Does the chromophore's ring move after photoexcitation of rhodopsin?

By comparing the shift of the absorption maxima when a visual pigment is converted to its lumirhodopsin photointermediate for two classes of pigments, we can infer whether or not the pigment's beta-ionone ring has left its binding site. We compare this shift for the long-wavelength sensitive visual pigment of chicken iodopsin (lambdamax = 571 nm), which has polar residues in the ring binding site that interact with the ring, with that for three pigments, which do not. We conclude that by the time the Lumi product of the pigment is formed, the ring has moved away from the ring binding site.     

Allelic characteristics of the β-amylase gene of barley varieties in Ukraine

Allelic diversity in a set of 99 spring and winter barley varieties intended for different use (malting, cereal, and valuable) was studied. PCR analysis with the use of the β-amylase DNA marker showed that genotypes of different barley varieties may include different alleles of the β-amy1 gene.     

Enzymatic production and in?situ separation of natural β-ionone from β-carotene

A biotechnological process concept for generation and in?situ separation of natural β-ionone from β-carotene is presented. The process employs carotenoid cleavage dioxygenases (CCDs), a plant-derived iron-containing nonheme enzyme family requiring only dissolved oxygen as cosubstrate and no additional cofactors. Organophilic pervaporation was found to be very well suited for continuous in?situ separation of β-ionone. Its application led to a highly pure product despite the complexity of the reaction solution containing cell homogenates. Among three different pervaporation membrane types tested, a polyoctylmethylsiloxane active layer on a porous polyetherimide support led to the best results. A laboratory-scale demonstration plant was set up, and a highly pure aqueous–ethanolic solution of β-ionone was produced from β-carotene. The described process permits generation of high-value flavor and fragrance compounds bearing the desired label “natural” according to US and European food and safety regulations and demonstrates the potential of CCD enzymes for selective oxidative cleavage of carotenoids.     

Roles of β-d-ribofuranose ring and the functional groups of the d-ribose moiety of adenosylcobalamin in the diol dehydratase reaction

Four analogs of adenosylcobalamin (AdoCbl) modified in the d-ribose moiety of the Coβ ligand were synthesized, and their coenzyme properties were studied with diol dehydratase of Klebsiella pneumoniae ATCC 8724. 2′-Deoxyadenosylcobalamin (2′-dAdoCbl) and 3′-deoxyadenosylcobalamin (3′-dAdoCbl) were active as coenzyme. 2′,3′-Secoadenosylcobalamin (2′,3′-secoAdoCbl), an analog bearing the same functional groups as AdoCbl but nicked between the 2′ and 3′ in the ribose moiety, and its 2′,3′-dialdehyde derivative (2′,3′-secoAdoCbl dialdehyde) were totally inactive analogs of the coenzyme. It is therefore evident that the β-d-ribofuranose ring itself, possibly its rigid structure, is essential and much more important than the functional groups of the ribose moiety for coenzyme function (relative importance; β-d-ribofuranose ring ⪢ 3′-OH ⪢ 2′-OH ⪢ ether group). With 2′-dAloCbl and 3′-dAdoCbl as enzymes. an absorption peak at 478 nm appeared during enzymatic reaction, suggesting homolysis of the CCo bound to form cob(II)alamin as intermediate. In the absence of substrate, the complexes of the enzyme with these active analogs underwent rapid inactivation by oxygen. This suggests that their CCo bond is activated even in the absence of substrate by binding to the apoprotein. No significant spectral changes were observed with 2′,3′-secoAdoCbl upon binding to the apoenzyme. In contrast, spectroscopic observation indicates that 2′,3′-secoAdoCbl dialdehyde, another inactive analog, underwent gradual and irreversible cleavage of the CCo bond by interaction with the apodiol dehydratase, forming the enzyme-bound cob(II)alamin without intermediates.     

Structural Determinants of the β-Selectivity of a Bacterial Aminotransferase

Chiral β-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure β-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-β-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a β-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the α-carboxylate group of an α-amino acid. A β-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both β- and α-amino acids.     

The performance of dogs in detecting α-ionone in the vapor phase

Summary Concentration-response functions for-ionone were established for four German shepherds (Figs. 3 and 4). The dogs were tested in a three choice behavioral apparatus (Fig. 2), and trained, by a restricted operant procedure, to establish an approach-avoidance discrimination between odor and air. Odorant concentrations were presented from an olfactometer calibrated by gas chromatography. Minimum detectable concentrations fell within the range 4.0×10^4.5–4.0×10^6.5 molecules/cm³. Concentration-response curves for three of the dogs show a clear double reversal in slope which is statistically significant and which divides the curve into a slowly descending upper limb, best fitted by a parabolic function, and a rapidly descending lower limb, best fitted by a cubic function (Figs. 5 and 6). There is evidence that this division might reflect a dual receptor mechanism. When performance during testing stabilized for a given concentration and the concentration was then lowered by a certain magnitude, the new performance level depended markedly on that magnitude. No evidence was found to suggest that adaptation to the test odor influenced performance. As defined by a differential threshold fraction (Flow/Flow) one dog's ability to discriminate between differences in flow rate fell in the range 0.12–0.08.We thank the U.S. Air Force, Air Force Office of Scientific Research for their generous support of this work (Grant No. 73-2425 to D.G.Moulton).-ionone was donated by Givaudan Inc. (Dr. J. Dorsky). We also thank Mr. Paul Phillips for able technical assistance and Dr. T. Burlingame for gas chromatographic measurements.     

Two species of the Rhodiola yunnanensis species complex distributed around the Sichuan Basin of China: Speciation in a ring?

A ring species consists of two reproductively isolated forms connected by a chain of intergrading populations encircling a geographic barrier. The mountains encircling the Sichuan Basin in southwestern China harbor great species diversity and endemism, and they are candidate regions for ring species. Here, we examined a potential ring species complex with a ring distribution surrounding the Sichuan Basinand the reproductive barrier between sibling species Rhodiola yunnanensis and R. henryi of the R. yunnanensis complex. In this study, we test the hypothesis that R. yunnanensis and R. henryi diverged by the ring-species model using an amplicon sequencing strategy targeting the introns of 27 single-copy nuclear genes and 14 chloroplast DNA sequences. Our studies indicated that the R. yunnanensis complex is monophyletic, originates at the late Miocene, and forms its current ring distribution pattern after the LIG. In addition, clear genetic intergradation was not found among R. henryi populations within the distribution ring. All these findings suggest that the divergence of two sibling species was not driven by the geographic isolation, and they were not originated from the ring-species model; however, the basin-surrounding distribution pattern and reproductive barrier between them meet some criteria for being a ring species.     

Methylation of the ß-positions of the furan ring in F-acids

Major incubation products in feeding experiments with the sodium salt of 7-(5-butyl-furan-2-yl)heptanoic acid (3) on suspension cultures of Saccharum spec. are the unusual F-acids (4a) and (4b). They possess in contrast to natural monomethyl substituted F-acids a methyl substituent in the 4-position of the furan ring. Unexpectedly, the dimethyl substituted F-acids (4c) and (4d) were found only in very small amounts. The detection and structure elucidation of the methylation products (4a)–(4d) was achieved predominantly by GC-MS analysis of the corresponding tetrahydrofuran derivatives (5a)–(5d).     

Preliminary studies on the characteristics of phenylalanine and β-methyl-glucoside transport in the tench intestine in vitro

• 1.1. The unidirectional transepithelial fluxes of L-phenylalanine, β-methyl-D-glucoside and sodium ions across emusculated sheets of tench mid-intestine were determined in flux chambers.
• 2.2. No net sodium flux was detectable, but phenylalanine was preferentially transferred from the mucosal to the serosal fluid.
• 3.3. There was also a net movement of β-methyl-glucoside towards the serosal medium, but it was much smaller than that of phenylalanine.
• 4.4. This transport was accompanied by an accumulation of each substrate from the mucosal medium into the tissue to a similar level and against a concentration gradient.
• 5.5. The poor transfer of the monosaccharide into the serosal medium could therefore be attributed to a low permeability of the baso-lateral membrane of the enterocyte for this substance.
• 6.6. The influx of L-phenylalanine and of β-methyl-d-glucoside into the epithelial cells of tench midintestine was examined by incubating slices of emusculated intestine in radioactively-labelled solutions of the substrate for 2 min.
• 7.7. The steady-state uptake was assessed after similar incubations lasting 45 min.
• 8.8. Phenylalanine influx obeys the Michaelis-Menten equation with a K_m of 2.9 mM and is dependent on the presence of sodium ions in the incubation medium.
• 9.9. β-Methyl-glucoside influx reveals the same characteristics with a K_m of 2.0 mM but a considerably lower V_max; in addition, it is inhibited by galactose.
• 10.10. The influx of both substrates is reduced by harmaline, which also inhibits the uptake of radioactive sodium by this preparation.
• 11.11. The steady-state uptake of β-methyl-glucoside is also inhibited by ouabain and by 2.4-dinitrophenol.
• 12.12. These results suggest that the mechanisms for sodium-dependent influx of monosaccharides and neutral amino-acids in the tench intestine are similar to those found in mammalian tissues.
• 13.13. The principal difference appears to involve the release of monosaccharides across the baso-lateral membrane of the enterocyte.

     

C4-Phenylthio β-lactams: Effect of the chirality of the β-lactam ring on antimicrobial activity

C4-phenylthio β-lactams are a new family of antibacterial agents that have activity against two phylogenetically distant bacteria – Mycobacterium tuberculosis (Mtb) and Moraxella catarrhalis (M. cat). These compounds are effective against β-lactamase producing Mtb and M. cat unlike the clinically relevant β-lactam antibiotics. The structure-activity relationship for the C4 phenylthio β-lactams has not yet been completely defined. Earlier efforts in our laboratories established that the C4-phenylthio substituent is essential for antimicrobial activity, while the N1 carbamyl substituent plays a more subtle role. In this present study, we investigated the role that the stereochemistry at C4 plays in these compounds’ antibacterial activity. This was achieved by synthesizing and testing the antimicrobial activity of diastereomers with a chiral carbamyl group at N1. Our findings indicate that a strict stereochemistry for the C4-phenylthio β-lactams is not required to obtain optimal anti-Mtb and anti-M. cat activity. Furthermore, the structure–bioactivity profiles more closely relate to the electronic requirement of the phenylthiogroup. In addition, the MICs of Mtb are sensitive to growth medium composition. Select compounds showed activity against non-replicating and multi-drug resistant Mtb.     

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (T_m). Mutation of His299 to Tyr increased the optimal temperature, the T_m, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures.     

Structural dynamics of the amyloid β-protein monomer folding nucleus

Alzheimer's disease (AD) is linked to the aberrant assembly of the amyloid β-protein (Aβ). The (21)AEDVGSNKGA(30) segment, Aβ(21-30), forms a turn that acts as a monomer folding nucleus. Amino acid substitutions within this nucleus cause familial forms of AD. To determine the biophysical characteristics of the folding nucleus, we studied the biologically relevant acetyl-Aβ(21-30)-amide peptide using experimental techniques (limited proteolysis, thermal denaturation, urea denaturation followed by pulse proteolysis, and electron microscopy) and computational methods (molecular dynamics). Our results reveal a highly stable foldon and suggest new strategies for therapeutic drug development.     

Structural Basis for the Function of the β-Barrel Assembly-Enhancing Protease BepA

The β-barrel assembly machinery (BAM) complex mediates the assembly of β-barrel membrane proteins in the outer membrane. BepA, formerly known as YfgC, interacts with the BAM complex and functions as a protease/chaperone for the enhancement of the assembly and/or degradation of β-barrel membrane proteins. To elucidate the molecular mechanism underlying the dual functions of BepA, its full-length three-dimensional structure is needed. Here, we report the crystal structure of full-length BepA at 2.6-Å resolution. BepA possesses an N-terminal protease domain and a C-terminal tetratricopeptide repeat domain, which interact with each other. Domain cross-linking by structure-guided introduction of disulfide bonds did not affect the activities of BepA in vivo, suggesting that the function of this protein does not involve domain rearrangement. The full-length BepA structure is compatible with the previously proposed docking model of BAM complex and tetratricopeptide repeat domain of BepA.     

Structural basis of specificity in tetrameric Kluyveromyces lactis β-galactosidase

β-Galactosidase or lactase is a very important enzyme in the food industry, being that from the yeast Kluyveromyces lactis the most widely used. Here we report its three-dimensional structure both in the free state and complexed with the product galactose. The monomer folds into five domains in a pattern conserved with the prokaryote enzymes of the GH2 family, although two long insertions in domains 2 and 3 are unique and related to oligomerization and specificity. The tetrameric enzyme is a dimer of dimers, with higher dissociation energy for the dimers than for its assembly. Two active centers are located at the interface within each dimer in a narrow channel. The insertion at domain 3 protrudes into this channel and makes putative links with the aglycone moiety of docked lactose. In spite of common structural features related to function, the determinants of the reaction mechanism proposed for Escherichia coli β-galactosidase are not found in the active site of the K. lactis enzyme. This is the first X-ray crystal structure for a β-galactosidase used in food processing.     

Genetics of Hemoglobin in the Deer Mouse, PEROMYSCUS MANICULATUS . I. Multiple α- and β-Globin Structural Loci

Genetic data, together with molecular structure studies, have demonstrated that the complex hemoglobin phenotypes in four subspecies of P. maniculatus are generated by at least four, and probably five, globin structural loci. The Hba and Hbc loci apparently arose by duplication of an ancestral α-type locus, while Hbb, Hbd, and Hbe apparently were derived from a β-type locus. The Hba and Hbb structural loci are electrophoretically monomorphic, while Hbc, Hbd, and Hbe are each polymorphic for at least two electrophoretic alleles. Alleles at Hbc and Hbd segregate independently. The globin products of Hbc and Hbd are electrophoretically indistinguishable; therefore, it is impossible to enumerate true gene frequencies in population surveys. Combined evidence from Peromyscus, Mus and Rattus indicates a remarkable similarity in the numbers of duplicated globin structural loci and in their linkage relationships to coat color loci.