l-THREONINE DEAMINASE IN MARINE PLANKTONIC ALGAE. V. KINETIC EVALUATION OF FEEDBACK EFFECTS FROM STRUCTURAL ANALOGS OF l-ISOLEUCINE ON THE DEAMINASE ACTIVITY OF SEVEN UNICELLULAR SPECIES AND ELUCIDATION OF THE ALLOSTERIC BASIS FOR ANOMALOUS FEEDBACK CONTROL SHOWN BY A DIATOM AND A CRYPTOMONAD1,2

The biosynthetic l -threonine deaminase of 2 cyanophytes, 1 rhodophyte, 2 cryptomonads, and 1 chlorophyte showed sensitive and powerful feedback inhibition from l -isoleucine, which effect was closely duplicated by its isosteric structural analog l -O-methylthreonine. The l -isoleucine inhibition was maximal in the pH range 7.0–8.5 and was rapidly weakened with more alkaline pH. Glycine, l -alanine, and d -isoleucine had no effect, while l -α-amino-n-butyric acid, l -norvaline, l -leucine, and l -valine caused insensitive partial inhibitions detectable only at high concentration (100-fold greater than l -isoleucine). Tests of these analogs at low concentration revealed the specific capacity of l -valine to invoke sensitive stimulation of enzyme activity from all the algal strains, and this effect was markedly enhanced by decrease in substrate concentration. Valine also counteracted the inhibitory influence of isoleucine. It was concluded from the reaction kinetics observed with these effectors that the algal threonine deaminases conform to Monad's K system of allosteric enzymes. The activity from the cryptomonad Hemiselmis virescens was exceptional in showing 2 closely spaced pH optima, which were related to marked difference in effects from isoleucine and valine at these pH values. It was inferred from various pH-related tests that this cryptomonad may contain 2 enzymes basically similar in biosynthetic function but differing only in pH optima and sensitivity to feedback regulation. The enzyme of the diatom Cyclotella nana was unique in showing insignificant feedback reaction from l -isoleucine and its structural analogs, excepting l -valine, which caused the strongest stimulation among the algae surveyed. It was concluded from the reaction kinetics that this deaminase is also of the biosynthetic type but that its feedback-inhibition site is modified or unavailable, presumably from having this site already filled by endogenous isoleucine from the diatom.     

Production of L-Isoleucine by AHV Resistant Mutants of Brevibacterium flavum

The growth of Brevibacterium flavum No. 2247A was inhibited by α-amino-β-hydroxy-valeric acid (AHV), and the inhibition was partially reversed by L-isoleucine.

AHV resistant strain ARI-129, which was isolated on a medium supplemented with 2 mg/ml of AHV, produced 11 g/liter of L-isoleucine.

No difference was observed in threonine dehydratase between No. 2247A and ARI–129. Homoserine dehydrogenase from ARI–129 was insensitive to the feedback inhibition by L-isoleucine and L-threonine.

O-Methyl-L-threonine resistant mutant, strain AORI–126, which was derived from ARI–129, produced 14.5 g/liter of L-isoleucine. Specific activity of threonine dehydratase from AORI–126 increased about two-fold higher than those from No. 2247A and ARI–129, whereas degree of inhibition of the enzyme by L-isoleucine was the same among three strains.

Among auxotrophic mutants derived from ARI–129, adenine and lysine auxotrophs produced more L-isoleucine than the parent did.

In the adenine auxotroph, L-isoleucine production was markedly reduced by the addition of excess adenine.     

The mechanism of growth inhibition by threonine inEuglena gracilis: Regulation of isoleucine-valine biosynthesis by 2-oxobutyrate

InEuglena gracilis the growth inhibition by threonine was accompanied by a rapid accumulation of isoleucine in the cells. Among threonine-catabolizing enzymes only threonine dehydratase was detected in high activity inEuglena, and 2-oxobutyrate, the dehydratase products of threonine, also inhibited as did threonine. Threonine dehydratase was located in the cytosol, and its activity was not affected by isoleucine and related amino acids. 2-Oxobutyrate strongly inhibited the synthesis of valine from pyruvate while augmented the synthesis of isoleucine in mitochondria.     

Screening of l-Isoleucine Producers among Ethionine Resistant Mutants of l-Threonine Producing Bacteria

Two types of l-isoleucine producing mutants were derived from l-threonine producers by the supplement of the resistance to ethionine.

Main control site in l-isoleucine biosynthetic pathway after threonine is threonine dehydratase. In case of Brevibacterium flavum No. 14083, l-isoleucine production was based on the insensitiveness of this key enzyme to feedback inhibition by l-isoleucine. As regards Brevibacterium flavum No. 168, it was based on the increase in the specific activity of this enzyme.

The former produced 11.3 g/liter of l-isoleucine and the latter produced 9.92 g/liter from glucose. The former showed a vigorous ability of acetic acid assimilation, but the latter did not.     

Engineering the homoserine dehydrogenase and threonine dehydratase control points to analyse flux towards L-isoleucine in Corynebacterium glutamicum

 The synthesis of L-isoleucine by Corynebacterium glutamicum involves 11 reaction steps, with at least 5 of them regulated in activity or expression. Using gene replacement we constructed a vector-free C. glutamicum strain having feedback-resistant aspartate kinase and feedback-resistant homoserine dehydrogenase activity. Isogenic strains carrying in addition one or several copies of feedback-resistant threonine dehydratase were made and their product accumulations compared. With strain SM1, with high threonine dehydratase activity, accumulation of 50 mM L-isoleucine was achieved, whereas with the parent strain only 4 mM L-isoleucine was obtained. Applying a closed-loop control fed-batch strategy to strain SM1 a final titre of 138 mM L-isoleucine was achieved with an integral molar yield of 0.11 mol/mol, and a maximal specific productivity of 0.28 mmol (g h)^-1. This shows that high L-isoleucine yields can be obtained in the presence of one copy of feedback-resistant homoserine dehydrogenase by applying the appropriate fermentation strategy. In addition, the specific profiles of 2-oxoglutarate and pyruvate accumulation during fermentation revealed a major transition of the metabolism of C. glutamicum during the fermentation process. Received: 16 October 1995/Received revision: 21 December 1995/Accepted: 8 January 1996     

MICROSPECTROPHOTOMETRY AS A METHOD TO IDENTIFY KLEPTOPLASTIDS IN THE NAKED FRESHWATER DINOFLAGELLATE GYMNODINIUM ACIDOTUM1

A relatively small number of freshwater dinoflagellates are involved in symbiotic association with cryptophytes. The chloroplasts of the cryptophytes are retained by the dinoflagellate and give it the characteristic phycobilin pigmentation, either phycoerythrin or phycocyanin. The pigment characterization of the retained chloroplasts can give precise and accurate information about the type of cryptophyte preyed upon by the dinoflagellate. For this purpose, we performed microspectrophotometric evaluation of the pigments of Gymnodinium acidotum Nygaard and three different cryptophytes present in samples collected from a tributary of the river Arno, in Tuscany (Italy). The comparison of the different spectroscopic data allowed us to discriminate effectively among the cryptophytes preyed upon by the dinoflagellate.     

UNIQUE LOCATION OF THE PHYCOBILIPROTEIN LIGHT-HARVESTING PIGMENT IN THE CRYPTOPHYCEAE1

The cryptophyte algae, or cryptomonads, comprise a small algal group with a unique photosynthetic apparatus. Both a chlorophyll a/c₂ light-harvesting complex and a phycobiliprotein antenna (which can be either phycoerythrin or phycocyanin) are present, with the phycobiliprotein playing the major role in harvesting light for photosynthesis. Longstanding circumstantial evidence suggested that, in cryptophytes, the phycobiliprotein is located in the intrathylakoid space (thylakoid lumen) rather than on the outer surface of the thylakoid as part of a phycobilisome as in other algae. We used immunogold labeling to show conclusively that 1) the phycoerythrin (PE) of the cryptophyte Rhodomonas lens Pascher and Ruttner is located within the intrathylakoid space, 2) the PE is not exclusively bound to the thylakoid membrane but instead is distributed across the thylakoid lumen and 3) a fraction of this PE is tightly associated with the thylakoid membrane. The thylakoids are not everted to compensate for this unusual arrangement. The location of the major light-harvesting pigment on the “wrong” side of the otherwise very normal photo-synthetic membrane is unexpected, unique to the cryptophytes, and, remarkably, does not impair the photosynthetic abilities of this organism. A model is presented which incorporates these results -with previous information to give a complete structural picture of the cryptophyte light-harvesting apparatus.     

Production of isoleucine by overexpression of ilvA in a Corynebacterium lactofermentum threonine producer

Overproduction of isoleucine, an essential amino acid, was achieved by amplification of the gene encoding threonine dehydratase, the first enzyme in the threonine to isoleucine pathway, in a Corynebacterium lactofermentum threonine producer. Threonine overproduction was previously achieved with C. lactofermentum ATCC 21799, a lysine-hyperproducing strain, by introduction of plasmid pGC42 containing the Corynebacterium hom ^dr and thrB genes (encoding homoserine dehydrogenase and homoserine kinase respectively) under separate promoters. The pGC42 derivative, pGC77, also contains ilvA, which encodes threonine dehydratase. In a shake-flask fermentation, strain 21799(pGC77) produced 15 g/l isoleucine, along with small amounts of lysine and glycine. A molar carbon balance indicates that most of the carbon previously converted to threonine, lysine, glycine and isoleucine was incorporated into isoleucine by the new strain. Thus, in our system, simple overexpression of wild-type ilvA sufficed to overcome the effects of feedback inhibition of threonine dehydratase by the end-product, isoleucine.     

Feedback inhibition of homoserine dehydrogenase and threonine deaminase in the genusBifidobacterium

Feedback inhibition of crude and purified extracts of homoserine dehydrogenase and threonine deaminase activities in the genusBifidobacterium was studied. Homoserine dehydrogenase was partially or completely inhibited byl-threonine, and a marked inhibitory effect byl-isoleucine on threonine deaminase was observed. In the speciesBifidobacterium cuniculi high levels ofl-valine reversed the inhibitory effect ofl-isoleucine. The -aminobutyric acid-resistant mutant Ru 326/106 of the speciesB. ruminale, overproducer ofl-isoleucine, had a derepressed homoserine dehydrogenase and a lesser feedback inhibition byl-threonine. Homoserine dehydrogenase appeared to be in bifids specifically NAD dependent. The regulatory mechanisms of aspartate family amino acid biosynthesis in bifidobacteria was discussed.     

Threonine dehydratases of Corynebacterium glutamicum with altered allosteric control: their generation and biochemical and structural analysis

Threonine dehydratase is the key enzyme in L-isoleucine synthesis, since it is allosterically feedback-inhibited by L-isoleucine. With the aim of obtaining regulatorily altered mutants of the threonine dehydratase of Corynebacterium glutamicum, amino acids were specifically exchanged and a new biological system of mutant selection was developed, based on the intoxication of Escherichia coli by ketobutyrate, which is the dehydratase reaction product. A collection of 19 mutant enzymes was generated and genetically and biochemically characterized comprising a whole range of regulatorily and catalytically altered enzymes. Of particular interest is the mutant Val-323-Ala, which is characterized by the fact that the L-isoleucine inhibition is entirely abolished so that the enzyme is always present in a relaxed, high-activity state. Correspondingly, the Hill coefficient is 1.4, in contrast to the value of 3.4 characteristic of the wild-type enzyme. Another peculiar mutant generated is the double mutant His-278-Arg-Leu-351-Ser. Here, again, L-isoleucine no longer inhibits catalytic activity, but the effector still promotes major structural changes of the protein, as ascertained from the L-isoleucine-dependent loss of pyridoxal-5 -phosphate from this mutant enzyme. Further enzymes obtained are reduced in L-isoleucine inhibition to a varying degree. Detailed studies on the structure of the enzyme revealed a partially very high similarity of the secondary structure to the mechanistically identical β-subunit of the tryptophan synthase. This provides further indications concerning the localization of the regulatory and catalytic domain of threonine dehydratase.     

Use of the 'Food Vacuole Content' Method to Estimate Grazing by the Mixotrophic Dinoflagellate Gyrodinium Galatheanum on Cryptophytes

We measured in situ grazing rates of the mixotrophic dinoflagellateGyrodinium galatheanum (Braarud) Taylor 1995 on populationsof phycoerythrin-containing cryptophytes in Chesapeake Bay.Rates were estimated from instantaneous food vacuole contents,in situ temperatures, cryptophyte abundances and experimentallydetermined digestion rates. Laboratory digestion experimentsshowed that specific digestion rate constants increased sigmoidallywith temperature, but were unrelated to the initial food vacuolecontent when it was <0.46 cryptophytes dinoflagellate^–1.These results allowed us to establish an empirical model toestimate in situ ingestion of cryptophyte prey by G. galatheanum.The estimated rates ranged from 0 to 0.26 cryptophytes dinoflagellate^–1day^–1, corresponding to daily ingestion of 0–12.29pg carbon, 0–2.48 pg nitrogen and 0–0.34 pg phosphorusdinoflagellate^–1. Estimated daily consumption of cryptophytebiomass by G. galatheanum was equivalent to 0–12% of bodycarbon, 0–13% of body nitrogen and 0–21% of bodyphosphorus. Estimated in situ clearance rates for cryptophytesranged from 0 to 0.27 µl dinoflagellate^–1 day^–1,representing daily removal of 0–4% of the cryptophytestanding stock. Although G. galatheanum may increase its growthrate through phagotrophy, it appears to have little grazingimpact on cryptophyte prey populations.     

DETECTION OF CHLOROPHYLL C1 AND MAGNESIUM-2,4-DIVINYLPHEOPORPHYRIN A5 MONOMETHYLESTER IN CRYPTOPHYTES1

Three different chlorophyll (chl) c-type pigments were isolated from two cryptophyte species by silica thin-layer chromatography or polyethylene high-performance liquid chromatography. Chroomonas sp. Hansgirg contained chl c₁ and magnesium-2,4-divinylpheoporphyrin a, mono-methylester; chl c₂ and magnesium-2,4-divinylpheoporphyrin a₅ monomethylester were found in Cryptomonas maculata (syn. Rhodomonas maculata Butcher). These identifications were based on spectral characteristics and on comparison with reference pigments isolated from the synurophycean Synura petersenii Korshikov and the prasinophyte Mantoniella squamata Manton & Park. Neither of the cryptophyte species contained chl c₁ and chl c₂. The significance of chl c₁ as a major pigment and the occurrence of magnesium-2,4-divinylpheoporphyrin a₅ monomethylester in cryptophytes are discussed.     

The l - and d-threonine dehydratases accompanying l-tyrosine phenol-lyase: selective decomposition of d-threonine in racemic mixture

Low-purity preparations from Escherichia intermedia A-21 and Citrobacter freundii 62 cells producing tyrosine phenol-lyase [l-tyrosine phenol-lyase (deaminating), EC 4.1.99.2] catalyse the decomposition of both threonine enantiomers to α-ketobutyric acid. Reactions with l-threonine and d-threonine are effected by two independent enzymes different from tyrosine phenol-lyase. The enzyme which acts on l-threonine has properties characteristic of biosynthetic threonine dehydratase [l-threonine hydro-lyase (deaminating), EC 4.2.1.16]. l-Isoleucine and dl-allothreonine are inhibitors of this enzyme, permitting a selective inhibition of biosynthetic threonine dehydratase and use of the preparations to act selectively on d-threonine in the racemate.     

Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase l-isoleucine production in Corynebacterium glutamicum

Threonine dehydratase and acetohydroxy acid synthase are critical enzymes in the l-isoleucine biosynthesis pathway of Corynebacterium glutamicum, but their activities are usually feedback-inhibited. In this study, we characterized a feedback-resistant threonine dehydratase and an acetohydroxy acid synthase from an l-isoleucine producing strain C. glutamicum JHI3-156. Sequence analysis showed that there was only a single amino acid substitution (Phe383Val) in the feedback-resistant threonine dehydratase, and there were three mutated amino acids (Pro176Ser, Asp426Glu, and Leu575Trp) in the big subunit of feedback-resistant acetohydroxy acid synthase. The mutated threonine dehydratase over-expressed in E. coli not only showed completely resistance to l-isoleucine inhibition, but also showed enhanced activity. The mutated acetohydroxy acid synthase over-expressed in E. coli showed more resistance to l-isoleucine inhibition than the wild type. Over-expression of the feedback-resistant threonine dehydratase or acetohydroxy acid synthase in C. glutamicum JHI3-156 led to increase of l-isoleucine production; co-expression of them in C. glutamicum JHI3-156 led to 131.7% increase in flask cultivation, and could produce 30.7g/L l-isoleucine in 72-h fed-batch fermentation. These results would be useful to enhance l-isoleucine production in C. glutamicum.     

The energy-yielding reactions of Peptococcus prévotii,their behaviour on starvation and the role and regulation of threonine dehydratase

The principal energy-yielding reactions of the strict anaerobe Peptococcus prévotii comprised the fermentation of l-serine and l-threonine via the enzymes threonine dehydratase, thioclastic enzyme, phosphotransacetylase and acetate kinase.Threonine dehydratase was purified 700-fold and shown to require pyridoxal 5-phosphate as co-enzyme, and a reducing agent for optimum activity. The ratio of threonine and serine dehydratase activities was unaltered during purification. The optimum pH was 8.5 to 9.5 and isoleucine did not inhibit.Lineweaver-Burk plots were linear at l-threonine concentrations above 1.35 mM and the K _m for threonine was 2.5 mM and for serine 29 mM. Below this concentration co-operativity occurred which was not nullified by individual adenine nucleotides: Hill plots were biphasic.However, the enzyme was controlled by the adenylate energy charge in a novel manner; only at very low threonine concentrations (<1 mM) was control manifest, when a high energy charge inhibited and a low energy charge stimulated activity.During starvation for 33 hrs in phosphate buffer, pH 6.8, viability fell to zero but, of the enzymes of the energy-generating sequence, only the total units and specific activity of threonine dehydratase decreased (by 35%), which was insufficient to explain the loss of ability to generate ATP.     

Mixotrophy in the phototrophic harmful alga Cochlodinium polykrikoides (Dinophycean): prey species, the effects of prey concentration, and grazing impact

We first reported here that the harmful alga Cochlodinium polykrikoides, which had been previously known as an autotrophic dinoflagellate, was a mixotrophic species. We investigated the kinds of prey species and the effects of the prey concentration on the growth and ingestion rates of C. polykrikoides when feeding on an unidentified cryptophyte species (Equivalent Spherical Diameter, ESD = 5.6 microm). We also calculated grazing coefficients by combining field data on abundances of C. polykrikoides and co-occurring cryptophytes with laboratory data on ingestion rates obtained in the present study. Cocholdinium polykrikoides fed on prey cells by engulfing the prey through the sulcus. Among the phytoplankton prey offered, C. polykrikoides ingested small phytoplankton species that had ESD's < or = 11 microm (e.g. the prymnesiophyte Isochrysis galbana, an unidentified cryptophyte, the cryptophyte Rhodomonas salina, the raphidophyte Heterosigma akashiwo, and the dinoflagellate Amphidinium carterae). It did not feed on larger phytoplankton species that had ESD's > or = 12 microm (e.g. the dinoflagellates Heterocapsa triquetra, Prorocentrum minimum, Scrippsiella sp., Alexandrium tamarense, Prorocentrum micans, Gymnodinium catenatum, Akashiwo sanguinea, and Lingulodinium polyedrum). Specific growth rates of C. polykrikoides on a cryptophyte increased with increasing mean prey concentration, with saturation at a mean prey concentration of approximately 270 ng C ml(-1) (i.e. 15,900 cells ml(-1)). The maximum specific growth rate (mixotrophic growth) of C. polykrikoides on a cryptophyte was 0.324 d(-1), under a 14:10 h light-dark cycle of 50 microE m(-2) s(-1), while its growth rate (phototrophic growth) under the same light conditions without added prey was 0.166 d(-1). Maximum ingestion and clearance rates of C. polykrikoides on a cryptophyte were 0.16 ng C grazer(-1)d(-1) (9.4 cells grazer(-1)d(-1)) and 0.33 microl grazer(-1)h(-1), respectively. Calculated grazing coefficients by C. polykrikoides on cryptophytes were 0.001-0.745 h(-1) (i.e. 0.1-53% of cryptophyte populations were removed by a C. polykrikoides population in 1 h). The results of the present study suggest that C. polykrikoides sometimes has a considerable grazing impact on populations of cryptophytes.     

A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K12 defective in the PheA gene

Three classes of mutant strains of Escherichia coli K12 defective in pheA, the gene coding for chorismate mutase/prephenate dehydratase, have been isolated: (1) those lacking prephenate dehydratase activity, (2) those lacking chorismate mutase activity, and (3) those lacking both activities. Chorismate mutase/prephenate dehydratase from the second class of mutants was less sensitive to inhibition by phenylalanine than wild-type enzyme and, along with the defective enzyme from the third class of mutants, could not be purified by affinity chromatography on Sepharosyl-phenylalanine. Pure chorismate mutase/prephenate dehydratase protein was prepared from two strains belonging to the first class. The chorismate mutase activity of these enzymes is kinetically similar to that of the wild-type enzyme except for a two- to threefold increase in both the K_a for chorismate and the K_is for inhibition by prephenate. In both cases only one change in the tryptic fingerprint was detected, resulting from a substitution of the threonine residue in the peptide Gln·Asn·Phe·Thr·Arg. This suggests that this residue is catalytically or structurally essential for the dehydratase activity.     

Dominance and recessiveness at the protein level in mutant x wildtype crosses in Sacchaomyces cerevisiae

Summary The is ₁-locus of the yeast Saccharomyces cerevisiae is the structural gene for threonine dehydratase. is ₁-mutants require isoleucine for growth and do not have active threonine dehydratase.Interallelic complementation is frequent among is ₁-mutants. This is indicative for an aggregate or multimeric structure of yeast threonine dehydratase.Complementing and non-complementing mutants were crossed to wildtype. Properties of threonine dehydratase were assayed in crude extracts of the resulting heterozygotes.Specific activities varied considerably between full wildtype activity and a level about 10% of that. The apparent Michaelis constants were increased in many heterozygotes. This effect was probably due to the aggregation of both mutant and wildtype subunits to form a hybrid threonine dehydratase with reduced substrate affinity in addition to pure wildtype enzyme. This notion is supported by the observation in one heterozygote of two enzyme fractions with increased Michaelis constants in addition to a wildtype-like fraction.The possible formation of hybrid enzymes with normal, reduced or no activity is considered to blur gene dosage relations.A given pair of alleles in a heterozygous cell can generate a new type of enzyme with properties not encountered in the corresponding two homozygous cells. This situation is not accounted for by the classical concepts of dominant-recessive or intermediate behaviour, because the difference between the heterozygotes and the homozygotes is not necessarily only quantitativ but also qualitative.We dedicate this publication to Prof. Dr. C. Auerbach on occasion of her official retirement in admiration for her pioneer work and many contribution to genetics.     

Amino acid metabolism in the thermophilic phototroph,Chloroflexus aurantiacus: properties and metabolic role of two l-threonine (l-serine) dehydratases

Two l-threonine (l-serine) dehydratases (EC 4.2.1.16) of the thermophilic phototrophic bacterium Chloroflexus aurantiacus Ok-70-fl were purified to electrophoretic homogeneity by procedures involving anion exchange and hydrophobic interaction chromatography. Only one of the two enzymes was sensitive to inhibition by l-isoleucine (K _i=2 M) and activation by l-valine. The isoleucine-insensitive dehydratase was active with l-threonine (K _m=20 mM) as well as with l-serine (K _m=10 mM) whereas the other enzyme, which displayed much higher affinity to l-threonine (K _m=1.3 mM), was inactivated when acting on l-serine. Both dehydratases contained pyridoxal-5-phosphate as cofactor. When assayed by gel filtration techniques at 20 to 25° C, the molecular weights of both enzymes were found to be 106,000±6,000. In sodium dodecylsulfate-polyacrylamide gel electrophoresis, the two dehydratases yielded only one type of subunit with a molecular weight of 55,000±3,000. The isoleucine-insensitive enzyme was subject to a glucose-mediated catabolite repression.Abbreviations A absorbance - ile isoleucine - PLP pyridoxal-5-phosphate - SDS sodium dodecyl sulfate - TDH threonine dehydratase - U unit     

The iron-sulfur-cluster-containing L-serine dehydratase from Peptostreptococcus asaccharolyticus. Stereochemistry of the deamination of L-threonine.

The stereochemistry of the deamination of L-threonine to 2-oxobutyrate, catalyzed by purified L-serine dehydratase of Peptostreptococcus asaccharolyticus, was elucidated. For this purpose the enzyme reaction was carried out with unlabelled L-threonine in 2H2O and in 3HOH, as well as with L-[3-3H]threonine in unlabelled water. Isotopically labelled 2-oxobutyrate thus formed was directly reduced in a coupled reaction with L- or D-lactate dehydrogenase and NADH. The (2R)- or (2S)-2-hydroxybutyrate species obtained were then subjected to configurational analyses of their labelled methylene group. The results from 1H-NMR spectroscopy and, after degradation of 2-hydroxybutyrate to propionate, the transcarboxylase assay consistently indicated that the deamination of L-threonine catalyzed by L-serine dehydratase of P. asaccharolyticus proceeds with inversion and retention in a 2:1 ratio. This partial racemization is the first ever to be observed for a reaction catalyzed by serine dehydratase, therefore confirming the distinction of the L-serine dehydratase of P. asaccharolyticus as an iron-sulfur protein from those dehydratases dependent on pyridoxal phosphate. For the latter enzymes exclusively, retention has been reported.