Francois R. Stoeber

Purification et propriétés de la d-mannonate hydrolyase d'Escherichia coli☆

Abstract 1. 1. d -Mannonate hydrolyase (EC 4.2.1.8), an enzyme of the hexuronate pathway, has been purified by ammonium sulfate precipitation, chromatography on DEAE-cellulose and Sephadex G-200 gel filtration. The overall procedure results in a 61-fold purification with a yield of 34%. 2. 2.Optimal conditions for enzyme assay have been established : glycylglycine buffer at pH 8.3, in the presence of ferrous sulfate and 2-mercaptoethanol as activators and protector. 3. 3.Mannonate hydrolyase is inducible by glucuronate and fructuronate, but not by galacturonate and tagaturonate. It has a maximum activity between pH 7 and 8.5. Among numerous compounds tested, only mannonate is a substrate with a K m of 20 mM. Several inhibitors of the enzyme have been studied, and the following three are competitive inhibitors: d -gluconate, d -mannitol and d -sorbitol. Some intermediates of the degradative hexuronate pathway activate the mannonate hydrolyase: d -glucuronate, d -galacturonate, d -tagaturonate and d -altronate. 4. 4.Thermal inactivation of mannonate hydrolyase in the presence of urea indicates that at last 85% of the activity of the enzyme is homogenous. The protection against heat denaturation by the competitive inhibitor d -gluconate confirms the nature of the inhibition. 5. 5.The enzyme is inactivated by 1 mM p -chloromercuribenzoate. 6. 6.The product of enzyme action on d -mannonate has been identified as 2-keto-3-deoxy- d -gluconate by paper chromatography.

Localisation génétique et caractérisation biochimique de mutations affectant le gène de structure de l'hydrolyase altronique chez Escherichia coli K 12

SummaryMutants of E. coli specifically deficient for the enzyme altronic hydrolyase have been isolated. These strains are unable to metabolize galacturonate but still normally grow on glucuronate; inducibility for the other galacturonate-induced enzymes is not modified.The position of these mutations called “uxaA” locus in relation to the argG, tolC and metC markers was established both by sexual crosses and by P1 transduction; evidence is presented that uxaA is located between argG and tolC.Biochemical characterization of uxaA mutants using thermosensible revertants or mutants was achieved by means of thermal inactivation and kinetic parameters determination. Experimental results strongly suggest that uxaA locus is the structural gene of the altronic hydrolyase enzyme.

Etude du rameau dégradatif commun des hexuronates chez Escherichia coli K 12: Purification, propriétés et individualité de la 2-Céto-3-Désoxy-D-Gluconokinase

Summary The investigation of hexuronate metabolism in E. coli K 12 has been completed by studying the KDG-kinase. This enzyme has been partially purified from glucuronate or galacturonate grown cells. We have established on 60 fold purified kinase fractions the basic biochemical properties (stability, optimal pH, cofactor, Km, specificity). Further, we have shown that the KDG-kinase which is induced by either galacturonate or glucuronate, is controlled by a single structural gene. This result is based on the following arguments: First, the mixture of purified kinase fractions from cells, which were separately induced by each hexuronate, is chromatographically not dissociable. Secondly, the kinetics of the thermal denaturation of each of the crude extracts and of their mixture are absolutely identical. Thirdly a KDG-kinase negative mutant has lost the faculty of utilizing both hexuronates, and the spontaneous reversion of this mutant on one of two hexuronates restores the growth on the other compound. These independent arguments show the uniqueness of the induction mechanism of KDG-kinase whatever the metabolized hexuronate.

[46] d-Mannonate and d-altronate dehydratases of Escherichia coli K12

Publisher Summary This chapter describes an assay method and the purification procedure for D-mannonate and D-altronate dehydratases of Escherichia coli K 12, which act, respectively, on D-mannonate and D-altronate and yield 2-keto-3-deoxy-D-gluconate. Mannonate and altronate dehydratase activities are estimated by formation of 2-keto-3-deoxy-D-gluconate (KDG) from D-mannonate and D-altronate, respectively; these methods show that determination of KDG complexed with thiobarbituric acid provides a sensitive measure of the reaction rate. The purification procedure of the enzyme involves preparation of crude extract, nucleic acid precipitation, ammonium sulfate fractionation, diethylaminoethyl (DEAE)-cellulose chromatography, and Sephadex gel filtration. At 52 ° C, the half-life of mannonate dehydratase in buffer III is 2 minutes, and at 59 ° C, the mannonate dehydratase in the crude extract is very rapidly inactivated. The mannonate dehydratase exhibits maximum activity and stability between pH 7.0 and 8.5; the altronate dehydratase exhibits maximum activity at pH 8.3. The mannonate dehydratase and the altronate dehydratase appear to be highly specific for o-mannonate and D-altronate, respectively.

Individualité des hydrolyases mannonique et altronique chez Escherichia coli K 12

Summary In Escherichia coli, mannonic and altronic hydrolyases act, respectively, on mannonate, the intermediate aldonate of the glucuronate branch, and on altronate the intermediate aldonate of the galacturonate branch of the hexuronate pathway, yielding 2-keto-3-deoxy-gluconate. Our results demonstrate that the two hydrolyases are two distinct proteins. First, each hydrolyase shows a different induction pattern. In addition, separate constitutive mutants for either hydrolyase have been obtained. Second, single mutants negatively affected for one of the activities but not the other have been isolated in each case. Third, comparative heat inactivation of both activities at 59°C shows mannonic hydrolyase to be clearly more thermosensitive than altronic hydrolyase. Furthermore the two enzymes also react differently to various effectors. Fourth, the two enzymes could be resolved on a DEAE cellulose column into two neighbouring but distinct peaks of activity, and a further purification yielded two pure hydrolyase fractions each being devoid of the activity of the other.

Individualité de la D-glucuronate-cétol isomerase d'Escherichia coli K 12

Summary The individuality of uronic isomerase (D-glucuronate-ketol isomerase) in Escherichia coli K 12, acting on both substrates: glucuronate and galacturonate, and induced either by the first of by the second hexuronate, is demonstrated on the basis of the following results: u — an identical thermostability of the isomerase activity toward either hexuronate and whatever the inducer may be — a constant ratio of purification between both activities measured through the successive steps: protamine sulfate, ammonium sulfate, heat and DEAE-Sephadex chromatography; occurence of a single and homogeneous enzyme peak after chromatography of the mixture of extracts from cells which have been induced by either hexuronate — a single mutation (mapped between arg G and met C) simultaneously suppresses both activities and their induction by both hexuronates; a reverse mutation restores both activities and their induction by both hexuronates.

Mutations affectant le gène de structure de la 2-céto-3-désoxy-6-P-gluconate aldolase chez E. coli K 12

SummaryIn order to determine the nature of KDPG-aldolase negative mutations (described in a recent paper) we have studied revertants to wild type. The structure of restored KDPG-aldolase in two revertants is very different with regard to wild type aldolase activity (modified thermosensibility, Km and VM). These restored aldolases like the wild type aldolase are under the control of the regulator gene (kdg R). The observation that one of these revertants maps in the eda locus demonstrates that this locus is the structural gene of KDPG-aldolase in E. coli K 12.

Dosages colorimétriques des oxydoréductase aldoniques d'Escherichia coli K 12 : Applications

Abstract Calorimetric estimations of aldonate oxidoreductases from Escherichia coli K 12 : Applications Altronate (EC 1.1.1.58) and mannonate oxidoreductases (EC 1.1.1.57), two NAD + enzymes involved in the hexuronate pathway of E. coli K 12, respectively catalyze the reversible dehydrogenation of altronate or mannonate into tagaturonate or fructuronate. 1. 1. The optimal conditions for quantitative colorimetric assays of these activities utilizing phenazine methosulfate as an intermediate agent for electron transfer from NADH to p -nitro blue tetrazolium have been determined. In 02 M glycylglycine buffer (pH 9.0) in the presence of gelatin (10 or 20 μg/ml), phenazine methosulfate (33μM) and 3,3′-(3,3′-dimethoxy-4,4′-biphenylene)-bis[2-( p -nitrophenyl)-5-phenyl-2H-tetrazolium] (0.25 or 05. mM), the apparent Michaelis constant for substrates are: K m (NAD + = 88 μ M and K m ( altronate ) = 90 μ M for altronate oxidoreductase; K m (NAD + ) = 1 mM and K m (mannonate) = 1.7 mM for mannonate oxidoreductase. 2. 2. Applications for these new methods in various fields such as aldonate estimations, rapid and visual detection of aldonate oxidoreductases and characterization specific mutants in the hexuronate pathway of E. coli are discussed.