Competition for L-glutamate between specialised and versatile Clostridium species

Clostridium cochlearium could be reproducibly enriched in an L-aspartate- and L-glutamate-limited, anaerobic chemostat inoculated with anaerobic sludge. L-glutamate, L-glutamine and L-histidine were the only fermentable substrates. Less specialised clostridia of the C. tetanomorphum type could only be isolated from batch enrichments with L-glutamate and L-aspartate as energy sources. Competition experiments with C. cochlearium and C. tetanomorphum in a L-glutamate-limited chemostat resulted in the selective elimination of the latter species. Addition of glucose to the medium resulted in coexistence of both species. The molar growth yields for L-glutamate at different dilution rates at 30°C were determined for both species. The maximum specific growth rates on L-glutamate were 0.55 h^-1 for C. cochlearium and 0.35 h^-1 for C. tetanomorphum.     

Growth inhibition by L-phenylalanine in Agmenellum quadruplicatum

Summary L-Phenylalanine is a potent inhibitor of growth in a marine species of blue-green bacteria, Agmenellum quadruplicatum. The growth inhibition is reversed by many amino acids when added to the culture medium simultaneously with L-phenylalanine. The most effective L-phenylalanine antagonists are L-tyrosine, L-alanine, L-leucine, L-methionine, L-tryptophan, and L-isoleucine. However, L-tyrosine is the only effective L-phenylalanine antagonist when growth is inhibited by L-phenylalanine for two or more hours prior to addition of an equimolar concentration of the compound tested as an antagonist. Various explanations that could account for inhibition of growth by L-phenylalanine are discussed. Inhibition of growth by L-phenylalanine is not a feature peculiar to the general physiology of blue-green bacteria. For example, the growth of Anacystis nidulans, a fresh water species, was not inhibited by L-phenylalanine, although a different pattern of metabolite sensitivity was found.     

BspA (CyuC) in Lactobacillus fermentum BR11 Is a Highly Expressed High-Affinity L-Cystine-Binding Protein

The BspA protein of Lactobacillus fermentum BR11 (BR11) is a cell envelope constituent that is similar to known solute-binding proteins and putative adhesins. BspA is required for L-cystine uptake and oxidative defense and is likely to be an L-cystine-binding protein. The aim of this study was to directly measure L-cystine-BspA binding and BspA expression. De-energized BR11 cells bound radiolabelled L-cystine with a Kd of 0.2 M. A bspA mutant could not bind L-cystine. L-cystine-BR11 binding was unaffected by large excesses of L-glutamine, L-methionine, or collagen, indicating L-cystine specificity. BR11 and the bspA mutant were identical in their abilities to bind L-cysteine, indicating that L-cysteine is not a BspA ligand. BspA expression levels were deduced from radiolabelled L-cystine binding and it was found that there are 1–2 × 10⁵ BspA molecules per cell, and that expression is slightly higher under oxidizing conditions. It is proposed that BspA be renamed CyuC.     

Induction of phenoloxidase in cell suspension cultures of Mucuna pruriens L.

The effects of limitating nitrogen-containing compounds in the medium and of adding the amino-acid analogues p-fluorophenylalanine and ethionine on both phenoloxidase activity and the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) are reported for cell suspension cultures of Mucuna pruriens. Nitrogen limitation of the cultures, or the addition of p-fluorophenylalanine or ethionine to the culture medium resulted in an increased phenoloxidase activity. There appeared to be an inverse relationship between phenoloxidase activity and the acccumulation of L-tyrosine into L-DOPA by alginate-entrapped cells occurred at a higher rate when phenoloxidase activity was increased.Abbreviations pFPA p-fluorophenylalanine - L-DOPA L-3,4-dihydroxyphenylalanine     

L-3-(3,4-Dihydroxyphenyl)alanine (<Emphasis Type="SmallCaps">L</Emphasis>-DOPA), an allelochemical exuded from velvetbean (<Emphasis Type="Italic">Mucuna pruriens</Emphasis>) roots

We investigated whether or not lettuce growth was inhibited by diffused L-3-(3,4-dihydroxyphenyl)alanine (L-DOPA), an allelochemical exuded from the roots of velvetbean (Mucuna pruriens (L.) DC. var. utilis) cultivars using a modified plant-box bioassay. For all the cultivars and one accession examined L-DOPA diffused from the roots and caused radicle and hypocotyl growth inhibition. A high correlation co-efficient (r = 0.838 to 0.982) was observed between L-DOPA concentration and lettuce seed sowing distance. L-DOPA diffused equally in all directions from roots at 0 mm position (close to root surface) in the plant-box, while the inhibition (%) of lettuce radicle growth gradually decreased with distance from the roots. For all cultivars the concentration of L-DOPA was significantly different at 0 mm position: being highest in cv. preta (167 g/ml) and lowest in cv. jaspeada and cv. ana (13 g/ml). The correlation between lettuce radicle growth inhibition and concentration of diffused L-DOPA was high (r = 0.856 to 0.966) in all cultivars and accession examined. However, the concentration of diffused L-DOPA did not correlate with the fresh weight concentration of L-DOPA measured in roots. The lettuce radicle growth inhibition from mucuna diffused L-DOPA was very similar that induced by synthetic L-DOPA, suggesting that diffused L-DOPA was the allelochemical responsible for growth inhibition.     

Transport of malic acid in Leuconostoc oenos strains defective in malolactic fermentation: a model to evaluate the kinetic parameters

Two Leuconostoc oenos mutant strains unable to metabolize malic acid were differentiated by [U-¹⁴C]-labelled L-malate transport assays into a malolactic-enzyme-deficient mutant and a malate-transport-defective mutant. A mathematical analysis of the data from L-malic acid uptake at three pH values (5.2, 4.5, and 3.2) in the malolactic-enzyme-deficient strains suggest two simultaneous uptake mechanisms, presumably a carrier-mediated transport and a passive diffusion for the anionic and the undissociated forms of the acid, respectively. The apparent affinity constant (K _m t) and the maximal rate (V _m t) values for L-malate active transport were, 12 mM and 43 mol L-malate·mg^–1·s^–1, respectively. Active transport was constitutive and strongly inhibited by protonophores and by ATPase inhibitors. L-Lactic acid appeared to inhibit L-malic acid transport, suggesting an L-lactate/L-malate exchange. At pH values of 4.5 or above, the passive diffusion of L-malic acid was negligible. However, at pH 3.2, the mean pH of wine, the permeability of the cells to the undissociated acid by simple diffusion could represent more than 50% of total L-malic acid uptake, with a diffusion constant (K _D) of 0.1 s^–1. Correspondence to: C. Divies     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

Characterization of an L-arabinose isomerase from Bacillus subtilis

An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of encoding a polypeptide of 496 amino acid residues. The gene was overexpressed in E. coli and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified enzyme showed the highest catalytic efficiency ever reported, with a k _cat of 14,504 min⁻¹ and a k _cat/K _m of 121 min⁻¹ mM⁻¹ for L-arabinose. A homology model of B. subtilis L-AI was constructed based on the X-ray crystal structure of E. coli L-AI. Molecular dynamics simulation studies of the enzyme with the natural substrate, L-arabinose, and an analogue, D-galactose, shed light on the unique substrate specificity displayed by B. subtilis L-AI only towards L-arabinose. Although L-AIs have been characterized from several other sources, B. subtilis L-AI is distinguished from other L-AIs by its high substrate specificity and catalytic efficiency for L-arabinose.     

Some physiological functions of the L-leucine dehydrogenase in Bacillus subtilis

Mutants of Bacillus subtilis constitutive for L-leucine dehydrogenase synthesis were selected. Using these mutants we could determine two functional roles for the L-leucine dehydrogenase. This enzyme liberates ammonium ions from branched chain amino acids when supplied as the sole nitrogen source. Another function is to synthesize from L-isoleucine, L-leucine, and L-valine the branched chain -keto acids which are precursors of branched chain fatty acid biosynthesis. These results together with the inducibility of the enzyme suggest that the L-leucine dehydrogenase has primarily a catabolic rather than an anabolic function in the metabolism of Bacillus subtilis.     

Chemical composition of Eubacterium nodatum cell wall peptidoglycan

The structure of Eubacterium nodatum cell wall peptidoglycan was investigated. The peptide subunit of E. nodatum peptidoglycan has the following structure: L-Ala-D-Glu (Gly)-L-Orn-D-Ala. The carboxyl group of alanine occupying position 4 is attached to the -amino group of ornithine of an other subunit by the cross-linking bridge L-Ala-L-Ala-L-Orn. All glycine molecules are connected with the -carboxyl group of glutamic acid with the ratio being 0.5–1. The hydrolysis of E. nodatum peptidoglycan by the S. albus G enzyme proceeds primarily due to the activity of alanyl-alanine endopeptidase, ornithyl-ornithine endopeptidase, ornithyl-alanine endopeptidase, N-acetyl-muramyl-alanine amidase, N-acetylmuramidase and N-acetylglucosaminidase.     

Transport of basic amino acids and regulation of aspartate kinase activity in thialysine andL-canavanine resistant strains ofSerratia marcescens

Growth ofSerratia marcescens was not inhibited by high concentrations ofL-lysine and its structural analogues,L-canavanine and S-(2-aminoethyl)-L-cysteine (thialysine). This insensitivity was not caused by deficient transport of basic amino acids, unlike in mutant strains ofEscherichia coli having the same properties. The tested strains showed a lack of regulation at the aspartate kinase level towardL-lysine and thialysine. The data indicate great intraspecific variability for aspartate kinase regulation inS. marcescens.     

Untersuchungen zur Entstehung von Dl-Milchsäure bei Lactobacillen und Charakterisierung einer Milchsäureracemase bei einigen Arten der Untergattung Streptobacterium

Zusammenfassung Eine kritische Überprüfung der von den verschiedenen Arten der Gattung Lactobacillus gebildeten Milchsäure ergab, daß zwar die D(-)-Laktatbildner reines D(-)-Isomer, die L(+)-Laktatbildner aber immer auch einige wenige Prozente des anderen Isomers bilden. Letzteres beruht auf der Anwesenheit einer sehr schwach aktiven NAD-abhängigen D-Laktatdehydrogenase neben der hochaktiven NAD-abhängigen L-Laktatdehydrogenase.Die Bildung von Dl-Laktat beruht entweder auf der Anwesenheit der beiden stereospezifisch verschiedenen Laktatdehydrogenasen oder auf der Bildung vo L(+)-Milchsäure und anschließender Racemisierung. In einigen Fällen ist die biochemische Grundlage der Racematbildung noch unklar. Bei fast allen Dl-Bildnern ist das Isomerenverhältnis von den Wachstumsbedingungen abhängig.Bei Lactobacillus curvatus, L. sake und L. casei ssp. pseudoplantarum wurde die für die Dl-Bildung verantwortliche Milchsäureracemase nachgewiesen und näher charakterisiert. Es handelt sich um ein induzierbares Enzym, dessen Induktor L(+)-Milchsäure ist. Die Induktion kann mit Actinomycin D gehemmt werden. Eine Trennung von Racemase und L-Laktatdehydrogenase gelang durch Zentrifugation im Saccharosedichtegradienten, da das Molekulargewicht der Racemase mit 52–60000 in einigen Fällen niedriger liegt als das der Laktatdehydrogenase.

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Formation of Dl-lactic acid by lactobacilli and characterization of a lactic acid racemase from several streptobacteria

Summary The isomer composition of the lactic acid formed by the various species of lactobacilli was enzymatically determined. It is shown, that the D(-)-lactate formers produce D(-)-lactate exclusively whereas all L(+)-lactate formers always produce a few per cent of the other isomer in addition. The latter is due to the presence of a NAD dependent D-LDH of very low activity. The formation of Dl-lactic acid is either caused by the presence of both stereospecific different LDHs or by the formation of L(+)-lactic acid followed by racemisation. In some species the biochemical basis of the racemate formation is still unknown.The racemase was isolated and characterised from Lactobacillus curvatus, L. sake and L. casei ssp. pseudoplantarum. This enzyme is induced by L(+)-lactic acid. The induction is inhibited by actinomycine D. A separation of lactic acid racemase and of L-LDH was achieved by the application of a sucrose density gradient.

     

Amino acid immobilization of fungal motile cells

Summary A 0.2 M mixture of L-leucine and L-lysine, a pair of amino acids which Machlis (1969) had shown could attract the zoospores of Allomyces in much lower concentrations, was found to immobilize zoospores by stopping flagellar motion. While the age of the spores does not affect the response to the amino acid mixture, the time for 100% immobilization does increase with increasing numbers of spores. Viability of the spores is not altered by treatment with the mixture of L-leucine and L-lysine and subsequent germling development is highly synchronized.Several other amino acid mixtures had a similar effect upon the Allomyces' flagellum. Indeed, L-lysine by itself seems to be the most effective compound tested. Immobilization of flagella in other fungi, algae, and one protozoan was also caused by treatment with L-leucine and L-lysine. Nothing is known of the mechanism of action of this amino acid treatment.     

Clostridium methylpentosum sp. nov.: a ring-shaped intestinal bacterium that ferments only methylpentoses and pentoses

An intestinal bacterium isolated from a human subject utilized only two methylpentoses (L-rhamnose and L-fucose) and two pentoses (L-lyxose and D-arabinose) as fermentable substrates, among many compounds tested. The isolate was obligately anaerobic and had a distinctive morphology, its cells being rods bent in the shape of rings with the ends slightly overlapping. Single ring-shaped cells and left-handed helical chains of cells were present in cultures. The cells were surrounded by large capsules which appeared as thick, fibrous masses when examined by electron microscopy. Capsules were formed by cells growing in media containing any one of the four fermentable substrates. Terminally located, heat-resistant endospores were formed on plates of an enriched agar medium supplemented with L-rhamnose. End products of L-rhamnose or L-fucose fermentation included acetate, propionate, n-propanol, CO₂, and H₂. The isolate represented a new species of Clostridium for which the name Clostridium methylpentosum (type strain R2. ATCC 43829) is proposed. This organism may participate in intestinal digestive processes by metabolizing rhamnose released via the enzymatic depolymerization of dietary pectin.Abbreviations G+C guanine plus cytosine - OD optical density - TEM transmission electron micrograph     

Production of ammonium dependent on basicL-amino acids byAnacystic nidulans

The incubation of the cyanobacteriumAnacystis nidulans withL-Arg,L-Lys orL-Orn, but neither with the correspondingD-isomers nor with other twentyL-amino acids, resulted in the production of large amounts of ammonium which accumulated in the outer medium. Relevant properties of thisin vivo ammonium production activity have been studied in cell suspensions treated with the glutamine synthetase inactivatorL-methionine-D,l-sulfoximine (MSX) to prevent assimilation by the cells of the resulting ammonium. In addition to its specificity for the basicL-amino acids, the system exhibited a set of properties (K _m value for substrates, requirement of oxygen which is taken up stoichiometrically with the production of ammonium, inhibition by o-phenanthroline and divalent cations) all of which are shared by a peculiarL-amino acid oxidase recently isolated fromA. nidulans. The data strongly suggest the participation of this enzyme in the production of ammonium from basic amino acids byA. nidulans, an activity that could account for the ability of this cyanobacterium to use arginine as a nitrogen source.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanide p-trifluoromethoxy-phenylhydrazone - MSX L-methionine-D,l-sulfoximine     

Effect of the interrupted adaptation to D-methionine on the efficiency of Rhizobium meliloti strains

Summary R. meliloti strains 107-1, 111 and 152 were adapted to D-methionine in three ways: a) consecutive transfer in the presence of increasing amounts of D-methionine, b) alternate transfer between D- and L-methionine-containing media followed by final cultivation in the presence of each isomer, c) alternate transfer between D-methionine and medium 79 followed by cultivation in medium 79 or in D-methionine-medium. At the end of the experiment efficiency of the strains was ascertained by a plant test.Strain 111 lost efficiency when it was adapted consecutively to 0.125% D-methionine or alternated between D-methionine and either L-methionine or medium 79-Strain 107-1 sucessively adapted to D-methionine lost efficiency within 16 weeks. On adaptation to D-methionine alternated with L-methionine, efficiency was retained in L-methionine medium and lost in D-methionine medium. On alternate adaptation between D-methionine and medium 79, strain 107-1 lost efficiency in the D-methionine-medium but not in medium 79. Efficiency of strain 152 was lost by adaptation to 0.125% D-methionine, but it was maintained on the alternate adaptation between D-methionine and L-methionine or medium 79.     

Die Aminosäuresequenz von 2,4-Diaminobuttersäure enthaltenden Mureinen bei verschiedenen coryneformen Bakterien und Agromyces ramosus

Zusammenfassung Bie 17 Stämmen von coryneformen Organismen wurde 2,4-Diaminobuttersäure als Bestandteil des Mureins gefunden. In 15 Fällen ergab die genauere Analyse die gleiche Aminosäuresequenz, wie sie schon früher von Perkins (1968) bei Corynebacterium insidiosum beschrieben wurde. In diesem Falle ist die L-2,4-Diaminobuttersäure ein Bestandteil der Peptiduntereinheit, während die D-2,4-Diaminobuttersäure die Quervernetzung zwischen dem Glutaminsäurerest und dem C-terminalen Alanin zweier benachbarter Peptiduntereinheiten herstellt. Das Murein gehört demnach zur Gruppe B nach Schleifer u. Kandler (1972). Die -Aminogruppe der L-2,4-Diaminobuttersäure ist in einigen Fällen acetyliert, in anderen Fällen ist sie frei.Das Murein der beiden anderen Stämme unterscheidet sich in seiner Primärstruktur dadurch, daß nur L-2,4-Diaminobuttersäure vorkommt. Im Falle von C. bovis ist wie bei einigen coryneformen pflanzenpathogenen Stämmen die Diaminosäure der Peptiduntereinheit durch Homoserin ersetzt und die Quervernetzung erfolgt durch das Dipeptid -Gly-L-Dab zwischen Glutaminsäure und D-Alanin. Dieses Murein gehört demnach ebenfalls zur Gruppe B. Dagegen ist das Murein von Arthrobacter sp. Ar 22 eine neue Variante der Gruppe A. Die L-2,4-Diaminobuttersäure ist hier ein Glied der Peptiduntereinheit und die Quervernetzung zwischen der -Aminogruppe der 2,4-Diaminobuttersäure und dem D-Alaninrest einer benachbarten Peptiduntereinheit wird durch das Pentapeptid -L-Asp-L-Ala-Gly-L-Ala-L-Ala gebildet. Außerdem ist die Position 1 der Peptiduntereinheit nicht mit L-Alanin, sondern mit Glycin besetzt. Letzteres ist bisher nur bei Mureinen der Gruppe B, aber nicht bei denen der Gruppe A gefunden worden. Ebenfalls neu ist das Vorkommen von L-Asparaginsäure anstelle der bisher gefundenen D-Form.

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The amino acid sequence of 2,4-diaminobutyric acid containing mureins of various coryneform bacteria and Agromyces ramosus

Summary In 17 strains of coryneform bacteria, 2,4-diaminobutyric acid was found to be a component of the murein (peptidoglycan). A detailed analysis showed that 15 strains contain a murein with the same amino acid sequence as that found in Corynebacterium insidiosum by Perkins (1968). In this case the L-2,4-diaminobutyric acid is a component of the peptite subunit while the D-2,4-diaminobutyric acid serves as interpetide bridge between D-glutamatic and the C-terminal D-alanine residue. Therefore this murein belongs to group B according to Schleifer and Kandler (1972). The -amino group of L-2,4-diaminobutyric acid is in some species acetylated, in others free.The murein of the remaining two strains differs by the lack of D-2,4-diaminobutyric acid. Only L-2,4-diaminobutyric acid is found. In the case of C. bovis, the diamino acid of the peptide subunit is replaced by L-homoserine as found in various plant pathogenic coryneform bacteria. The interpeptide bridge consists of the dipeptide -Gly-2,4-Dab. It connects the D-glutamic acid of one peptide subunit with the C-terminal D-alanine residue of an adjacent peptide subunit. Therefore this murein belongs also to group B.The murein of Arthrobacter sp. Ar 22 is a new varition of group A, however. Here the L-2,4-diaminobutyric acid is a component of the peptide subunit. The interpeptide bridge consists of the pentapeptide -L-Asp-L-Ala-Gly-L-Ala-L-Ala. It connects the -amino group of L-2,4-diaminobutyric acid and the C-terminal D-alanine residue of two peptide subunits. Position 1 of the peptide subunit is occupied by glycine instead of L-alanine as found in all the other mureins of group A so far. Another new feature of this murein is the occurrence of the L-form instead of the D-form of aspartic acid.

     

Chemical composition of Eubacterium alactolyticum cell wall peptidoglycan

The mechanism of lysis of Eubacterium alactolyticum cell walls by Streptomyces albus G enzyme was studied. The analysis of the peptide terminal groups and peptide subunits isolated from the cell wall digest, released during solubilization of the cell walls, revealed that lytic action of S. albus G enzyme was mainly due to D-alanyl-A₂pm endopeptidase, N-acetylmuramyl-L-alanine amidase, N-acetylmuramidase and N-acetylglucosaminidase. E. alactolyticum cell wall peptidoglycan is composed mainly of glucosamine, muramic acid, D-glutamic acid, L- and D-alanine, meso-diaminopimelic acid and glycine. The peptide subunit consists of L-alanyl-D-glutamyl-meso-A₂pm-D-alanine. D-Alanine is connected directly with the amino group of the meso-A₂pm residue of another peptide subunit. All of the L-amino groups of meso-diaminopimelic acid are involved in cross-linking.The possible structure of the peptide moiety of E. alactolyticum cell wall peptidoglycan is presented.     

Cloning, characterization, and engineering of fungal L-arabinitol dehydrogenases

L-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD⁺ reduction in fungal L-arabinose catabolism. It is an important enzyme in the development of recombinant organisms that convert L-arabinose to fuels and chemicals. Here, we report the cloning, characterization, and engineering of four fungal LADs from Penicillium chrysogenum, Pichia guilliermondii, Aspergillus niger, and Trichoderma longibrachiatum, respectively. The LAD from P. guilliermondii was inactive, while the other three LADs were NAD⁺-dependent and showed high catalytic activities, with P. chrysogenum LAD being the most active. T. longibrachiatum LAD was the most thermally stable and showed the maximum activity in the temperature range of 55–65°C with the other LADs showed the maximum activity in the temperature range of 40–50°C. These LADs were active from pH 7 to 11 with an optimal pH of 9.4. Site-directed mutagenesis was used to alter the cofactor specificity of these LADs. In a T. longibrachiatum LAD mutant, the cofactor preference toward NADP⁺ was increased by 2.5 × 10⁴-fold, whereas the cofactor preference toward NADP⁺ of the P. chrysogenum and A. niger LAD mutants was also drastically improved, albeit at the expense of significantly reduced catalytic efficiencies. The wild-type LADs and their mutants with altered cofactor specificity could be used to investigate the functionality of the fungal L-arabinose pathways in the development of recombinant organisms for efficient microbial L-arabinose utilization.     

Lysine degradation in Candida maltosa: occurrence of a novel enzyme,acetyl-CoA: L-lysine N-acetyltransferase

The yeast Candida maltosa can utilize L-lysine as sole nitrogen and sole carbon source accompanied by accumulation of -N-acetyl-L-lysine, indicating that lysine is metabolized by way of N-acetylated intermediates. A novel lysine acetyltransferase catalyzing the first step in this pathway, the N-acetylation of the -amino group of L-lysine, was found in this yeast. The enzyme, acetyl-CoA:L-lysine N-acetyltransferase, is strongly induced in cells grown on L-lysine as sole carbon source. The enzyme is specific for both L-lysine and acetyl-CoA. The K _m values are 10 mM for L-lysine and 0.33 mM for acetyl-CoA. The enzyme has a maximum activity at pH 8.1.Dedicated to Prof. Dr. F. Böttcher in occasion of his 60th birthday