Synthesis of β-alanine from l-aspartate using l-aspartate-α-decarboxylase from Corynebacterium glutamicum

β-Alanine is mainly produced by chemical methods in current industrial processes. Here, panD from Corynebacterium glutamicum encoding l-aspartate-α-decarboxylase (ADC) was cloned and expressed in Escherichia coli BL21(DE3). ADC _C.g catalyzes the α-decarboxylation of l-aspartate to β-alanine. The purified ADC _C.g was optimal at 55 °C and pH 6 with excellent stability at 16–37 °C and pH 4–7. A pH–stat directed, fed-batch feeding strategy was developed for enzymatic synthesis of β-alanine to keep the pH value within 6–7.2 and thus attenuate substrate inhibition. A maximum conversion of 97.2 % was obtained with an initial 5 g l-aspartate/l and another three feedings of 0.5 % (w/v) l-aspartate at 8 h intervals. The final β-alanine concentration was 12.85 g/l after 36 h. This is the first study concerning the enzymatic production of β-alanine by using ADC.     

Expression of Bacterial l-aspartate-α-decarboxylase in Tobacco Increases β-Alanine and Pantothenate Levels and Improves Thermotolerance

l-Aspartate-α-decarboxylase catalyzes the decarboxylation of l-aspartate to generate β-alanine and carbon dioxide. This is an unusual pyruvoyl-dependent enzyme unique to prokaryotes that undergoes limited self-processing. The Escherichia coli panD gene encoding l-aspartate-α-decarboxylase was expressed under a constitutive promoter in transgenic tobacco. Transgene expression was verified by assays based on RNA blots, immunoblots and enzyme activity in vitro. The panD lines had increased levels of leaf β-alanine (1.2- to 4-fold), pantothenate (3.2- to 4.1-fold) and total free amino acids (up to 3.7-fold) compared to wild-type and vector controls. Growth of homozygous lines expressing E. coli l-aspartate-α-decarboxylase was less affected than that of the control lines when the plants were stressed for 1 week at 35 °C. When transferred from 35 to 30 °C for 3 weeks, the PanD transgenic lines recovered significantly (P ≤ 0.001) better than the controls: PanD lines had on an average 54% and 84% greater fresh and dry weights respectively, compared to the controls. Homozygous lines expressing E. coli l-aspartate-α-decarboxylase had significantly greater thermotolerance (P ≤ 0.05) during germination. At 42 °C, 95% of two T3 PanD transgenic line seeds germinated after 12 days compared to 73% for the wild-type seeds. Our results indicated that E. colil-aspartate-α-decarboxylase was correctly processed and active in the transgenic eukaryotic host and its expression resulted in increased thermotolerance in tobacco. This is Florida Agricultural Experiment Station journal series number R-10355. W.M.F. was supported by the Egypt Development Training fellowship and by the UF College of Agriculture and Life Sciences assistantship.     

α-Trifluoromethylhistamine: A mechanism-based inhibitor of mammalian histidine decarboxylase

α-Trifluoromethylhistamine (1), proposed as a suicide inhibitor of histidine decarboxylase, has been prepared from β-trifluoromethyl-β-alanine. Histidine decarboxylase from hamster placenta is inhibited in a time-dependent manner by 1; however, the adduct formed between inhibitor and enzyme is labile. 1 inhibits stomach histidine decarboxylase activity in vivo in rats, but has no antisecretory effect in the pyloric-ligated stomach of the mouse.     

Approaches to the simultaneous inactivation of metallo- and serine-β-lactamases

A series of cephalosporin-derived reverse hydroxamates and oximes were prepared and evaluated as inhibitors of representative metallo- and serine-β-lactamases. The reverse hydroxamates showed submicromolar inhibition of the GIM-1 metallo-β-lactamase. With respect to interactions with the classes A, C, and D serine β-lactamases, as judged by their correspondingly low K_m values, the reverse hydroxamates were recognized in a manner similar to the non-hydroxylated N–H amide side chains of the natural substrates of these enzymes. This indicates that, with respect to recognition in the active site of the serine β-lactamases, the OC–NR–OH functionality can function as a structural isostere of the OC–NR–H group, with the N–O–H group presumably replacing the amide N–H group as a hydrogen bond donor to the appropriate backbone carbonyl oxygen of the protein. The reverse hydroxamates, however, displayed k_cat values up to three orders of magnitude lower than the natural substrates, thus indicating substantial slowing of the hydrolytic action of these serine β-lactamases. Although the degree of inactivation is not yet enough to be clinically useful, these initial results are promising. The substitution of the amide N–H bond by N–OH may represent a useful strategy for the inhibition of other serine hydrolases.     

Nuclear magnetic resonance investigation of the conformation of δ-haemolysin bound to dodecylphosphocholine micelles

δ-Haemolysin in mixed micelles with perdeuterated dodecylphosphocholine was investigated with two-dimensional proton nuclear magnetic resonance experiments at 500 MHz. A single set of resonance lines was observed for the micelle-bound polypeptide, indicating that δ-haemolysin adopts a single conformation in this environment. Nearly complete, sequence-specific assignments were obtained for the segment 5–23 of this 26 residue polypeptide chain. From the sequential connectivities and numerous medium-range nuclear Overhauser effects this central portion of the molecule was found to form an extended helix with pronounced amphipathic distribution of polar and nonpolar amino acid side-chains.     

Glucocorticoids relieve collectin-driven suppression of apoptotic cell uptake in murine alveolar macrophages through downregulation of SIRPα

The lung environment actively inhibits apoptotic cell (AC) uptake by alveolar macrophages (AM?s) via lung collectin signaling through signal regulatory protein α (SIRPα). Even brief glucocorticoid (GC) treatment during maturation of human blood monocyte-derived or murine bone marrow-derived macrophages (M?s) increases their AC uptake. Whether GCs similarly impact differentiated tissue M?s and the mechanisms for this rapid response are unknown and important to define, given the widespread therapeutic use of inhaled GCs. We found that the GC fluticasone rapidly and dose-dependently increased AC uptake by murine AM?s without a requirement for protein synthesis. Fluticasone rapidly suppressed AM? expression of SIRPα mRNA and surface protein, and also activated a more delayed, translation-dependent upregulation of AC recognition receptors that was not required for the early increase in AC uptake. Consistent with a role for SIRPα suppression in rapid GC action, murine peritoneal M?s that had not been exposed to lung collectins showed delayed, but not rapid, increase in AC uptake. However, pretreatment of peritoneal M?s with the lung collectin surfactant protein D inhibited AC uptake, and fluticasone treatment rapidly reversed this inhibition. Thus, GCs act not only by upregulating AC recognition receptors during M? maturation but also via a novel rapid downregulation of SIRPα expression by differentiated tissue M?s. Release of AM?s from inhibition of AC uptake by lung collectins may, in part, explain the beneficial role of inhaled GCs in inflammatory lung diseases, especially emphysema, in which there is both increased lung parenchymal cell apoptosis and defective AC uptake by AM?s.     

Identification of an “α-helix-extended segment-α-helix” conformation of the sixth transmembrane domain in DMT1

DMT1 (divalent metal ion transporter 1) is one member of a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions. A pair of mutation-sensitive and highly conserved histidines in the sixth transmembrane domain (TM6) of DMT1 was found to be important for proton-metal ion cotransport. In the present work, we investigate the structures and locations of the peptides from TM6 of DMT1 and its H267A and H272A mutants in SDS micelles by CD and NMR methods. The circular dichroism studies show that the α-helix is a predominant conformation for the wildtype peptide and H267A mutant in SDS micelles, whereas the helicity is evidently decreased for H272A mutant. The pH value has little effect on the α-helical contents of the three peptides. The NMR studies indicate that the wildtype peptide in SDS micelles forms an “α-helix-extended segment-α-helix” structure in which the His267 locates near the central part of the extended segment, while the His272 is involved in the α-helical folding. Both histidines are buried in SDS micelles as evidenced by their pK_a values. The structure of the wildtype peptide is evidently changed by the mutations of H267A and H272A. The H267A mutant forms an ordered structure consisting of an α-helix from the C-terminus to the central part and continuous turns in the residual part. The extended structure in the central part of the wildtype peptide is abolished by H267A mutation. The H272A mutation mainly induces unfolding of the short helix in the N-terminal side, while the short helix in the C-terminal side and unordered conformation in the central part remain. All the three peptides are embedded in SDS micelles, and the H267A mutant is inserted more deeply due to increasing hydrophobicity in the central part of the peptide. The specific “α-helix-extended segment-α-helix” structure of TM6 may have an important implication for the binding of the transporter to H⁺ and metal ions and the conformation change induced by the mutations of two highly conserved histidines may be correlated to the deficiency of the transport activity of DMT1.     

The unusual conformation of cross-strand disulfide bonds is critical to the stability of β-hairpin peptides

The cross-strand disulfides (CSDs) found in β-hairpin antimicrobial peptides (β-AMPs) show a unique disulfide geometry that is characterized by unusual torsion angles and a short Cα-Cα distance. While the sequence and disulfide bond connectivity of disulfide-rich peptides is well studied, much less is known about the disulfide geometry found in CSDs and their role in the stability of β-AMPs. To address this, we solved the nuclear magnetic resonance (NMR) structure of the β-AMP gomesin (Gm) at 278, 298, and 310 K, examined the disulfide bond geometry of over 800 disulfide-rich peptides, and carried out extensive molecular dynamics (MD) simulation of the peptides Gm and protegrin. The NMR data suggests Cα-Cα distances characteristic for CSDs are independent of temperature. Analysis of disulfide-rich peptides from the Protein Data Bank revealed that right-handed and left-handed rotamers are equally likely in CSDs. The previously reported preference for right-handed rotamers was likely biased by restricting the analysis to peptides and proteins solved using X-ray crystallography. Furthermore, data from MD simulations showed that the short Cα-Cα distance is critical for the stability of these peptides. The unique disulfide geometry of CSDs poses a challenge to biomolecular force fields and to retain the stability of β-hairpin fold over long simulation times, restraints on the torsion angles might be required.     

On the conformation of proteins: The handedness of the β-strand-α-helix-β-strand unit

The β-strand-α-helix-β-strand unit consists of two parallel, but not necessarily adjacent, β-strands which lie in a β-pleated sheet and are connected by one or more α-helices. This unit, which occurs in 17 functionally different globular proteins, may adopt a right- or a left-handed conformation. An analysis of the distribution shows that 57 out of the 58 units are right-handed. If the unit had no right-handed preference, the probability of observing such a distribution by chance is 10^?16. This may be explained in terms of the twist of the β-sheet which is shown to favour a right-handed unit, as otherwise steric hindrance occurs in the loop regions. We show that the right-handed strand-helix-strand unit determines the sense of the super-secondary structure found in the dehydrogenases and of related folds found in other structures. The evolutionary relationships between proteins containing this unit are re-evaluated in terms of this preference. The high probability that the unit will fold with a right-handed conformation has implications for the prediction of tertiary structure.     

A single amino acid of human immunodeficiency virus type 2 capsid protein affects conformation of two external loops and viral sensitivity to TRIM5α

We previously reported that human immunodeficiency virus type 2 (HIV-2) carrying alanine or glutamine but not proline at position 120 of the capsid protein (CA) could grow in the presence of anti-viral factor TRIM5α of cynomolgus monkey (CM). To elucidate details of the interaction between the CA and TRIM5α, we generated mutant HIV-2 viruses, each carrying one of the remaining 17 possible amino acid residues, and examined their sensitivity to CM TRIM5α-mediated restriction. Results showed that hydrophobic residues or those with ring structures were associated with sensitivity, while those with small side chains or amide groups conferred resistance. Molecular dynamics simulation study revealed a structural basis for the differential TRIM5α sensitivities. The mutations at position 120 in the loop between helices 6 and 7 (L6/7) affected conformation of the neighboring loop between helices 4 and 5 (L4/5), and sensitive viruses had a common L4/5 conformation. In addition, the common L4/5 structures of the sensitive viruses were associated with a decreased probability of hydrogen bond formation between the 97th aspartic acid in L4/5 and the 119th arginine in L6/7. When we introduced aspartic acid-to-alanine substitution at position 97 (D97A) of the resistant virus carrying glutamine at position 120 to disrupt hydrogen bond formation, the resultant virus became moderately sensitive. Interestingly, the virus carrying glutamic acid at position 120 showed resistance, while its predicted L4/5 conformation was similar to those of sensitive viruses. The D97A substitution failed to alter the resistance of this particular virus, indicating that the 120th amino acid residue itself is also involved in sensitivity regardless of the L4/5 conformation. These results suggested that a hydrogen bond between the L4/5 and L6/7 modulates the overall structure of the exposed surface of the CA, but the amino acid residue at position 120 is also directly involved in CM TRIM5α recognition.     

Antithrombin conformation and the catalytic role of heparin. II. Is the heparin-induced conformational change in antithrombin required for rapid inactivation of thrombin?

The role of antithrombin conformation in heparin-catalyzed inhibition of thrombin was investigated using antithrombins modified with the tryptophan reagent dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNB). Affinity fractionation of HNB-labeled antithrombin (0.6-0.7 mol of HNB/mol of protein) on heparin-Sepharose using a linear salt gradient allowed separation of three singly labeled protein species and a fourth HNB-antithrombin species which co-eluted with unlabeled protein. Conformational alterations induced by heparin binding to each of the labeled antithrombins were assessed by spectroscopic techniques, including protein fluorescence, difference spectroscopy in the ultraviolet-visible range, and circular dichroism. Comparison of spectra of the labeled proteins in the presence and absence of added heparin indicated changes to occur in protein conformation at the sites of the bound HNB moieties and at aromatic amino acid residues within the protein matrix. These spectroscopic alterations mimicked changes induced by heparin in the native protein, but were reduced in magnitude. Rates of thrombin inactivation by the labeled antithrombins were measured over a wide range in both heparin concentration and inhibitor concentration to determine maximal rates of protease inactivation. The kinetic analysis indicated that each of these HNB-antithrombin derivatives, which undergo the heparin-induced changes to varying extents, can react with thrombin at the same maximal rate. Thus, this series of chemically modified antithrombin species demonstrated that the conformational change which is induced in antithrombin by heparin does not render the protein intrinsically more reactive toward thrombin.     

Genetic Engineering of Tobacco with Double Resistance to Both Virus and Insect

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Engineering of a fungal β-galactosidase to remove product inhibition by galactose

β-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent in most world regions. The gene encoding an acid-stable β-galactosidase potentially suited for use as a digestive supplement was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition, a model of the enzyme structure was generated based on a Penicillium sp. β-galactosidase crystal structure with bound β-galactose. This led to targeted mutagenesis of an Asp²⁵⁸-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant β-galactosidase enzyme with reduced sensitivity to inhibition by galactose (K _i of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased K _m (3.76 mM compared to 2.21 mM) and reduced V _max (110.8 μmol min⁻¹ mg⁻¹ compared to 172.6 μmol min⁻¹ mg⁻¹) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties for treatment of lactose intolerance.     

Complexation of peptide with Cu2+ responsible to inducing and enhancing the formation of α-helix conformation

Role of some metal ions on the conformations of peptides was examined by using a series of short alanine-based peptides with single Trp-His (W-H) interaction in different environments. Circular dichroism (CD), Trp (W) fluorescence emission, and Fourier transform infrared (FTIR) spectroscopy revealed that there is a conformational role of Cu²⁺ in inducing and enhancing the formation of -helix conformation. The complexation of the peptide with Cu²⁺ is responsible to the conformational effect because the chelation is able to stabilize peptide with an -helix conformation. The possible factors affecting the role of Cu²⁺ are discussed in the paper. The results in this paper are useful to understand the important structural role of Cu²⁺ in protein folding and the possible mechanism in some neurodegenerative diseases such as Alzheimer's disease.     

Proteolytic inactivation of 1, 4-α-d-glucan 6-α-d-glucosyltransferase purified from Aspergillus niger R-27

An extracellular 1,4-α-d-glucan 6-α-d-glucosyltransferase [d-glucosyltransferase, 1,4-α-d-glucan:1,4-α-d-glucan(d-gluco 6-α-d-glucosyltransferase, EC 2.4.1.24] from Aspergillus niger R-27 has been purified and the kinetics of its proteolytic inactivation with subtilisin studied. The purified enzyme was shown to be homogeneous using disc polyacrylamide gel electrophoresis. It contained 16.0% mannose, 0.19% glucose and 2.95% 2-acetamido-2-deoxy-d-glucose. The characteristic feature of the proteolytic degradation of glucosyltransferase is rapid hydrolysis of ～12 peptide bonds per mol and the formation of an active intermediate product which is more resistant to further proteolysis, but is easily heat-inactivated. The isolation and some properties of glucosyltransferase are also described.     

Synergistic inactivation of AXL: a (cross)road to cure ovarian cancer?

Inhibition of the receptor tyrosine kinase AXL, a key molecular driver of ovarian cancer, has recently been highlighted as promising therapeutic strategy. In this issue of EMBO Reports, Antony et al 1 have identified a novel mechanism of inhibition of AXL, wherein the GPI‐anchored tumour suppressor OPCML sequesters AXL into specialised plasma membrane domains where the phosphatase PTPRG is located, therefore facilitating AXL dephosphorylation. This attenuation of AXL signalling has translational implications for the design of synergistic therapies, to target the kinase for this aggressive malignancy.     

4,4,14α-trimethyl 9β,19-cyclo-5α-26-homocholesta-24,26-dien-3β-ol: a potent mechanism-based inactivator of Δ- to Δ-sterol methyl transferase

The title compound (4A) was synthesized and tested as a mechanism-based inactivator of the sterol methyl transferase (SMT) enzyme from Prototheca wickerhamii. Using cycloartenol as substrate, 4A was found to exhibit time-dependent inactivation kinetics, generating a K_i value of 30 μM and K_inact value of 0.30 min⁻¹.