Β-hydroxyl Group Research Articles

Structural and functional studies of a trans-acyltransferase polyketide assembly line enzyme that catalyzes stereoselective α- and β-ketoreduction.

While the cis-acyltransferase modular polyketide synthase assembly lines have largely been structurally dissected, enzymes from within the recently discovered trans-acyltransferase polyketide synthase assembly lines are just starting to be observed crystallographically. Here we examine the ketoreductase (KR) from the first polyketide synthase module of the bacillaene nonribosomal peptide synthetase/polyketide synthase at 2.35-Å resolution. This KR naturally reduces both α- and β-keto groups and is the only KR known to do so during the biosynthesis of a polyketide. The isolated KR not only reduced an N-acetylcysteamine-bound β-keto substrate to a D-β-hydroxy product, but also an N-acetylcysteamine-bound α-keto substrate to an L-α-hydroxy product. That the substrates must enter the active site from opposite directions to generate these stereochemistries suggests that the acyl-phosphopantetheine moiety is capable of accessing very different conformations despite being anchored to a serine residue of a docked acyl carrier protein. The features enabling stereocontrolled α-ketoreduction may not be extensive since a KR that naturally reduces a β-keto group within a cis-acyltransferase polyketide synthase was identified that performs a completely stereoselective reduction of the same α-keto substrate to generate the D-α-hydroxy product. A sequence analysis of trans-acyltransferase KRs reveals that a single residue, rather than a three-residue motif found in cis-acyltransferase KRs, is predictive of the orientation of the resulting β-hydroxyl group.

"Head-to-side-chain" cyclodepsipeptides of marine origin.

Since the late 1980s, a large number of depsipeptides that contain a new topography, referred to as “head-to-side-chain” cyclodepsipeptides, have been isolated and characterized. These peptides present a unique structural arrangement that comprises a macrocyclic region closed through an ester bond between the C-terminus and a β-hydroxyl group, and terminated with a polyketide moiety or a more simple branched aliphatic acid. This structural pattern, the presence of unique and complex residues, and relevant bioactivity are the main features shared by all the members of this new class of depsipeptides, which are reviewed herein.

Structures Required of Polyphenols for Inhibiting Advanced Glycation end Products Formation

The properties of polyphenols as AGEs formation inhibitors have attracted great interest among researchers. This review discusses the antiglycation activities of polyphenols and focuses on the relationship between the AGEs formation inhibitory activities and their chemical structures. The molecular structures influence the inhibition in the following ways: (1) The hydroxylation on both A ring and B ring improved the inhibitory activity on AGEs formation, while hydroxylation on C ring decreased the activity. (2) The methylation generally reduced the anti-AGEs activity of flavonoids, except for the 3-O-methylation of flavonols. (3) The glycosylation of hydroxyls of flavonoids tended to decrease the inhibitory activities on inhibiting AGEs formation, although contradictionary results were existed. (4) Hydrogenation of the C2=C3 double bond of flavones slightly weakened their activities. (5) A 5,7-dihydroxy structure was favorable to the activity of isoflavones. (6) Proanthocyanidins dimer or trimers showed a stronger inhibitory activity than catechins, and the glucosides of anthocyanidin had higher activities than their rutinosides. (7) The hydroxylation on B ring and the methylation of stilbenes decreased the inhibitory activity. (8) Presence of galloyl groups was important for the activity of catechins, and α-hydroxyl group at C-3 was much more effective than β-hydroxyl group at C-3. (9) The phenolic acids with multiple hydroxyls showed strong inhibition against AGEs formation, and an ortho or meta dihydroxyl structure on the benzene ring was vital to the anti-AGEs activity of anthraquinone. (10) Both ellagic acids and ellagitannins showed potent inhibitory activities on AGEs formation, and hydroxylation increased the activities but methylation decreased them.

The orientation of the β-hydroxyl group controls the diastereoselectivity during the hydride reduction and Grignard reaction of inososes

A comparison of the results of the Grignard reaction and the hydride reduction of the carbonyl group of epi- and scyllo-inososes reveals that the extent of diastereoselectivity of these reactions is decided by the orientation of the β-hydroxyl group (or its derivative). Presence of an axial β-hydroxyl group generally results in the formation of relatively larger amount of the axial alcohol as a result of the reduction of the carbonyl group. The possible reasons for the observed differences in diastereoselectivity between the reactions of these isomeric epi- and scyllo-inososes have been discussed. The sequence of reactions reported here provides convenient access to C-alkylated inositols, such as iso-laminitol and iso-mytilitol as well as 2-O-methyl epi-inositol, an epimer of the naturally occurring ononitol.

Comparative evaluation of in-vitro cytotoxicity, antiviral and antioxidant activities of different soyasapogenols from soybean saponin

Objectives The aim of this study was to evaluate comparative and structure–activity relationships of in-vitro cytotoxicity, antiviral and antioxidant activities of soyasapogenols A, B, D and F (SSA, SSB, SSD and SSF) together against the total soyasaponin extract (TSSE) itself. Methods The cytotoxicity of soyasapogenols and TSSE against human colon carcinoma cell line (HCT-116), liver carcinoma cell line (Hep-G2), human breast carcinoma cell line (MCF-7) and normal human melanocytes (HFB-4) cell lines was assessed using sulforhodamine B assay. Their antiviral activities were investigated against Rift Valley fever virus (RVFV), hepatitis C virus model (vesicular stomatitis virus, VSV), and hepatitis A virus (HAV). The antioxidant activity of soyasapogenols and TSSE was assessed using a stable DPPH free radical. Results and conclusion The results obtained showed that both TSSE and soyasapogenols have a potent cytotoxic effect on Hep-G2, HCT-116, MCF-7 and HBF-4 cell lines in a concentration-dependent manner. SSA and SSF showed the highest cytotoxic activities against tested cell lines. Analysis of the three-dimensional structure of the measured soyasapogenols indicated that if the β-hydroxyl group at C-21 or C-22 was aligned with the plane of the molecule, a marked increase in the cytotoxic activity of the soyasapogenol was produced. Their antiviral activities against RVFV, VSV and HAV showed significant inhibition activities compared with both TSSE and interferon. SSB showed the best activity against RVFV and HAV, whereas SSA was the best inhibitor against VSV. It was concluded that the hydroxylation at C-21 as well as the presence of a double bond in ring D might enhance anti-VSV activity, whereas they may not be essential for anti-RVFV and anti-HAV activities. On the other hand, the tested soyasapogenols and TSSE did not show good antioxidant activities.

Mechanism for folate‐independent aldolase reaction catalyzed by serine hydroxymethyltransferase

Serine hydroxymethyltransferase catalyzes the cleavage of β-hydroxyamino acids into glycine and aldehydes in the absence of tetrahydrofolate. The enzyme accepts various β-hydroxyamino acids as the substrate of this reaction. The reaction rate varies depending on the substituent and stereochemistry at the Cβ atom: the erythro forms and the β-phenyl substituent are preferred over the threo forms and the β-methyl substituent, respectively. Although several mechanisms have been proposed, what determines the substrate preference remains unclear. We first performed quantum mechanical calculations to assess the validity of the reaction mechanisms. The results indicate that the retro-aldol mechanism starting with abstraction of the proton from the β-hydroxyl group is plausible. This also suggests that Cα-Cβ bond cleavage is the rate-limiting step. We next measured the dependence of the rate constants on temperature with four representative substrates and calculated the activation energies and pre-exponential factors from the Arrhenius plots. The activation energies of the erythro forms were lower than those of the threo forms. The β-phenyl substituent lowered the activation energy in the threo form, whereas it did not alter the activation energy but increased the pre-exponential factor in the erythro form. We present a unified model to explain the origin of the substituent and stereochemical preferences by combining the theoretical and experimental results. A possible biological role of the tetrahydrofolate-independent activity in thermophiles is also discussed.

C-Lactam Derivatives of Oleanolic Acid. The Synthesis of C-lactam by Beckmann Rearrangement of C-oxime

Oleanolic acid, one of the most known triterpenes, was subjected to different chemical transformations within C-3 beta-hydroxyl group, a double bond between C-12 and C-13, and a carboxyl function at C-17 in order to obtain new derivatives. The key compound consists of four six-membered rings (A, B, D, E) and one enlarged ring (C ring) containing a nitrogen atom and a carbonyl function - lactam. This type of derivative can be obtained by Beckmann rearrangement of the appropriate oxime. The lactam can be transformed into thiolactam with the use of Lavesson's reagent. The method is also presented for new derivatives synthesis, as well as their structure elucidation by spectroscopic means.

Beta Hydroxylation of Glycolipids from Ustilago maydis and Pseudozyma flocculosa by an NADPH-Dependent β-Hydroxylase

Flocculosin and ustilagic acid (UA), two highly similar antifungal cellobiose lipids, are respectively produced by Pseudozyma flocculosa, a biocontrol agent, and Ustilago maydis, a plant pathogen. Both glycolipids contain a short-chain fatty acid hydroxylated at the β position but differ in the long fatty acid, which is hydroxylated at the α position in UA and at the β position in flocculosin. In both organisms, the biosynthesis genes are arranged in large clusters. The functions of most genes have already been characterized, but those of the P. flocculosa fhd1 gene and its homolog from U. maydis, uhd1, have remained undefined. The deduced amino acid sequences of these genes show homology to those of short-chain dehydrogenases and reductases (SDR). We disrupted the uhd1 gene in U. maydis and analyzed the secreted UA. uhd1 deletion strains produced UA lacking the β-hydroxyl group of the short-chain fatty acid. To analyze the function of P. flocculosa Fhd1, the corresponding gene was used to complement U. maydis Δuhd1 mutants. Fhd1 was able to restore wild-type UA production, indicating that Fhd1 is responsible for β hydroxylation of the flocculosin short-chain fatty acid. We also investigated a P. flocculosa homolog of the U. maydis long-chain fatty-acid alpha hydroxylase Ahd1. The P. flocculosa ahd1 gene, which does not reside in the flocculosin gene cluster, was introduced into U. maydis Δahd1 mutant strains. P. flocculosa Ahd1 neither complemented the U. maydis Δahd1 phenotype nor resulted in the production of β-hydroxylated UA. This suggests that P. flocculosa Ahd1 is not involved in flocculosin hydroxylation.

Construction of AE ring system for the C19-diterpenoid alkaloids with a 5β-hydroxyl group

An AE azabicyclic fragment, with a β-hydroxyl group at C-5 and a substituent at C-1, of the C19-diterpenoid alkaloids, was synthesized. The key reactions include an intramolecular Claisen-type condensation, double Mannich reaction, and stereoselective nucleophilic addition of carbonyl group with the assistance of steric effect of 1β-substituent.

Excited-State Intramolecular Proton Transfer in Hydroxyoxime-Based Chemical Sensors

The electronic structure of a series of β-hydroxy-oximes, with different aromatic cores (naphthalene, pyrene, coumarin, pyridine) between the oxime and the hydroxyl groups, has been investigated by time-dependent density functional theory (TDDFT) and of the naphthalene-based oxime, in addition, by resolution-of-identity second-order perturbative coupled cluster (RICC2) calculations with basis sets up to augmented triple-ζ quality. The particular systems have been proposed as fluorescent sensors of organophosphorus (OP) nerve agents, with enhancement of fluorescence accompanying the sensing of OP agents. It is found that the experimentally observed fluorescence quenching of the oxime sensors in their initial form can be attributed to intramolecular proton transfer upon excitation from the β-hydroxyl group to the nitrogen atom, thus forming a weakly emitting hydroxylaminoquinoid.

ChemInform Abstract: General Stereoselective Synthesis of (E)-exo-Alkylidene Tetrahydrofurans via Base-Mediated Cyclization of Hydroxyl Propargylic Sulfones.

Abstract The base-promoted cyclization of a number of phenyl propargylic sulfones possessing a β-hydroxyl group afforded 5-substituted (E)-2-[(benzenesulfonyl)methylene]tetrahydrofurans 3 in >80% yield. The phenyl allenic sulfone, formed by isomerization of the phenyl propargylic sulfone under the basic conditions, is attacked by the internal alkoxide to give an allylic anion which undergoes protonation to form the conjugated exo double bond.

Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15

The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3) were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD+-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienyl)serine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO2.

The effect of stereochemistry on the thermodynamic characteristics of the binding of fenoterol stereoisomers to the β2-adrenoceptor

The binding thermodynamics of the stereoisomers of fenoterol, (R,R′)-, (S,S′)-, (R,S′)-, and (S,R′)-fenoterol, to the β2-adrenergic receptor (β2-AR) have been determined. The experiments utilized membranes obtained from HEK cells stably transfected with cDNA encoding human β2-AR. Competitive displacement studies using [3H]CGP-12177 as the marker ligand were conducted at 4, 15, 25, 30 and 37°C, the binding affinities calculated and the standard enthalpic (ΔH°) and standard entropic (ΔS°) contribution to the standard free energy change (ΔG°) associated with the binding process determined through the construction of van’t Hoff plots. The results indicate that the binding of (S,S′)- and (S,R′)-fenoterol were predominately enthalpy-driven processes while the binding of (R,R′)- and (R,S′)-fenoterol were entropy-driven. All of the fenoterol stereoisomers are full agonists of the β2-AR, and, therefore, the results of this study are inconsistent with the previously described “thermodynamic agonist–antagonist discrimination”, in which the binding of an agonist to the β-AR is entropy-driven and the binding of an antagonist is enthalpy-driven. In addition, the data demonstrate that the chirality of the carbon atom containing the β-hydroxyl group of the fenoterol molecule (the β-OH carbon) is a key factor in the determination of whether the binding process will be enthalpy-driven or entropy-driven. When the configuration at the β-OH carbon is S the binding process is enthalpy-driven while the R configuration produces an entropy-driven process.

Computational Methods for Mapping Ligand Induced Conformational Changes in GPCRs

The active conformation of a G-Protein Coupled Receptor (GPCR) is influenced by the chemical structure and efficacy of the bound ligand. Insight into the active conformation and activation pathway for ligands with different efficacies is critical in designing functionally specific drugs for GPCRs. Using the computational method LITiCon, we have predicted the activation pathways for full, partial and inverse agonists of β2 Adrenergic Receptor, which are in agreement with fluorescence intensity lifetime measurements. Starting from the inactive state, the full agonists take the receptor to a stable intermediate in an energy downhill step (fast step), followed by a barrier crossing leading to the active state (slow step). The reasons for the energy barrier are the disruption of a HB between N293 on TM6 and S204 on TM5 as well as the one between D113 on TM3 and the β-hydroxyl group on the ligand. During MD simulation of norepinephrine bound β2-AR, water polarization helps to disrupt the HB between D113 and β-hydroxyl thus facilitating the barrier crossing. In contrast with the full agonists, the activation pathway for the partial agonist salbutamol involves only a slow step, which stems from steric interactions between the ligand and the aromatic residues on TM6.We have further refined the LITiCon method to optimize the backbone tilts and kinks of the transmembrane helices in the presence of a bound ligand. We will present the details of the method and the results on validation of rhodopsin activation by all-trans retinal, and β2-AR bound to various agonists to investigate the modulation of helical kinks and tilts due to agonist binding.

Characterization of Thr-354 in the human sodium/iodide symporter (NIS) by site-directed mutagenesis

Sodium/iodide symporter (NIS) is the key molecule concentrating iodide in the thyroid gland. The first-described human NIS (hNIS) mutation to cause a complete iodide transport defect was the T354P mutation. The Thr-354 lies in the midst of the putative ninth transmembrane segment which is well-conserved within the members of the SLC5A transporter family. Here we have investigated the molecular function of Thr-354 using site-directed mutagenesis and found that T354S and T354A mutations result in significantly decreased iodide transport activity, 50 % and 2 % of wild-type hNIS. Our findings indicate that whereas Thr-354 is indispensable for the complete NIS activity, the β-hydroxyl group accounts for half, and the α-helical structure alone contributes for one-fiftieth of wild-type hNIS activity.

Efficient and stereoselective synthesis of threo-β-hydroxy- l-glutamic acid via a tandem ( Z)-olefination-conjugate addition

threo-β-Hydroxy- l-glutamic acid 1 is an attractive target as a biologically active compound and as a chiral synthon. The required β-hydroxyl group in 1 was efficiently and stereoselectively introduced via an intramolecular conjugate addition of the N-hydroxymethyl group of γ -amino -α ,β-unsaturated ( Z) -ester 4. While the corresponding ( E)-ester 3 gave a lower selectivity of ca. 5:1 in the intramolecular conjugate addition, a selectivity of up to 70:1 was shown with ( Z)-ester 4. The tandem ( Z)-olefination-conjugate addition could be achieved by simply changing the reaction conditions to give a selectivity of >20:1. Thus, the target compound 1 was obtained as its hydrochloride salt in 70% overall yield over four steps from lactol 2.

Hydroxy Oximes as Organophosphorus Nerve Agent Sensors

Find and destroy: A series of oximes were constructed to simultaneously detect and detoxify organophosphorus-based nerve agents. They function as optical sensors employing the oxime reactivity and incorporating a β-hydroxyl group to undergo an intramolecular cyclization from the intermediate oxime–organophosphorus species. The generated isoxazole produces an enhanced fluorescent signal that reports on the presence and destruction of the nerve agent.

Analysis of Two Additional Signaling Molecules in Streptomyces coelicolor and the Development of a Butyrolactone-Specific Reporter System

gamma-Butyrolactone bacterial hormones regulate antibiotic production and morphological differentiation in Streptomyces species. One gamma-butyrolactone, SCB1, has been previously characterized in Streptomyces coelicolor. Here we report the characterization of two additional gamma-butyrolactones, named SCB2 (2-[1'-hydroxyoctyl]-3-hydroxymethylbutanolide) and SCB3 (2-[1'-hydroxy-6'-methyloctyl]-3-hydroxymethylbutanolide), possessing an antibiotic stimulatory activity. To elucidate the specificity determinants of these ligands for the receptor protein, ScbR, 30 chemically synthesized gamma-butyrolactone analogs were tested by utilizing the release of ScbR from DNA upon binding to a gamma-butyrolactone, which can be detected by kanamycin resistance. The butyrolactone detection method developed here revealed that ScbR shows preference toward a ligand possessing a 7-10 carbon C-2 side chain, a C-1'-beta-hydroxyl group, and a C-6'-methyl branch that coincides with SCB3. Moreover, this method was successfully used to screen for potential gamma-butyrolactone producers from commercial-antibiotic-producing Streptomyces.

Asparaginyl β-Hydroxylation of Proteins Containing Ankyrin Repeat Domains Influences Their Stability and Function

Recent reports have provided evidence that the β-hydroxylation of conserved asparaginyl residues in ankyrin repeat domain (ARD) proteins is a common posttranslational modification in animal cells. Here, nuclear magnetic resonance (NMR) and other biophysical techniques are used to study the effect of asparaginyl β-hydroxylation on the structure and stability of ‘consensus’ ARD proteins. The NMR analyses support previous work suggesting that a single β-hydroxylation of asparagine can stabilize the stereotypical ARD fold. A second asparaginyl β-hydroxylation causes further stabilization. In combination with mutation studies, the biophysical analyses reveal that the stabilizing effect of β-hydroxylation is, in part, mediated by a hydrogen bond between the asparaginyl β-hydroxyl group and the side chain of a conserved aspartyl residue, two residues to the N-terminal side of the target asparagine. Removal of this hydrogen bond resulted in reduced stabilization by hydroxylation. Formation of the same hydrogen bond is also shown to be a factor in inhibiting binding of hydroxylated ARDs to factor-inhibiting hypoxia-inducible factor (FIH). The effects of hydroxylation appear to be predominantly localized to the target asparagine and proximal residues, at least in the consensus ARD protein. The results reveal that thermodynamic stability is a factor in determining whether a particular ARD protein is an FIH substrate; a consensus ARD protein with three ankyrin repeats is an FIH substrate, while more stable consensus ARD proteins, with four or five ankyrin repeats, are not. However, NMR studies reveal that the consensus protein with four ankyrin repeats is still able to bind to FIH, suggesting that FIH may interact in cells with natural ankyrin repeats without resulting hydroxylation. Overall, the work provides novel biophysical insights into the mechanism by which asparaginyl β-hydroxylation stabilizes the ARD proteins and reduces their binding to FIH.

Conformational structure of propranolol: A β-adrenergic blocking drug studied by NMR and PCILO methods

The conformational structure of propranolol, a β-adrenergic blocking drug, has been investigated by pcilo calculations and 270-MHz proton nuclear magnetic resonance in D2O solution. The molecules coexist in at least two conformational states in solution with a low energy barrier. Both preferred conformations have extended structures which allow a three-point drug-receptor binding involving the aromatic moiety, the β-hydroxyl group, and -NH+2 groups of propranolol. The previously postulated “rigid” bicyclic structure does not exist to an appreciable extent in D2O solution.