Equilibrium of the Cis-Trans Isomerisation of the Peptide Bond with N-alkyl Amino Acids Measured by 2D NMR

Summary The conformationalcis-trans equilibrium around the peptide bond in model tripeptides has been determined by 2D NMR methods (HOHAHA, ROESY). The study was limited to three different N-substituted amino acids in position 2, namely Pro (proline), Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), and N-MePhe (N-methylphenylalanine). In all cases the amino acid in position 1 was tyrosine and in position 3, phenylalanine. The results of our studies show that thecis-trans ratio depends mostly on the configuration of the amino acids forming the peptide bond undergoing thecis-trans isomerisation. The amino acid following the sequence (in position 3) does not have much influence on thecis-trans isomerisation, indicating that there is no interaction of the side chains between these amino acids. The model peptides with the L-Tyr-L-AA-(L-or D-)Phe (where AA is N-substituted amino acid) chiralities give 80–100% more of thecis form in comparison to the corresponding peptides with the D-Tyr-L-AA-(L-or D-)Phe chiralities. These results indicate that the incorporation of N-substituted amino acids in small peptides with the same chirality as the precedent amino acid involved in the peptide bound undergoing thecis/trans isomerisation moves the equilibrium to a significant amount of thecis form.     

Effect of Pseudomonas fluorescens on the root exudates of two tomato mutants differently sensitive to Fe chlorosis

Plants with different Fe-mobilization properties are known to differ in the amount and kind of Fe-reducing and Fe-chelating compounds exuded by their roots. Although rhizosphere bacteria are known to affect the exudation of organic compounds by the plant roots, their effect on the root exudates of plants differing in Fe-mobilization properties is not known. We studied the effect of Pseudomonas fluorescens, on the exudation of sugars and organic and amino acids by roots of an iron chlorosis-resistant (T3238FER) and a chlorosis-susceptible (T3238fer) tomato mutant. Under sterile conditions two tomato mutants grew equally well and did not differ in the total amount of sugars and organic acid exuded by their roots. More amino acids, however, were exuded by the roots of T3238FER than T323fer. Mutants differed in the amount of oxalic acid and the amino acids Ala, Asp, Gaba, Gln, Gly, His, Hyl, Ile, Leu, Lys, Phe, Pro, and Val exuded by their roots into sterile rooting media. Addition of P. fluorescens to the rooting medium did not affect the growth of T3238FER but stimulated the root growth of chlorosis-susceptible T3238fer, reduced the amounts of glucose, arabinose and fructose but increased the amount of sucrose, reduced the amounts of fumaric, malic and oxalic acid but increased the amounts of citric and succinic acid in the rooting media of both mutants. P. fluorescens resulted in the following changes in the amino acids in the rooting media: reduced the amounts of Gly, Leu, and Lys in T3238FER, and of Asp, Gln, Hyp, and Ile in T3238fer, and increased the amounts of Cys, Glu, His, Hyp, Ile, Phe and Tyr in T3238FER and of Ala, Glu, His, Phe, and Ser in T323fer—in cases more than 40-fold. These differential effects of P. fluorescens in altering the pattern of organic and amino acids compounds with some Fe-chelating properties detected in the rooting medium of these two mutants may indicate that the differences in Fe-chlorosis susceptibility of these tomato mutants may be the result of, or modified by, the interactions between plant roots and rhizosphere microorganisms. We postulate that the Fe-chlorosis susceptibility in plants may be the product of the interactions between soil microorganisms and plant roots, and may not be solely related to the plant per se.     

Role of the phenylalanine 260 residue in defining product profile and alcoholytic activity of the α-amylase AmyA from Thermotoga maritima

Some α-amylases besides catalyzing the hydrolysis of α-1,4 glycosidic bonds in starch are also capable of carrying out some transglycosylation activity. The importance of aromatic residues near the catalytic site in determining the ratio of these two competing activities has been remarked in the past. In the present work we investigated the role of residue 260 in the product profile of the α-amylase AmyA from Thermotoga maritima. This phenylalanine residue, two positions after the glutamic acid/base catalyst was substituted by both tryptophan and glycine residues, showing opposite behaviors. The tryptophan mutant displayed a very similar product profile pattern to that of the wild-type enzyme; while the mutant Phe260Gly showed a higher transglycosylation/hydrolysis ratio. When the Phe260Trp mutation was constructed in the context of His222Gln, a mutant we have already reported with an increased transglycosylation/hydrolysis ratio and a higher alcoholysis activity, the resultant enzyme showed an apparent higher hydrolysis/transglycosylation ratio and a change to shorter products pattern than the single mutant enzyme, still maintaining the increased alcoholytic activity provided by the His222Gln mutation. The mutant Phe260Gly, on the other hand showed by itself a higher alcoholytic activity, similar to that of the His222Gln mutant.     

Involvement of the nadA gene in formation of G-group aflatoxins in Aspergillus parasiticus

The nadA gene is present at the end of the aflatoxin gene cluster in the genome of Aspergillus parasiticus as well as in Aspergillus flavus. RT-PCR analyses showed that the nadA gene was expressed in an aflatoxin-inducible YES medium, but not in an aflatoxin-non-inducible YEP medium. The nadA gene was not expressed in the aflR gene-deletion mutant, irrespective of the culture medium used. To clarify the nadA gene’s function, we disrupted the gene in aflatoxigenic A. parasiticus. The four nadA-deletion mutants that were isolated commonly accumulated a novel yellow-fluorescent pigment (named NADA) in mycelia as well as in culture medium. When the mutants and the wild-type strain were cultured for 3 days in YES medium, the mutants each produced about 50% of the amounts of G-group aflatoxins that the wild-type strain produced. In contrast, the amounts of B-group aflatoxins did not significantly differ between the mutants and the wild-type strain. The NADA pigment was so unstable that it could non-enzymatically change to aflatoxin G₁ (AFG₁). LC–MS measurement showed that the molecular mass of NADA was 360, which is 32 higher than that of AFG₁. We previously reported that at least one cytosol enzyme, together with two other microsome enzymes, is necessary for the formation of AFG₁ from O-methylsterigmatocystin (OMST) in the cell-free system of A. parasiticus. The present study confirmed that the cytosol fraction of the wild-type A. parasiticus strain significantly enhanced the AFG₁ formation from OMST, whereas the cytosol fraction of the nadA-deletion mutant did not show the same activity. Furthermore, the cytosol fraction of the wild-type strain showed the enzyme activity catalyzing the reaction from NADA to AFG₁, which required NADPH or NADH, indicating that NADA is a precursor of AFG₁; in contrast, the cytosol fraction of the nadA-deletion mutant did not show the same enzyme activity. These results demonstrated that the NadA protein is the cytosol enzyme required for G-aflatoxin biosynthesis from OMST, and that it catalyzes the reaction from NADA to AFG₁, the last step in G-aflatoxin biosynthesis.     

The relationship of biomass,polysaccharide and H2 formation in the wild-type and nifA/nifB mutants of Rhodobacter capsulatus

The production of biomass, polysaccharide storage material and H₂ from malate was studied in the wild-type and mutants RdcI, RdcII and RdcI/cII of Rhodobacter capsulatus. The mutants are defective in either copy I, copy II or both copies of the nitrogenase genes nifA and nifB. Stationary phase levels of biomass, polysaccharide and H₂ were determined in phototrophic batch cultures grown with 30 mM of d,l-malate and either 2, 5, or 8 mM of ammonium or 7 mM of glutamate. Calculation of the amounts of malate converted into the three products revealed that, at 8 mM of ammonium and 7 mM of glutamate, malate consumption and product formation were balanced. But with decreasing ammonium concentrations malate not converted into biomass was utilized with decreasing efficiency in polysaccharide and H₂ formation. This suggests formation of unknown products at the lower ammonium concentrations. Under conditions of optimal N supply, 80% of the malate not used for biomass production was converted by the wild-type and strain RdcII to H₂ and CO₂. Mutant RdcI exhibited slightly decreased H₂ production. The double mutant did not evolve H₂ but accumulated increased amounts of polysaccharide. However, the amounts of polysaccharide were lower than should be expected if all of the spare malate, not utilized by the double mutant for H₂ production, was converted into storage material. This and incomplete conversion of malate into known products at low ammonium supplies suggests that polysaccharide accumulation does not compete with the process of H₂ formation for malate.     

Molecular cloning and sequence analysis of an Azospirilium brasilense indole-3-pyruvate decarboxylase gene

Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid p0.2, isolated from an A. brasilense Sp245 genome library in pLAFR1, complements the Tn5-induced mutant SpM7918 of A. brasilense Sp6 which excretes reduced amounts of IAA. Restriction mapping and gene expression studies identified a BglII-EcoRI 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of IAA production in mutant SpM7918. Tn5 mutagenesis localized the gene responsible on a 1.8 kb SmaI fragment. Nucleotide sequence analysis revealed that this fragment contains one complete open reading grame. The predicted protein sequence shows extensive homology with the indole-3-pyruvate decarboxylase of Enterobacter cloacae and the pyruvate decarboxylases of Saccharomyces cerevisiae and Zymomonas mobilis. The A. brasilense mutant Sp245a, constructed by homogenotization of a Tn5 insertion derivative of the 1.8 kb SmaI fragment, also displayed reduced IAA production. Introduction of the cloned wild-type gene into Rhizobium meliloti 1021 resulted in increased IAA production. Cell-free extracts prepared from R. meliloti and A. brasilense transconjugants harboring this gene could convert indole-3-pyruvic acid to indole-3-acetaldehyde and tryptophol. These results clearly demonstrate that IAA production in A. brasilense is mediated by indole-3-pyruvate decarboxylase.     

Fusarinines and dimerum acid, mono- and dihydroxamate siderophores from Penicillium chrysogenum, improve iron utilization by strategy I and strategy II plants

Cucumber, as a strategy I plant, and Maize as a strategy II plant, were cultivated in hydroponic culture in the presence of a ferrated siderophore mixture (1 M) from a culture of Penicillium chrysogenumisolated from soil. The siderophore mixture significantly improved the iron status of these plants as measured by chlorophyll concentration to the same degree as a 100-fold higher FeEDTA supply. Analysis of the siderophore mixture from P. chrysogenum by HPLC and electrospray mass spectrometry revealed that besides the trihydroxamates, coprogen and ferricrocin, large amounts of dimerum acid and fusarinines were present which represent precursor siderophores or breakdown products of coprogen. In order to prove the iron donor properties of dimerum acid and fusarinines for plants, purified coprogen was hydrolyzed with ammonia and the hydrolysis products consisting of dimerum acid and fusarinine were used for iron uptake by cucumber and maize. In short term experiments radioactive iron uptake and translocation rates were determined using ferrioxamine B, coprogen and hydrolysis products of coprogen. While the trihydroxamates revealed negligible or intermediate iron uptake rates by both plant species, the fungal siderophore mixture and the ammoniacal hydrolysis products of coprogen showed high iron uptake, suggesting that dimerum acid and fusarinines are very efficient iron sources for plants. Iron reduction assays using cucumber roots or ascorbic acid also showed that iron bound to hydrolysis products of coprogen was more easily reduced compared to iron bound to trihydroxamates. Ligand exchange studies with epi-hydroxymugineic acid and EDTA showed that iron was easily exchanged between coprogen hydrolysis products and phytosiderophores or EDTA. The results indicate that coprogen hydrolysis products are an excellent source for Fe nutrition of plants.     

Isolation and characterization of a Na+/H+ antiporter gene from the halophyte Atriplex gmelini

With a homologous gene region we successfully isolated a Na⁺/H⁺ antiporter gene from a halophytic plant, Atriplex gmelini, and named it AgNHX1. The isolated cDNA is 2607 bp in length and contains one open reading frame, which comprises 555 amino acid residues with a predicted molecular mass of 61.9 kDa. The amino acid sequence of the AgNHX1 gene showed more than 75% identity with those of the previously isolated NHX1 genes from glycophytes, Arabidopsis thaliana and Oryza sativa. The migration pattern of AgNHX1 was shown to correlate with H⁺-pyrophosphatase and not with P-type H⁺-ATPase, suggesting the localization of AgNHX1 in a vacuolar membrane. Induction of the AgNHX1 gene was observed by salt stress at both mRNA and protein levels. The expression of the AgNHX1 gene in the yeast mutant, which lacks the vacuolar-type Na⁺/H⁺ antiporter gene (NHX1) and has poor viability under the high-salt conditions, showed partial complementation of the NHX1 functions. These results suggest the important role of the AgNHX1 products for salt tolerance.     

Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots

Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA₁ and GA₄. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA₉- as well as GA₂₀-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod ^– and fix ^– mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.Abbreviations EIA enzyme immunoassay - GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - Me methyl ester - RIA radioimmunoassay - TLC thin-layer chromatography     

Effects of N-starvation and C-source on Bradyrhizobium japonicum exopolysaccharide production and composition, and bacterial infectivity to soybean roots

The exopolysaccharide (EPS) is an extracellular molecule that in Bradyrhizobium japonicum affects bacterial efficiency to nodulate soybean. Culture conditions such as N availability, type of C-source, or culture age can modify the amount and composition of EPS. To better understand the relationship among these conditions for EPS production, we analyzed their influence on EPS in B. japonicum USDA 110 and its derived mutant ΔP22. This mutant has a deletion including the 3′ region of exoP, exoT, and the 5′ region of exoB, and produces a shorter EPS devoid of galactose. The studies were carried out in minimal media with the N-source at starving or sufficient levels, and mannitol or malate as the only C-source. Under N-starvation there was a net EPS accumulation, the levels being similar in the wild type and the mutant with malate as the C-source. By contrast, the amount of EPS diminished in N-sufficient conditions, being poyhydroxybutyrate accumulated with culture age. Hexoses composition was the same in both N-situations, either with mannitol or malate as the only C-source, in contrast to previous observations made with different strains. This result suggests that the change in EPS composition in response to the environment is not general in B. japonicum. The wild type EPS composition was 1 glucose:0.5 galactose:0.5 galacturonic acid:0.17 mannose. In ΔP22 the EPS had no galactose but had galacturonic acid, thus indicating that it was not produced from oxidation of UDP-galactose. Infectivity was lower in ΔP22 than in USDA 110. When the mutant infectivity was compared between N-starved or N-sufficient cultures, the N-starved were not less infective, despite the fact that the amounts of altered EPS produced by this mutant under N-starvation were higher than in N-sufficiency. Since this altered EPS does not bind soybean lectin, the interaction of EPS with this protein was not involved in increasing ΔP22 infectivity under N-starvation.     

Development of natto with germination-defective mutants of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> (<Emphasis Type="Italic">natto</Emphasis>)

The effects of cortex-lysis related genes with the pdaA, sleB, and cwlD mutations of Bacillus subtilis (natto) NAFM5 on sporulation and germination were investigated. Single or double mutations did not prevent normal sporulation, but did affect germination. Germination was severely inhibited by the double mutation of sleB and cwlD. The quality of natto made with the sleB cwlD double mutant was tested, and the amounts of glutamic acid and ammonia were very similar to those in the wild type. The possibility of industrial development of natto containing a reduced number of viable spores is presented.     

Biochemical studies of citric acid production and accumulation by Aspergillus niger mutants

The biochemical rationale for the inhibition of citric acid fermentation by Aspergillus niger in the presence of Mn²⁺ ions has been investigated using high citric acid-yielding, Mn²⁺ ion-sensitive as well as Mn²⁺ ion-tolerant mutant strains of A. niger. In the presence of Mn²⁺ (1.5 mg/l), citric acid production by the Mn²⁺ ion-sensitive strain (KCU 520) was reduced by about 75% with no apparent effect on citric acid yield by the Mn²⁺ ion-tolerant mutant strain (GS-III) of A. niger. The significantly increased level of the Mn²⁺ ion-requiring NADP⁺-isocitrate dehydrogenase activity in KCU 520 cells and the lack of effect on the activity level of the enzyme in GS-III mutant cells by Mn²⁺ ions during fermentation seem to be responsible for the Mn²⁺ ion inhibition of citric acid production by the KCU 520 strain and the high citric acid yield by the mutant strain GS-III of A. niger even in the presence of Mn²⁺.     

Simple device to monitor aerobic biotransformations by in situ 1H-NMR

A simple device for taking in situ proton NMR measurements in ¹H₂O is described. This allows aeration of reactions in a 10 mm diameter NMR tube without modifying the magnet or the probe head. With this device, aerobic biotransformations can be monitored in the NMR-tube placed in the spectrometer. It allows in situ analyses of the transformations, separating the aeration period temporally from the measurement time, not unlike traditional Warburg respiratory experiments. Two reactions determining kinetic and stoichieometric parameters: (i) a biotransformation by a growing Pseudomonas putida culture and (ii) l-phenylalanine oxidation catalysed by l-amino acid oxidase [E.C. 1.4.3.2]; both incubations were contained in the magnet.     

IAA-overproducer mutants of Hebeloma cylindrosporum Romagnesi mycorrhizal with Pinus pinaster (Ait.) Sol. and P. sylvestris L. in hydroponic culture

Indole-3-acetic acid (IAA) is thought to play a role in the regulation of ectomycorrhiza development, and vigorous mycorrhiza formers such as Pisolithus and Laccaria have previously been shown to accumulate large amounts of IAA in the culture medium in vitro, particularly in the presence of tryptophan. Recently, 5-fluoroindole-resistant and IAA-overproducing mutant strains of Hebeloma cylindrosporum Romagnesi have been developed and described by Durand et al. (1992). We have used some of these and corresponding wild-type strains as mycobionts on seedlings of Pinus pinaster (Ait.) Sol. and P. sylvestris L. in semi-hydroponic culture in an attempt to study IAA effects independent of species-specific differences. However, no significant differences between strains were found in host growth rate, shoot carbohydrate concentration, root morphology, root IAA concentration or mycorrhizal biomass. Since previous work showed a stimulation by these and other mutants and strains on mycorrhiza formation in Petri dish and test tube cultures, we assume that a semi-hydroponic culture system prevents the build up of tryptophan of fungal origin, which is most likely a precondition for enhanced IAA production.     

Stems of the <Emphasis Type="Italic">Arabidopsis pin1-1</Emphasis> mutant are not deficient in free indole-3-acetic acid

The pin1-1 mutant of Arabidopsis thaliana has been pivotal for studies on auxin transport and on the role of auxin in plant development. It was reported previously that when whole shoots were analysed, levels of the major auxin, indole-3-acetic acid (IAA) were dramatically reduced in the mutant, compared with the WT (Okada et al. 1991). The cloning of PIN1, however, provided evidence that this gene encodes a facilitator of auxin efflux, raising the question of how the pin1-1 mutation might reduce overall IAA levels as well as IAA transport. We therefore re-examined IAA levels in individual parts of pin1-1 and WT plants, focusing on inflorescence stems. Our data show that there is in fact no systemic IAA deficiency in the mutant. The previously reported difference between mutant and WT may have been due to the inclusion of reproductive structures in the WT harvest: we show here that the inflorescence itself contains high levels of IAA. We reconcile the normal IAA levels of pin1-1 inflorescence stems with their (previously-reported) reduced ability to transport IAA by presenting evidence that the auxin in mutant stems is not imported from their apical portion. Our data also indicate that levels of another auxin, indole-3-butyric acid (IBA), are very low in stems of the genotypes used in this study.     

Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University     

Production of l-DOPA by Mutants of Pseudomonas melanogenum

Several kinds of mutants of Pseudomonas melanogenum were derived by mutational treatment with N-methyl-N’-nitro-N-nitrosoguanidine, and selected for 3,4-dihydroxyphenyl-l-alanine (l-DOPA) production by newly devised screening method which was carried out on agar plates based on violet-black colour formation by the reaction of l-DOPA with iron ion. Mutants tested were; glucose-insensitive mutant, cysteine-insensitive mutant, 3-amino-tyrosine-resistant mutant and p-fluorophenylalanine-resistant mutant. Some colonies isolated by monocolony procedure without mutagenic treatment were also tested. Among the 3-aminotyrosine-resistant mutants many good l-DOPA producers were found.

An 3-aminotyrosine-resistant mutant, strain ATN–36, produced 14 to 15 mg/ml of l-DOPA from 26 mg/ml of l-tyrosine (68 % in molar conversion ratio). When the cell concentration in reaction mixture was increased to 4-times the concentration of culture broth, l-DOPA production reached to 21 mg/ml from 52 mg/ml of tyrosine. An enzymatic basis of the high l-DOPA productivity of the improved mutants was found to be due to the increased tyrosinase activity (150 to 160% of the parental strain) of the mutants.     

HPLC separation of enterobactin and linear 2,3-dihydroxybenzoylserine derivatives: a study on mutants ofEscherichia coli defective in regulation (fur), esterase (fes) and transport (fepA)

Reversed-phase HPLC separation of enterobactin and its 2,3-dihydroxybenzoylserine derivatives was used for a comparative analysis of mutants of Escherichia coli, defective in the regulation of enterobactin biosynthesis (fur), enterobactin transport (fepA) and enterobactin esterase (fes). A complete separation of all 2,3-dihydroxybenzoylserine compounds was achieved: the monomer (DHBS), the linear dimer (DHBS)₂ and trimer (DHBS)₃, the cyclic trimer, enterobactin, as well as 2,3-dihydroxybenzoic acid. The production of all these compounds was followed after ethylacetate extraction from acidified culture fluids. Enterobactin was found to be the predominant product in all mutant strains. The mutant strains behaved differently with regard to the breakdown products. All degradation products, such as DHBS, (DHBS)₂ and (DHBS)₃, were detected in the overproducing fur mutant where both transport and esterase are still functioning, while only the monomer, DHBS, was detected in the fepA mutant and no degradation was found in the esterase-deficient fes mutant. From the pattern of breakdown products it may be inferred that the esterase acts in two different ways, depending on whether transport is functioning or not. Thus, esterolytic cleavage of ferric enterobactin after entering the cells results in a mixture of all three hydrolysis products, i.e. DHBS, (DHBS)₂ and (DHBS)₃, while cleavage of iron-free enterobactin subsequent to its biosynthesis yields only the monomer. Thus, the results of quantitative HPLC analysis of enterobactin and its breakdown products show that different enterobactin esterase products arise, depending on whether iron is bound to enterobactin or not.     

Vanillic acid metabolism by the white-rot fungus Sporotrichum pulverulentum

Vanillic acid metabolism was studied in wild-type Sporotrichum pulverulentum and three different mutants. Vanillic acid was found to be oxidatively decarboxylated to methoxyhydroquinone (MHQ) and simultaneously reduced to vanillin and vanillyl alcohol to different degrees depending upon the cultivation conditions. The reducing pathway cannot be utilized unless the fungus has access to an easily metabolized carbon source such as glucose or cellobiose, while decarboxylation takes place in cultures with only vanillic acid present. Polymerization reactions also occurred in the culture solutions. Some evidence for reoxidation of vanillin and vanillyl alcohol was obtained in vivo, and in vitro experiments using horseradish peroxidase.Using vanillic acids labelled in the carboxyl, methoxyl and the aromatic ring it was shown that decarboxylation occures before ring-cleavage, which in turn takes place earlier than the release of ¹⁴CO₂ from O¹⁴CH₃-vanillate. The ¹⁴CO₂ evolution from the methoxyl group is repressed by 1% cellobiose as compared to 0.25% cellobiose, but is stimulated by 26 mM nitrogen (as asparagine plus NH₄NO₃) compared to 2.6 mM nitrogen. Since S. pulverulentum appears to require three hydroxyl groups attached to the benzene ring before ring-cleavage can occur, preparation for ring-cleavage is apparently achieved by hydroxylation rather than by demethylation.A scheme for metabolism of vanillic acid by S. pulverulentum based upon these results is proposed.Non-Standard Abbreviations WT wild type Sporotrichum pulverulentum - MHQ methoxyhydroquinone - MQ methoxyquinone - NKM Norkrans medium - DMS dimethylsuccinate - DHP dehydropolymer of coniferyl alcohol     

Mutational analysis of potential pore-lining amino acids in TM IV of the Na/H exchanger

The Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. In this study we examined the effect of site-specific mutagenesis on the pore-lining amino acid Phe161 and effects of mutagenesis on the charged amino acids Asp159 and Asp172. There was no absolute requirement for a carboxyl side chain at amino acid Asp159 or Asp172. Mutation of Asp159 to Asn or Gln maintained or increased the activity of the protein. Similarly, for Asp172, substitution with a Gln residue maintained activity of the protein, even though substitution with an Asn residue was inhibitory. The Asp172Glu mutant possessed normal activity after correction for its aberrant expression and surface targeting. Replacement of Phe161 with a Leu demonstrated that it was not irreplaceable in NHE1 function. However, the mutation Phe161lys inhibited NHE1 function, while the Phe161Ala mutation caused altered NHE1 targeting and expression levels. Our results show that these three amino acids, while being important in NHE1 function, are not irreplaceable. This study demonstrates that multiple substitutions at a single amino acid residue may be necessary to get a clearer picture membrane protein function.