Search

We have shown for the first time that a natural protein (human insulin) can be acylated at the N‐terminus with a β‐amino acid (H‐β3hAla‐), in a process catalyzed by the β‐peptidyl aminopeptidase 3‐2W4‐BapA. This selective modification, which could also be applied for protein labeling and tagging, should be generally useful, also to protect peptides and proteins from attack by common aminopeptidases. [ABSTRACT FROM AUTHOR]

Rationale Oxygen isotope fractionation of molecular O2 is an important process for the study of aerobic metabolism, photosynthesis, and formation of reactive oxygen species. The latter is of particular interest for investigating the mechanism of enzyme-catalyzed reactions, such as the oxygenation of organic pollutants, which is an important detoxification mechanism. Methods We developed a simple method to measure the δ18O values of dissolved O2 in small samples using automated split injection for gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). After creating a N2 headspace, the dissolved O2 partitions from aqueous solution to the headspace, from which it can be injected into the gas chromatograph. Results In aqueous samples of 10 mL and in diluted air samples, we quantified the δ18O values at O2 concentrations of 16 μM and 86 μM, respectively. The chromatographic separation of O2 and N2 with a molecular sieve column made it possible to use N2 as the headspace gas for the extraction of dissolved O2 from water. We were therefore able to apply a rigorous δ18O blank correction for the quantification of 18O/16O ratios in 20 nmol of injected O2. Conclusions The successful quantification of 18O-kinetic isotope effects associated with enzymatic and chemical reduction of dissolved O2 illustrates how the proposed method can be applied for studying enzymatic O2 activation mechanisms in a variety of (bio)chemical processes. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Although the production and use of technical hexachlorocyclohexane ( HCH) and lindane (the purified insecticidal isomer γ- HCH) are prohibited in most countries, residual concentrations still constitute an immense environmental burden. Many studies describe the mineralization of γ- HCH by bacterial strains under aerobic conditions. However, the metabolic fate of the other HCH isomers is not well known. In this study, we investigated the transformation of α-, β-, γ-, δ-, ε- HCH, and a heptachlorocyclohexane isomer in the presence of varying ratios of the two enzymes that initiate γ- HCH degradation, a dehydrochlorinase ( LinA) and a haloalkane dehalogenase ( LinB). Each substrate yielded a unique metabolic profile that was strongly dependent on the enzyme ratio. Comparison of these results to those of in vivo experiments with different bacterial isolates showed that HCH transformation in the tested strains was highly optimized towards productive metabolism of γ- HCH and that under these conditions other HCH-isomers were metabolized to mixtures of dehydrochlorinated and hydroxylated side-products. In view of these results, bioremediation efforts need very careful planning and toxicities of accumulating metabolites need to be evaluated. [ABSTRACT FROM AUTHOR]

β-Peptides and their derivates are usually stable to proteolysis and have an increased half-life compared with α-peptides. Recently, β-aminopeptidases were described as a new enzyme class that enabled the enzymatic degradation and formation of β-peptides. As an alternative to the existing chemical synthesis routes, the aim of the present work was to develop a whole-cell biocatalyst for the synthesis and production of β-peptides using this enzymatic activity. For the optimization of the reaction system we chose the commercially relevant β, α-dipeptidel-carnosine ( β-alanine-l-histidine) as model product. We were able to show that different recombinant yeast and bacteria strains, which overexpress a β-peptidase, could be used directly as whole-cell biocatalysts for the synthesis ofl-carnosine. By optimizing relevant reaction conditions for the best-performing recombinant Escherichia coli strain, such as pH and substrate concentrations, we obtained highl-carnosine yields of up to 71%. Long-time as well as biocatalyst recycling experiments indicated a high stability of the developed biocatalyst for at least five repeated batches. Application of the recombinant E. coli in a fed-batch process enabled the accumulation ofl-carnosine to a concentration of 3.7 g l−1. [ABSTRACT FROM AUTHOR]

We previously discovered that BapA, a bacterial β-peptidyl aminopeptidase, is able to hydrolyze two otherwise metabolically inert β-peptides [Geueke B, Namoto K, Seebach D & Kohler H-PE (2005) J Bacteriol 187, 5910–5917]. Here, we describe the purification and characterization of two distinct bacterial β-peptidyl aminopeptidases that originated from different environmental isolates. Both bapA genes encode a preprotein with a signal sequence and were flanked by ORFs that code for enzymes with similar predicted functions. To form the active enzymes, which had an (αβ)4 quaternary structure, the preproteins needed to be cleaved into two subunits. The two β-peptidyl aminopeptidases had 86% amino acid sequence identity, hydrolyzed a variety of β-peptides and mixed β/α-peptides, and exhibited unique substrate specificities. The prerequisite for peptides being accepted as substrates was the presence of a β-amino acid at the N-terminus; peptide substrates with an N-terminal α-amino acid were not hydrolyzed at all. Both enzymes cleaved the peptide bond between the N-terminal β-amino acid and the amino acid at the second position of tripeptidic substrates of the general structure H-βhXaa-Ile-βhTyr-OH according to the following preferences with regard to the side chain of the N-terminal β-amino acid: aliphatic and aromatic > OH-containing > hydrogen, basic and polar. Experiments with the tripeptides H-d-βhVal-Ile-βhTyr-OH and H-βhVal-Ile-βhTyr-OH demonstrated that the two BapA enzymes preferred the peptide with thel-configuration of the N-terminal β-homovaline residue as a substrate. [ABSTRACT FROM AUTHOR]

The sulfate-reducing bacteria Desulfobacterium autotrophicum. Desulfobulbus propionicus and Archaeoglobus fulgidus (VC-16) and the sulfur-metabolizing archaebacteria Desulfurolobus ambivalens and Thermoplasma acidophilum were found to contain considerable amounts of corrinoids, that were isolated and crystallized in their Coβ-cyano form. In three other sulfur-metabolizing archaebacteria, Thermoproteus neutrophilus, Pyrodictium occultum and Staphylothermus marinus significant amounts of corrinoids were not detected under the isolation methods used. The samples from the three sulfate-reducers were identified with Coα-[α-(5'-methylbenzimidazolyl)]-Coβcyanocobamide. This corrinoid was also obtained from a 5-methylbenzimidazole-supplemented Propionibacterium fermentation and was structurally characterized by ultraviolet/visible, CD, fast-atom-bombardment MS, ¹H- and 13C-NMR spectroscopy. Also the major corrinoid from T. acidophilum was (tentatively) analyzed as a 5'-methylbenzimidazolyl-cobamide, whereas the main corrinoid from D. ambivalens was indicated to be vitamin B12 (a 5',6'-dimethylbenzimidazolyl-cobamide). The 5'-methylbenzimidazolylcobamides are found here as the common corrins of some sulfate-reducing and sulfur-metabolizing bacteria. The structural diversity due to the differing nucleotide bases of the coffins examined here and in methanogenic and acetogenic bacteria appears not to correlate to the biological function(s) of the corrins, but rather to be determined by biosynthetic properties of these organisms under natural growth conditions. [ABSTRACT FROM AUTHOR]

2-Hydroxybiphenyl 3-monooxygenase (HbpA; EC 1.14.13.44) from Pseudomonas azelaica HBP1 was produced in Escherichia coli both as native and SeMet-labelled protein. The two enzymes were purified to homogeneity and crystallized by the hanging-drop vapour diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 108.6, b = 196.8, c = 79.3 Å, β = 97.7° for the native protein and a = 108.3, b = 196.8, c = 79.0 Å, β = 97.8° for SeMet HbpA. Crystal-packing considerations led to the assumption of two HbpA subunits per asymmetric unit, which corresponds to a V[sub M] value of 3.3 ų Da[sup -1] and a solvent content of 62%. The crystals were radiation-sensitive and only had a lifespan of about 120 s when exposed to synchrotron radiation on an undulator beamline. To obtain complete data sets, data were collected from 23 native and 26 derivative crystals. The high-resolution limit was 2.0 Å for native and 2.25 Å for SeMet HbpA. [ABSTRACT FROM AUTHOR]

β-Peptides and their derivates are usually stable to proteolysis and have an increased half-life compared with α-peptides. Recently, β-aminopeptidases were described as a new enzyme class that enabled the enzymatic degradation and formation of β-peptides. As an alternative to the existing chemical synthesis routes, the aim of the present work was to develop a whole-cell biocatalyst for the synthesis and production of β-peptides using this enzymatic activity. For the optimization of the reaction system we chose the commercially relevant β,α-dipeptide L-carnosine (β-alanine-L-histidine) as model product. We were able to show that different recombinant yeast and bacteria strains, which overexpress a β-peptidase, could be used directly as whole-cell biocatalysts for the synthesis of L-carnosine. By optimizing relevant reaction conditions for the best-performing recombinant Escherichia coli strain, such as pH and substrate concentrations, we obtained high l-carnosine yields of up to 71%. Long-time as well as biocatalyst recycling experiments indicated a high stability of the developed biocatalyst for at least five repeated batches. Application of the recombinant E. coli in a fed-batch process enabled the accumulation of l-carnosine to a concentration of 3.7 g l(-1)., (© 2009 The Authors. Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.)