retinal and are responsible for the initiation of visual excitation. Retinochrome-like opsins in the RPE are bound to all-trans-retinal and play an important role in chromophore metabolism. The retinal G protein-coupled receptor (RGR) of the RPE and Müller cells is an abundant opsin that generates 11-cis-retinal by stereospecific photoisomerization of its bound all-trans-retinal chromophore. We have analyzed a 32-kDa protein (p32) that co-purifies with bovine RGR from RPE microsomes. The co-purified p32 was identified by mass spectrometric analysis as 11-cis-retinol dehydrogenase (cRDH), and enzymatic assays have confirmed the isolation of an active cRDH. The co-purified cRDH showed marked substrate preference to 11-cis-retinal and preferred NADH rather than NADPH as the cofactor in reduction reactions. cRDH did not react with endogenous all-trans-retinal bound to RGR but reacted specifically with 11-cis-retinal that was generated by photoisomerization after irradiation of RGR. The reduction of 11-cis-retinal to 11-cis-retinol by cRDH enhanced the net photoisomerization of all-trans-retinal bound to RGR. These results indicate that cRDH is involved in the processing of 11-cis-retinal after irradiation of RGR opsin and suggest that cRDH has a novel role in the visual cycle.     

11 Beta-hydroxysteroid dehydrogenase type 1 from human liver: dimerization and enzyme cooperativity support its postulated role as glucocorticoid reductase

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a microsomal enzyme that catalyzes the reversible interconversion of receptor-active 11-hydroxy glucocorticoids (cortisol) to their receptor-inactive 11-oxo metabolites (cortisone). However, the physiological role of 11beta-HSD 1 as prereceptor control device in regulating access of glucocorticoid hormones to the glucocorticoid receptor remains obscure in light of its low substrate affinities, which is in contrast to low glucocorticoid plasma levels and low Kd values of the receptors to cortisol. To solve this enigma, we performed detailed kinetic analyses with a homogeneously purified 11beta-HSD 1 from human liver. The membrane-bound enzyme was successfully obtained in an active state by a purification procedure that took advantage of a gentle solubilization method as well as providing a favorable detergent surrounding during the various chromatographic steps. The identity of purified 11beta-HSD 1 was proven by determination of enzymatic activity, N-terminal amino acid sequencing, and immunoblot analysis. By gel-permeation chromatography we could demonstrate that 11beta-HSD 1 is active as a dimeric enzyme. The cDNA for the enzyme was cloned from a human liver cDNA library and shown to be homologous to that previously characterized in human testis. Interestingly, 11beta-HSD 1 exhibits Michaelis-Menten kinetics with cortisol and corticosterone (11beta-dehydrogenation activity) but cooperative kinetics with cortisone and dehydrocorticosterone (11-oxoreducing activity). Accordingly, this enzyme dynamically adapts to low (nanomolar) as well as to high (micromolar) substrate concentrations, thereby providing the fine-tuning required as a consequence of great variations in circadian plasma glucocorticoid levels.     

Cloning and expression of the bovine 11beta-hydroxysteroid dehydrogenase type-2

The bovine 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD-2) cDNA was cloned from three overlapping PCR fragments using primers based on the human and ovine 11β-HSD-2 cDNA sequences. Both cDNA ends were obtained by a modified RACE (Rapid Amplification of cDNA Ends) method. The bovine 11β-HSD-2 cDNA is 1878 bp long, excluding the poly(A) tail. It consists of a 5′-untranslated region of 133 bp, an open reading frame of 1215 bp and a 3′-untranslated region of 530 bp. Bovine 11β-HSD-2 cDNA is highly homologous to that of the sheep (92%) and less related to the human (67%), rabbit (65%), rat (52%) and mouse (45%) cDNA. The predicted bovine 11β-HSD-2 protein contains 404 amino acid residues with a calculated mol wt of 43,985. It is homologous to the sheep (98%) and human (88%) protein, and less related to that of the rabbit (76%), rat (80%) and mouse (77%). The cloned 11β-HSD-2 cDNA was transfected into CHOP cells and the enzymatic characteristics determined. The enzyme functions primarily as an oxidase, uses NAD⁺ and is more active with corticosterone as a substrate than with cortisol or dexamethasone. It is expressed in high concentrations in kidney, adrenal and colon, and in small concentrations in liver, heart and lung. In conclusion, the 11β-HSD-2 enzyme of cattle is very similar to that of other species in its structure and enzymatic characteristics.     

Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11

The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes phytate (myo-inositol hexakisphosphate, IP6) to less phosphorylated myo-inositol phosphates in the presence of Ca2+. In this report, we discuss the unique Ca2+-dependent catalytic properties of the phytase and its specific substrate requirement. Initial rate kinetic studies of the phytase indicate that the enzyme activity follows a rapid equilibrium ordered mechanism in which binding of Ca2+ to the active site is necessary for the essential activation of the enzyme. Ca2+ turned out to be also required for the substrate because the phytase is only able to hydrolyze the calcium-phytate complex. In fact, both an excess amount of free Ca2+ and an excess of free phytate, which is not complexed with each other, can act as competitive inhibitors. The Ca2+-dependent catalytic activity of the enzyme was further confirmed, and the critical amino acid residues for the binding of Ca2+ and substrate were identified by site-specific mutagenesis studies. Isothermal titration calorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding. The pH dependence of the Vmax and Vmax/Km consistently supported these observations by demonstrating that the enzyme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate. The Ca2+-dependent activation of enzyme and substrate was found to be different from other histidine acid phytases that hydrolyze metal-free phytate.     

Mapping the ligand binding pocket in the cellular retinaldehyde binding protein

Retinoid interactions determine the function of the cellular retinaldehyde binding protein (CRALBP) in the rod visual cycle where it serves as an 11-cis-retinol acceptor for the enzymatic isomerization of all-trans- to 11-cis-retinol and as a substrate carrier for 11-cis-retinol dehydrogenase (RDH5). Based on preliminary NMR studies suggesting retinoid interactions with Met and Trp residues, human recombinant CRALBP (rCRALBP) with altered Met or Trp were produced and analyzed for ligand interactions. The primary structures of the purified proteins were verified for mutants M208A, M222A, M225A, W165F, and W244F, then retinoid binding properties and substrate carrier functions were evaluated. All the mutant proteins bound 11-cis- and 9-cis-retinal and therefore were not grossly misfolded. Altered UV-visible spectra and lower retinoid binding affinities were observed for the mutants, supporting modified ligand interactions. Altered kinetic parameters were observed for RDH5 oxidation of 11-cis-retinol bound to rCRALBP mutants M222A, M225A, and W244F, supporting impaired substrate carrier function. Heteronuclear single quantum correlation NMR analyses confirmed localized structural changes upon photoisomerization of rCRALBP-bound 11-cis-retinal and demonstrated ligand-dependent conformational changes for residues Met-208, Met-222, Trp-165, and Trp-244. Furthermore, residues Met-208, Met-222, Met-225, and Trp-244 are within a region exhibiting high homology to the ligand binding cavity of phosphatidylinositol transfer protein. Overall the data implicate Trp-165, Met-208, Met-222, Met-225, and Trp-244 as components of the CRALBP ligand binding cavity.     

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion

Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.     

Identification of the key residues determining the product specificity of isomerohydrolase

The efficient recycling of the chromophore of visual pigments, 11-cis-retinal, through the retinoid visual cycle is an essential process for maintaining normal vision. RPE65 is the isomerohydrolase in retinal pigment epithelium and generates predominantly 11-cis-retinol (11cROL) and a minor amount of 13-cis-retinol (13cROL), from all-trans-retinyl ester (atRE). We recently identified and characterized novel homologues of RPE65, RPE65c, and 13-cis-isomerohydrolase (13cIMH), which are expressed in the zebrafish inner retina and brain, respectively. Although these two homologues have 97% identical amino acid sequences, they exhibit distinct product specificities. Under the same assay conditions, RPE65c generated predominantly 11cROL, similar to RPE65, while 13cIMH generated exclusively 13cROL from atRE substrate. To study the impacts of the key residues determining the isomerization product specificity of RPE65, we replaced candidate residues by site-directed mutagenesis in RPE65c and 13cIMH. Point mutations at residues Tyr58, Phe103, and Leu133 in RPE65c resulted in significantly altered isomerization product specificities. In particular, our results showed that residue 58 is a primary determinant of isomerization specificity, because the Y58N mutation in RPE65c and its reciprocal N58Y mutation in 13cIMH completely reversed the respective enzyme isomerization product specificities. These findings will contribute to the elucidation of molecular mechanisms underlying the isomerization reaction catalyzed by RPE65.