Birgit Geueke

Bacterial β‐peptidyl aminopeptidases with unique substrate specificities for β‐oligopeptides and mixed β,α‐oligopeptides

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 Bacteriol187, 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 the l‐configuration of the N‐terminal β‐homovaline residue as a substrate.

A Novel β-Peptidyl Aminopeptidase (BapA) from Strain 3-2W4 Cleaves Peptide Bonds of Synthetic β-Tri- and β-Dipeptides

A novel bacterial strain that was capable of growing on the β-tripeptide H-βhVal-βhAla-βhLeu-OH as the sole carbon and nitrogen source was isolated from an enrichment culture. On the basis of physiological characterization, partial 16S rRNA sequencing, and fatty acid analysis, strain 3-2W4 was identified as a member of the family Sphingomonadaceae. Growth on the β-tripeptide and the β-dipeptide H-βhAla-βhLeu-OH was observed, and emerging metabolites were characterized. Small amounts of a persisting metabolite, the N-acetylated β-dipeptide, were identified in both media. According to dissolved organic carbon measurements, 74 to 80% of the available carbon was dissimilated. The β-peptide-degrading enzyme was purified from the crude cell extract of cells from strain 3-2W4 grown on complex medium. The enzyme was composed of two subunits, and the N-terminal sequences of both were determined. With this information, it was possible to identify the complete nucleotide sequence and to deduce the primary structure of the gene bapA. The gene encoded a β-peptidyl aminopeptidase (BapA) of 402 amino acids that was synthesized as preprotein with a signal sequence of 29 amino acids. The enzyme was cleaved into two subunits (residues 30 to 278 and 279 to 402). It belonged to the N-terminal nucleophile (Ntn) hydrolase superfamily.

Biotransformation of Hexabromocyclododecanes (HBCDs) with LinB--an HCH-converting bacterial enzyme.

Hexabromocyclododecanes (HBCDs) and hexachlorocyclohexanes (HCHs) are polyhalogenated hydrocarbons with similar stereochemistry. Both classes of compounds are considered biologically persistent and bioaccumulating pollutants. In 2009, the major HCH stereoisomers came under regulation of the Stockholm convention. Despite their persistence, HCHs are susceptible to bacterial biotransformations. Here we show that LinB, an HCH-converting haloalkane dehalogenase from Sphingobium indicum B90A, is also able to transform HBCDs. Racemic mixtures of α-, β-, and γ-HBCDs were exposed to LinB under various conditions. All stereoisomers were converted, but (-)α-, (+)β-, and (+)γ-HBCDs were transformed faster by LinB than their enantiomers. The enantiomeric excess increased to 8 ± 4%, 27 ± 1%, and 20 ± 2% in 32 h comparable to values of 7.1%, 27.0%, and 22.9% as obtained from respective kinetic models. Initially formed pentabromocyclododecanols (PBCDOHs) were further transformed to tetrabromocyclododecadiols (TBCDDOHs). At least, seven mono- and five dihydroxylated products were distinguished by LC-MS so far. The widespread occurrence of HCHs has led to the evolution of bacterial degradation pathways for such compounds. It remains to be shown if LinB-catalyzed HBCD transformations in vitro can also be observed in vivo, for example, in contaminated soils or in other words if such HBCD biotransformations are important environmental processes.

Kinetic Resolution of Aliphatic β-Amino Acid Amides by β-Aminopeptidases

Access to enantiopure β‐amino acids: β‐Aminopeptidases are hydrolases that possess the unique ability to cleave N‐terminal β‐amino acids from peptides and amides. Hydrolysis of racemic β‐amino acid amides catalyzed by these enzymes displays enantioselectivity with strong preference for substrates with the L‐configuration, and gives access to various aliphatic β‐amino acids of high enantiopurity.

Bacterial Cell Penetration by β3‐Oligohomoarginines: Indications for Passive Transfer through the Lipid Bilayer

Recent studies on the mechanisms of uptake of cell-penetrating peptides (CPPs) by mammalian cells provide evidence that one possible pathway for peptide entry involves initial cell-surface binding of the peptide carrying positively charged side chains followed by endocytosis and cytoplasmic trafficking. 3] This endocytotic uptake mechanism has been verified with various techniques, such as fluorescent peptide probes, flow cytometry, and confocal laser scanning microscopy (CLSM). 4, 5] However, we have also become aware of some reports showing that various putative endocytosis inhibitors, low temperature, or cell-energy-depletion conditions could not effectively suppress peptide uptake; this suggests a passive, direct transfer through the plasma membrane. With regard to these findings, it is noteworthy that counteranions and the electric potential across the biological membrane play an important role in the cell penetration of polycationic compounds. Although an increasing number of reports have appeared, in which the occurrence of misleading artifacts due to cell fixation or fluorescence bound to the cell surface have been described, 10–12] the existence of alternative transport mechanisms can not be excluded. Such pathways will have significant implications for understanding the fundamental functions of biological membranes as well as for designing novel, medicinally valuable peptide–drug conjugates based upon CPPs as molecular transporting vehicles. The goal at the outset of the present study was to shed new light on alternative uptake pathways. To this end, we incubated our fluorescently labeled CPPs comprised of b-homoarginine with selected strains of bacteria that lack the normally indigenous endocytotic mechanism associated with mammalian cells. Little was known about the uptake of CPPs by microorganisms until recently, when it was reported that CPPs improve drug delivery into bacteria and fungi. Our experiments were specifically designed to avoid potential artifacts by cell fixation and to discriminate the internalized portion of peptide from the extracellular surface-bound portion. Thus, in addition to conventional fluorescence microscopic techniques, we employed confocal laser scanning microscopy (CLSM) to gain more precise and direct information on the peptide distribution within unfixed cells. This approach was further complemented with spectrofluorometric assays by using NBD-labeled b-oligoarginines as quenchable fluorescent probes (Figure 1). The fluorescent dye 7-nitrobenzo-2-oxa-1,3-

Enzymatic Conversion of ε-Hexachlorocyclohexane and a Heptachlorocyclohexane Isomer, Two Neglected Components of Technical Hexachlorocyclohexane

α-, β, γ-, and δ-Hexachlorocyclohexane (HCH), the four major isomers of technical HCH, are susceptible to biotic transformations, whereby only α- and γ-HCH undergo complete mineralization. Nevertheless, LinA and LinB catalyzing HCl elimination and hydrolytic dehalogenations, respectively, as initial steps in the mineralization also convert β- and δ-HCH to a variety of mainly hydroxylated metabolites. In this study, we describe the isolation of two minor components of technical HCH, ε-HCH, and heptachlorocyclohexane (HeCH), and we present data on enzymatic transformations of both compounds by two dehydrochlorinases (LinA1 and LinA2) and a haloalkane dehalogenase (LinB) from Sphingobium indicum B90A. In contrast to reactions with α-, γ-, and δ-HCH, both LinA enzymes converted ε-HCH to a mixture of 1,2,4-, 1,2,3-, and 1,3,5-trichlorobenzenes without the accumulation of pentachlorocyclohexene as intermediate. Furthermore, both LinA enzymes were able to convert HeCH to a mixture of 1,2,3,4- and 1,2,3,5-tetrachlorobenzene. LinB hydroxylated ε-HCH to pentachlorocyclohexanol and tetrachlorocyclohexane-1,4-diol, whereas hexachlorocyclohexanol was the sole product when HeCH was incubated with LinB. The data clearly indicate that various metabolites are formed from minor components of technical HCH mixtures. Such metabolites will contribute to the overall toxic potential of HCH contaminations and may constitute serious, yet unknown environmental risks and must not be neglected in proper risk assessments.

Metabolomics of hexachlorocyclohexane (HCH) transformation: ratio of LinA to LinB determines metabolic fate of HCH isomers

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.

Simple enzymatic procedure for L-carnosine synthesis: whole-cell biocatalysis and efficient biocatalyst recycling.

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.

Sphingomicrobium lutaoense gen. nov., sp. nov., isolated from a coastal hot spring

A yellowish pigmented, Gram-negative, rod-shaped, non-spore-forming bacterium (strain CC-TBT-3(T)), was isolated on marine agar 2216 from a coastal hot spring of Green Island (Lutao), located off Taituang, Taiwan. 16S rRNA gene sequence analysis of strain CC-TBT-3(T) showed a relatively low similarity (<95.5 %) to representatives of the genera Novosphingobium, Sphingosinicella and Sphingomonas of the Sphingomonadaceae, with the most related strain being the type strain of Novosphingobium soli. In addition to the relatively low 16S rRNA gene sequence similarity to members of established species, the isolate also showed some unique chemotaxonomic features, including the presence of some glycolipids with unusual chromatographic behaviour. The major components of the polar lipid profile were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid and three unidentified glycolipids. The major respiratory quinone was ubiquinone Q-10. The polyamine pattern was characterized by the triamine sym-homospermidine as a major component. Although the predominant fatty acids were C(18:1)ω7c and summed feature 3 (C(16:1)ω7c and/or iso-C(15:0) 2-OH), the isolate did not show the typical hydroxyl fatty acids, such as C(14:0) 2-OH, C(15:0) 2-OH and C(16:0) 2-OH, found in members of the genera Novosphingobium, Sphingomonas and Sphingosinicella, but showed instead high amounts of C(18:1) 2-OH (12.0 %). The DNA G+C content of strain CC-TBT-3(T) was 63.4 mol%. 16S rRNA gene sequence, chemotaxonomic and physiological analyses revealed that strain CC-TBT-3(T) represents a novel species in a new genus in the family Sphingomonadaceae for which the name Sphingomicrobium lutaoense gen. nov., sp. nov. is proposed; the type strain is of the type species S. lutoaense, CC-TBT-3(T) ( = DSM 24194(T) = CCM 7794(T)).

β-Aminopeptidase-Catalyzed Biotransformations of β2-Dipeptides: Kinetic Resolution and Enzymatic Coupling

We have previously shown that the β‐aminopeptidases BapA from Sphingosinicella xenopeptidilytica and DmpA from Ochrobactrum anthropi can catalyze reactions with non‐natural β3‐peptides and β3‐amino acid amides. Here we report that these exceptional enzymes are also able to utilize synthetic dipeptides with N‐terminal β2‐amino acid residues as substrates under aqueous conditions. The suitability of a β2‐peptide as a substrate for BapA or DmpA was strongly dependent on the size of the Cα substituent of the N‐terminal β2‐amino acid. BapA was shown to convert a diastereomeric mixture of the β2‐peptide H‐β2hPhe‐β2hAla‐OH, but did not act on diastereomerically pure β2,β3‐dipeptides containing an N‐terminal β2‐homoalanine. In contrast, DmpA was only active with the latter dipeptides as substrates. BapA‐catalyzed transformation of the diastereomeric mixture of H‐β2hPhe‐β2hAla‐OH proceeded along two highly S‐enantioselective reaction routes, one leading to substrate hydrolysis and the other to the synthesis of coupling products. The synthetic route predominated even at neutral pH. A rise in pH of three log units shifted the synthesis‐to‐hydrolysis ratio (vS/vH) further towards peptide formation. Because the equilibrium of the reaction lies on the side of hydrolysis, prolonged incubation resulted in the cleavage of all peptides that carried an N‐terminal β‐amino acid of S configuration. After completion of the enzymatic reaction, only the S enantiomer of β2‐homophenylalanine was detected (ee>99 % for H‐(S)‐β2‐hPhe‐OH, E>500); this confirmed the high enantioselectivity of the reaction. Our findings suggest interesting new applications of the enzymes BapA and DmpA for the production of enantiopure β2‐amino acids and the enantioselective coupling of N‐terminal β2‐amino acids to peptides.