Side-chain OH Research Articles

An Atom-Economic Inverse Solid-Phase Peptide Synthesis Using Bn or BcM Esters of Amino Acids.

An atom-economic N-to-C-directed solid-phase peptide synthesis is reported that uses benzyl (Bn) or (benzhydryl-carbamoyl)-methyl (BcM) esters of amino acids as the building blocks, which facilitate efficient hydrazinolysis, convenient conversion to acyl azide, and robust amidation with the next amino acid ester. This method is free of coupling reagents and free of protection on the side-chain OH, CO2H, CONH2, etc., therefore exhibiting a significantly improved atom economy compared to those of BOC- or Fmoc-based C-to-N-directed approaches.

NMR Observation of Intermolecular Hydrogen Bonds between Protein Tyrosine Side-Chain OH and DNA Phosphate Groups.

Hydrogen bonds between protein side-chain hydroxyl (OH) and phosphate groups are one of the most common types of intermolecular hydrogen bonds in protein-DNA/RNA complexes. Using NMR spectroscopy, we identified and characterized the hydrogen bonds between tyrosine side-chain OH and DNA phosphate groups in a protein-DNA complex. These OH groups exhibited relatively slow hydrogen-exchange rates and sizable scalar couplings between hydroxyl 1H and DNA phosphate 31P nuclei across the hydrogen bonds. Information about intermolecular hydrogen bonds facilitates investigations of the DNA/RNA recognition by the protein.

Acetylation of Bombyx mori silk fibroin and their characterization in the dry and hydrated states using 13C solid-state NMR

Acetylation of Bombyx mori silk fibroin (SF) was accomplished by chemical reaction of the side chain OH groups in Ser and Tyr residues with acetic anhydrate to increase hydrophobic character of SF. A combination of three kinds of 13C solid-state NMR techniques was used to elucidate the effect of acetylation in the dry and hydrated states for [3-13C]Ser, [3-13C]Tyr and [3-13C]Ala enriched-SF powder. The mobilities of Tyr and Ala residues in the amorphous region of acetylated 13C-labeled SF powder increased slightly and only very small amounts of the Ala residue with high mobility was observed by hydration. On the other hand, there are essentially no effect of water on the β-sheet region of these residues and acetylated Ser residues. A similar tendency toward the effect of hydration for acetylated SF powder was observed for acetylated SF fiber. The remarkable improvement of the dimensional stability in water was attained although the tensile strengths of fibers were slightly lower by the acetylation of SF fiber. Thus, acetylation proved very effective to achieve water-proofing of SF materials and the effect of acetylation on the local structure and dynamics of SF could be clarified using 13C solid-state NMR.

Quantitative chiral analysis of amino acids in solution using enantiomer-selective photodissociation of cold gas-phase tryptophan via chiral recognition

To explore the origin of biomolecule homochirality in interstellar molecular clouds, enantiomer-selective photodissociation via chiral recognition between amino acids in the gas phase was examined using a tandem mass spectrometer containing an electrospray ionization source and a cold ion trap. Ultraviolet photodissociation mass spectra of cold gas-phase noncovalent complexes of sodiated l-tryptophan ion, Na+(l-Trp), with an amino acid such as serine (Ser), threonine (Thr), or alanine (Ala) were obtained by the photo-excitation of l-Trp in the noncovalent complexes. Dissociation of l-Trp via CO2 loss occurred when it was noncovalently complexed with d-Ser or d-Thr in the presence of Na+. For the l-enantiomers, the energy absorbed by l-Trp was released through evaporation of l-Ser or l-Thr, and dissociation of the amino acids was suppressed. In contrast, the enantiomer-selective phenomenon was not observed in the noncovalent complex with Ala, suggesting that a side-chain OH group plays an important role in chiral recognition and enantiomer-selective photodissociation. The enantiomer-selective photodissociation was applied to the quantitative chiral analysis of amino acids. The enantiomeric excess of Ser and Thr in solution could be determined by measuring the relative abundance ratio of the enantiomer-selective photodissociation of Trp to amino acid evaporation in a single photodissociation mass spectrum obtained by photo-excitation of l-Trp used as a chiral probe in cold gas-phase noncovalent complexes with the analyte amino acids, and by referring to the linear relationships established in this work.

Introduction of a Hydrogen Bond between Phylloquinone PhQA and a Threonine Side-Chain OH Group in Photosystem I

The phylloquinone acceptor PhQ(A) in photosystem I binds to the protein through a single H-bond to the backbone nitrogen of PsaA-L722. Here, we investigate the effect of this H-bond on the electron transfer (ET) kinetics by substituting threonine for PsaA-L722. Room temperature spin-polarized transient EPR measurements show that in the PsaA-L722T mutant, the rate of PhQ(A)(-) to F(X) ET increases and the hyperfine coupling to the 2-methyl group of PhQ(A) is much larger than in the wild type. Molecular dynamics simulations and ONIOM type electronic structure calculations indicate that it is possible for the OH group of the Thr side chain to form an H-bond to the carbonyl oxygen atom, O(4) of the phylloquinone, and that this results in an increase in the 2-methyl hyperfine couplings as observed in the transient EPR data. The Arrhenius plot of the PhQ(A)(-) to F(X) ET in the PsaA-L722T mutant suggests that the increased rate is probably the result of a slight change in the electronic coupling between PhQ(A)(-) and F(X). The strong deviation from Arrhenius behavior observed at ∼200 K can be reproduced using a semiclassical model, which takes the zero-point energy of the mode coupled to the ET into account. However, since the change in slope of the Arrhenius plot occurs at the protein glass transition temperature, it is argued that it could be the result of a change in the protein relaxation dynamics at this temperature rather than quantum mechanical effects.

Kinetics of hydrolysis and mutational analysis of N,N‐diethyl‐m‐toluamide hydrolase from Pseudomonas putida DTB

The initial step in the biodegradation pathway of N,N-diethyl-m-toluamide (DEET) in Pseudomonas putida strain DTB is catalyzed by DEET hydrolase (DthA), which hydrolyzes the amide bond to yield 3-methylbenzoic acid and diethylamine. In order to extend our understanding of DthA, the enzyme was purified and characterized. The enzyme is most active at pH 7.9, and is probably a tetramer in its native state. The kinetic parameters of the wild-type enzyme are K(m) = 10.2 ± 0.8 μm, k(cat) = 5.53 ± 0.09 s(-1) , and k(cat) /K(m) = (5.4 ± 0.4) × 10(5) m(-1) ·s(-1) . Mild substrate inhibition was observed with DEET concentrations over 500 μm. A homology model of DthA was used to guide mutational analysis of the active site, confirming that the catalytic triad is formed by Ser166, Ap292, and His320. The oxyanion hole is formed by the side chain OH of Tyr84 and the backbone amide of Trp167, with the Tyr84 OH being essential for enzyme activity. The DthA model also revealed a hydrophobic substrate-binding pocket comprosed of Trp167, Met170, and Trp214. W167A and M170A mutations decreased enzymatic activity and exacerbated substrate inhibition, whereas Trp214, which probably plays a role in substrate recognition, was essential for enzymatic activity. The pH rate profile of DthA was fitted to two ionizable groups (pK(a1) = 6.1 and pK(a2) = 9.9) that probably correspond to Nε of His320 and the OH of Tyr84, respectively. In addition to catalyzing the hydrolysis of DEET, DthA hydrolyzed a variety of esters and amides.

Fine tuning of the redox function of Pseudomonas aeruginosa cytochrome c551 through structural properties of a polypeptide loop bearing an axial Met residue

Pseudomonas aeruginosa cytochrome c551 (PA) possesses a long polypeptide loop near its heme, and a unique hydrogen bond network among Ser52, axial Met61, and the heme 13-propionate side chain, i.e., Ser52 amide NH is hydrogen bonded to axial Met61 carbonyl CO, Met61 amide NH to Ser52 carbonyl CO, and Ser52 side chain OH to the heme 13-propionate side chain, contributes to stabilization of the structure of the loop [Y. Matsuura, T. Takano, R.E. Dickerson, J. Mol. Biol. 156 (1982) 389–409]. In this study, the structure and redox function of S52N and S52G mutants were characterized in order to elucidate the role of Ser52 in functional regulation of the protein. We found that the redox function of PA was hardly affected by an S52N mutation, but was slightly by an S52G one. The functional similarity between the wild-type protein and the S52N mutant demonstrated that Asn52 in the mutant plays a similar pivotal role in the formation of the unique hydrogen bond network that stabilizes the structure of the loop as Ser52 in the wild-type protein does. On the other hand, the functional alteration induced by the S52G mutation can be attributed to a structural change of the loop due to the lack of the hydrogen bond between the Gly52 and heme 13-propionate side chain in the mutant. Thus, this study demonstrated that the function of the protein can be tuned through the structural properties of the polypeptide loop near its heme.

IR spectra of stigmastane series brassinosteroids differing in configuration and number of diol groups

IR spectra of steroid phytohormones of the stigmastane series (22R, 23R)-28-homocastasterone and (22R,23R)-28-homosecasterol and their isomers (22S,23S)-28-homocastasterone and (22S,23S)-28-homosecasterol have been analyzed. The 28-homocastasterone molecule contains diol groups in ring A and in the side chain whereas that of 28-homosecasterol has one diol group in the side chain. The lack of two OH groups in ring A of homosecasterol compared to homocastasterone results in the appearance of stretching vibrational bands of H-C= (ν max = 3025 cm -1 ) and -C=C (ν max = 1656 cm -1 ) groups of ring A. Substantial changes are observed in the area of OH stretching vibrations. Homocastasterones pressed in KBr possess twice as many OH groups as homosecasterols such that absorption band total intensities in IR spectra of both isomers caused by H-bonds of the diol groups in the side chain amount to 65% whereas the share of the 2α,3α group is only 35% of the total intensity. Hence the contribution from the side-chain OH groups of the studied brassinosteroids to the integral optical density of the bands exceeds that from the ring-A OH groups. In dilute CHCl3 solutions of the brassinosteroids, the conformations of the brassinosteroid side chains are not the same. As a result, intramolecular H-bonds of different energy are created. The optical density D max in band maxima of free OH groups for homocastasterones is three times higher than that for the corresponding band maxima of homosecasterol. This implies that D max for bands of free OH groups of the homocastasterone ring-A diol group is greater, in contrast with the relatively greater D max for bands of homosecasterol side-chain OH groups bound by an intermolecular H-bond. The homocastasterone diol groups also form intramolecular Hbonds more actively. The lack of the diol group in ring A of the homosecasterols does not affect the frequencies of the C=O stretching vibrations. This leads to the conclusion that the C=O group forms intermolecular H-bonds only with the side-chain OH groups of brassinosteroids pressed in KBr.

Conformers of gaseous threonine†

Following an extensive search on the potential energy surfaces (PES) of the natural amino acid L-threonine (Thr) and its allotropic form L-allo-threonine (aThr), 56 and 61 conformers of Thr and aThr, respectively, have been located with the help of density functional theory (DFT). Accurate structures, relative energies, rotational as well as quartic and sextic centrifugal distortion constants, dipole moments, 14N nuclear quadrupole coupling constants, anharmonic vibrational frequencies and double-harmonic infrared intensities have been determined from ab initio electronic structure calculations for the five most stable Thr and aThr conformers. The global minimum, Thr-I, has a cyclic triple H-bond motif with strong OH ··· N, C=O ··· HO, and a weaker NH ··· OH H-bond, where the latter two involves the side chain OH, and an energetically unfavourable trans-COOH arrangement. The best relative energies of the conformers, accurate within ±1 kJ mol−1, have been determined through the first-principles composite focal-point analysis (FPA) approach. There are four and three conformers of Thr and aThr, respectively, within a relative energy of 5 kJ mol−1. Similarly to other amino acids investigated, lower levels of electronic structure theory, especially the Hartree–Fock level, are unable to determine the correct relative energies of the conformers. The rotational, the quartic and sextic centrifugal distortion, and the 14N nuclear quadrupole coupling constants as well as the anharmonic vibrational fundamentals and double-harmonic infrared intensities, all determined using DFT, should aid identification and characterization of the conformers of threonine and allo-threonine by rotational and vibrational spectroscopies, respectively. †This paper is dedicated to Professor Fritz Schaefer on the occasion of his 65th birthday.

Effect of pyridyl nitrogen on the conformational landscape of 7-azaserotonin: A computational study

The conformational topology of gaseous neutral 7-azaserotonin has been investigated by employing systematic ab initio calculations. The whole conformational landscape has been explored by varying four dihedral angles which define the position of ethyl amine side chain and phenolic OH group with respect to the 7-azaindole plane. A total of 22 local minima have been located by geometry optimization of 216 possible trial structures at the B3LYP/6-31+G * level of theory. With the exception of a few conformers, in most cases the OH- syn conformers were more stable than their OH- anti counterparts. Vibrational frequency analysis for all 22 conformers computed at B3LYP/6-31+G * shows some interesting features in the pyrrole and pyridine C H stretches. In some of the conformers the presence of intramolecular blue shifted H-bonding is confirmed using the atoms in molecules (AIM) analysis. The AIM analysis explains the differences in the relative energy order of various conformers compared to that observed in the case of serotonin. It also explains the behavior of the pyrrole and pyridine C H stretches observed for some of the conformers. The conformational distribution of 7-azaserotonin at various temperatures is computed based on simple thermodynamic principle. At lower temperature of about 100 K only six conformers were significantly populated according to their electronic energies while at higher temperature of about 400 K the populations were determined by their vibrational partition functions.

Complexation of boric acid with vitamin C

Mono-chelate (1 : 1) and bis-chelate (1 : 2) anionic complexes of boric acid with vitamin C (L-ascorbic acid, H2A) were isolated from aqueous solutions in salt form with Li+, Na+ and Ca2+ ions. The complexes were characterized by FTIR, 13C and 11B MAS (magic angle spinning) NMR techniques. The spectral data agreed with the calculated structures that H2A complexes with boric acid through its cis-enediol groups by forming five-membered chelate rings. Side-chain OH groups do not participate in complexation as corroborated by the results of the test experiments conducted with iso-propylideneascorbic acid (i-H2A) where the side chain is blocked. Empirical relations, that can be used as diagnostics for the 1 : 1 and 1 : 2 ascorbatoborate complexes, were derived from the observed chemical shift values. Bis-chelate complexes were found to have higher thermal and hydrolytic stabilities than their mono-chelate homologs. The effect of the cation on the stabilization of the complexes was also investigated. The observed relative stability of the 1 : 1 Na–ascorbatoborate complex with respect to the analogous Ca complex, correlated with the recent reports about the role of alkali metal ions in the stabilization of ribose in the prebiotic world.

Roles of adenine anchoring and ion pairing at the coenzyme B12-binding site in diol dehydratase catalysis

The X-ray structure of the diol dehydratase-adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Seralpha224, and of 6-NH(2), N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the epsilon-NH(2) group of Lysbeta135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Seralpha224 and Lysbeta135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Salpha224A, Salpha224N and Kbeta135E mutants were 19-2% as active as the wild-type enzyme, whereas the Kbeta135A, Kbeta135Q and Kbeta135R mutants retained 58-76% of the wild-type activity. The presence of a positive charge at the beta135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme-cobalamin interaction. The Salpha224A and Salpha224N mutants showed a k(cat)/k(inact) value that was less than 2% that of the wild-type, whereas for Lysbeta135 mutants this value was in the range 25-75%, except for the Kbeta135E mutant (7%). Unlike the wild-type holoenzyme, the Salpha224N and Salpha224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Seralpha224 is important for cobalt-carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Salpha224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5'-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one.

Probing the Lower Size Limit for Protein-Like Fold Stability: Ten-Residue Microproteins With Specific, Rigid Structures in Water

Mutational optimization of two long-range interactions first observed in Ac-WINGKWT-NH2, (a) bifurcated H-bonding involving the threonine amide H(N) and side chain OH and the N-terminal acetyl carbonyl and (b) an H-bond between the entgegen-H(N) of the C-terminal amide and the indole ring of Trp6 that stabilizes a face-to-edge indole/indole interaction between Trp1 and Trp6, has afforded < or = 10 residue systems that yield a remarkably stable fold in water. Optimization was achieved by designing a hydrophobic cluster that sequesters these H-bonds from solvent exposure. The structures and extent of amide H/D exchange protection for CH3CH2CO-WI pGXWTGPS (p = D-Pro, X = Leu or Ile) were determined. These two systems are greater than 94% folded at 298 K (97.5% at 280 K) with melting temperatures > 75 degrees C. The fold appears to display minimal fluxionality; a well-converged NMR structure rationalizes all of the large structuring shifts observed, and we suggest that these designed constructs can be viewed as microproteins.

Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase

An extremely low-field signal (at approximately 18 p.p.m.) in the (1)H NMR spectrum of rhamnogalacturonan acetylesterase (RGAE) shows the presence of a short strong hydrogen bond in the structure. This signal was also present in the mutant RGAE D192N, in which Asp192, which is part of the catalytic triad, has been replaced with Asn. A careful analysis of wild-type RGAE and RGAE D192N was conducted with the purpose of identifying possible candidates for the short hydrogen bond with the 18 p.p.m. deshielded proton. Theoretical calculations of chemical shift values were used in the interpretation of the experimental (1)H NMR spectra. The crystal structure of RGAE D192N was determined to 1.33 A resolution and refined to an R value of 11.6% for all data. The structure is virtually identical to the high-resolution (1.12 A) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop. Searches of the Cambridge Structural Database were conducted to obtain information on the donor-acceptor distances of different types of hydrogen bonds. The short hydrogen-bond interactions found in RGAE have equivalents in small-molecule structures. An examination of the short hydrogen bonds in RGAE, the calculated pK(a) values and solvent-accessibilities identified a buried carboxylic acid carboxylate hydrogen bond between Asp75 and Asp87 as the likely origin of the 18 p.p.m. signal. Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB. The shortest hydrogen bonds in RGAE are all located close to the active site and short interactions between Ser and Thr side-chain OH groups and backbone carbonyl O atoms seem to play an important role in the stability of the protein structure. These results illustrate the significance of short strong hydrogen bonds in proteins.

2-(3-Hydroxypropyl)isoindoline-1,3-dione: competition among hydrogen-bond acceptors

The title compound, C(11)H(11)NO(3), has two molecules in the asymmetric unit, which differ in the orientation of their side-chain OH groups, allowing them to form intermolecular O-H...O hydrogen bonds to different acceptors. In one case, the acceptor is the OH group of the other molecule, and in the other case it is an imide O=C group. This is the first example in the N-substituted phthalimide series in which independent molecules have different types of acceptor. Molecular-orbital calculations place the greatest negative charge on the OH group.

Low-Calcemic, Highly Antiproliferative, 23-Oxa Ether Analogs of the Natural Hormone 1α,25-Dihydroxyvitamin D3: Design, Synthesis, and Preliminary Biological Evaluation

Eight new side-chain allylic, benzylic, and propargylic ether analogs of the natural hormone calcitriol have been rationally designed and easily synthesized. Three of these 23-oxa ether analogs lacking the typical side-chain OH group are more antiproliferative in vitro and desirably less calcemic in vivo than the natural hormone. One of these three 23-oxa analogs has transcriptional potency almost as high as that of calcitriol, even though it binds to the human vitamin D receptor only about 1% as well as calcitriol.

Competitive H-bonds in vacuo and in aqueous solution for N-protonated adrenaline and its monohydrated complexes

The conformational landscape of the N-protonated form of adrenaline (epinephrine in the U.S.) has been considered at both the B3LYP/6-31G* and 6-311++G** levels, with full geometry optimization in the gas phase and in aqueous solution, using the integral equation formalism for the polarizable continuum model (IEF-PCM). All stable orientations of the two catecholic hydroxy groups as well as the interconversion pathways between them have been taken into account. The dependence of the potential energy profile for the −[NH 2CH 3] + group rotation on the catechol ring and side-chain OH group orientation has been also examined. B3LYP/6-31G* stationary points have been confirmed with frequency calculations (at 310 K and 1 atm) allowing thermal corrections and IR spectra to be evaluated. Basis set effects do not alter the relative stability of the various forms that is maintained also when MP2 correlation corrections are included. The role of the internal structures either embedding in aqueous solution the conformers kept rigid at their in vacuo geometries or allowing the structure to relax has been examined at the IEF-PCM/B3LYP/6-31G* and 6-311++G** levels and compared to the IEF-PCM/MP2/6-31G* results. In vacuo the G1 conformers are favored with respect to the T and G2 ones, while in water the stability ordering changes, T becoming significantly more stable than both gauche forms. This occurs even for the gas-phase structures, including or not thermal corrections. The addition of a single water molecule to N-protonated adrenaline caused the intramolecular H-bond within the side chain to weaken till formation of two H-bonds with water. This fact, coupled to the strain relaxation, further stabilized the G1 and T conformers. The maximum intensity of the IR spectra increases in solution, especially for monohydrated adducts, where a significant red shift in the vibration of the NH involved in a H-bond to the water O is found.

Hydroxyl-bearing poly(α-hydroxy acid)-type aliphatic degradable polyesters prepared by ring opening (co)polymerization of dilactones based on glycolic, gluconic and l-lactic acids

The synthesis of HO-protected poly(glycolic acid- co-gluconic acid) and poly( l-lactic acid- co-glycolic acid- co-gluconic acid) by copolymerisation of l-lactide and 3-(1,2-3,4-tetraoxobutyl-diisopropylidene)-1,4-dioxane-2,5-dione (DIPAGYL) is reported. The resulting polymers were characterized by size exclusion chromatography, FT Infrared, nuclear magnetic resonance, differential scanning calorimetry and X-ray diffractometry. The composition of reaction media and the reactivity ratios of the two cyclic monomers were determined at low conversion and indicated random distributions of lactyl and gluconoglycolyl constitutive units. X-ray diffraction data showed semi-crystalline morphology, as observed for poly (lactide) stereocopolymers containing more than 90% of l-enantiomer. Deprotection of the isopropylidene-protected side chain OH was possible under acidic conditions and yielded copolymers with various degrees of hydroxylation. Deprotection of the 5–6 OH groups was fast and complete whereas that of 3–4 ones was partial and occurred at the expenses of partial degradation of the aliphatic polyester chains. T g increased with the number of hydroxyl functions, a feature attributed to the formation of hydrogen bonds. Comparison is made with features reported previously for analogs derived from dl-lactide.

Structural Scaffold of 18-Crown-6 Tetracarboxylic Acid for Optical Resolution of Chiral Amino Acid: X-Ray Crystal Analyses of Complexes of D- and L-Isomers of Serine and Glutamic Acid

(+)-18-crown-6 tetracarboxylic acid (18C6H4) has been used as a chiral selector for D/L-amino acids in HPLC, where L-isomer is usually eluted prior to D-isomer, except for the case of serine. To clarify why serine exhibits the reverse order for the elusion, the chiral interactions of D- and L-serines with (+)-18C6H4 were investigated by the X-ray single crystal analyses, together with the case of D- and L-glutamic acids, which exhibit the usual elution order in HPLC. The backbone structures (amino, Calpha-H and carboxyl groups) of these four amino acids showed the nearly same interaction with (+)-18C6H4 despite their different chirality. In contrast, the hydroxyl group of L-serine side chain formed a hydrogen bond with the carboxyl group of (+)-18C6H4, whereas such a interaction was not formed for the side chain of D-serine and D- and L-glutamic acids. Thus, it was shown that the exception of D/L-serine from the first elution rule of L-isomer in HPLC is due to the presence and absence of a hydrogen bond formation of its side chain OH group.

The Crystal Structure of Guinea Pig 11β-Hydroxysteroid Dehydrogenase Type 1 Provides a Model for Enzyme-Lipid Bilayer Interactions

The metabolic reduction of 11-keto groups in glucocorticoid steroids such as cortisone leads to the nuclear receptor ligand cortisol. This conversion is an example of pre-receptor regulation and constitutes a novel pharmacological target for the treatment of metabolic disorders such as insulin resistance and possibly other derangements observed in the metabolic syndrome, such as hyperlipidemia, hypertension, and lowered insulin secretion. This reaction is carried out by the NADPH-dependent type 1 11beta-hydroxysteroid dehydrogenase (11beta-HSD1), an enzyme attached through an integral N-terminal transmembrane helix to the lipid bilayer and located with its active site within the lumen of the endoplasmic reticulum. Here we report the crystal structure of recombinant guinea pig 11beta-HSD1. This variant was determined in complex with NADP at 2.5 A resolution and crystallized in the presence of detergent and guanidinium hydrochloride. The overall structure of guinea pig 11beta-HSD1 shows a clear relationship to other members of the superfamily of short-chain dehydrogenases/reductases but harbors a unique C-terminal helical segment that fulfills three essential functions and accordingly is involved in subunit interactions, contributes to active site architecture, and is necessary for lipid-membrane interactions. The structure provides a model for enzyme-lipid bilayer interactions and suggests a funneling of lipophilic substrates such as steroid hormones from the hydrophobic membrane environment to the enzyme active site.