Formation and cleavage of 2-keto-3-deoxygluconate by 2-keto-3-deoxygluconate aldolase of Aspergillus niger.

2-Keto-3-deoxygluconate aldolase of Aspergillus niger, an enzyme that has not been reported previously, was purified 468-fold. Maximal activity was obtained at pH 8.0 and 50 C. The enzyme exhibited relative stereochemical specificity with respect to glyceraldehyde. The Km values for 2-keto-3-deoxygluconate, glyceraldehyde, and pyruvate were 10, 13.3, and 3.0 mM, respectively. The effects of some compounds and inhibitors on enzyme activity were examined. Stability of the enzyme under different conditions was investigated. The equilibrium constant was about 0.33 X 10(-3) M.     

Specificity of 2-keto-3-deoxygluconate-6-P aldolase for open chain form of 2-keto-3-deoxygluconate-6-P.

2-Keto-3-deoxygluconate-6-P exists as an euqilibrium of three forms at 25 degrees measurable by 13C NMR: alpha-furanose anomer (41%), beta-furanose anomer (50%), and open chain keto (9%). The three forms are interconverted rapidly (greater than 0.5 s-1) so that the unidirectional rates of furanose ring opening and closing can be quantitated by an NMR line broadening method. The 2-keto-3-deoxygluconate aldolase is specific for only one of these forms, the open chain keto form. The rates for ring opening calculated from the rapid kinetic enzyme system compare closely with those obtained with the NMR method.     

2-keto-3-deoxygluconate transport system in Erwinia chrysanthemi.

In Erwinia chrysanthemi, the gene kdgT encodes a transport system responsible for the uptake of ketodeoxyuronates. We studied the biochemical properties of this transport system. The bacteria could grow on 2,5-diketo-3-deoxygluconate but not on 2-keto-3-deoxygluconate. The 2-keto-3-deoxygluconate entry reaction displayed saturation kinetics, with an apparent Km of 0.52 mM (at 30 degrees C and pH 7). 5-Keto-4-deoxyuronate and 2,5-diketo-3-deoxygluconate appeared to be competitive inhibitors, with Kis of 0.11 and 0.06 mM, respectively. The 2-keto-3-deoxygluconate permease could mediate the uptake of glucuronate with a low affinity. kdgT was cloned on an R-prime plasmid formed by in vivo complementation of a kdgT mutation of Escherichia coli. After being subcloned, it was mutagenized with a mini-Mu-lac transposable element able to form fusions with the lacZ gene. We introduced a kdgT-lac fusion into the E. chrysanthemi chromosome by marker exchange recombination and studied its regulation. kdgT product synthesis was not induced by external 2-keto-3-deoxygluconate in the wild-type strain but was induced by galacturonate and polygalacturonate. Two types of regulatory mutants able to grow on 2-keto-3-deoxygluconate as the sole carbon source were studied. Mutants of one group had a mutation in the operator region of kdgT; mutants of the other group had a mutation in kdgR, a regulatory gene controlling kdgT expression.     

Enzymatic synthesis of 2-keto-3-deoxy-d-glucose from D-gluconate

2-Keto-3-deoxy-d-gluconate (KDG¹) was prepared from d-gluconate with a cell-free extract of Clostridium pasteurianum containing gluconate dehydratase. This enzyme is present in gluconate-grown cells in high activity. Because of the heat tolerance of the dehydratase KDG synthesis could be carried out at 50°C. Sodium-KDG was isolated from the reaction mixture as crude product with a yield of 96%. The crystalline free acid was prepared from the sodium salt in 40% yield.     

On the interaction of 2-keto-3-deoxygalactonate-6P with 2-keto-3-deoxygluconate-6P aldolase ofPseudomonas putida. Evidence for enzyme-mediated,noncatalytic C-C bond turnover

2-Keto-3-deoxygluconate-6P (KDPG) aldolase ofPseudomonas putida mediates the cleavage of, as well as the condensation of, pyruvate andd-glyceraldehyde-3P (GaP) yielding, 2-keto-3-deoxygalactonate-6P (KDPGal) as side reactions of normal catalysis. These are visualized at high levels of aldolase. KDPGal cleavage occurs with aV _max that is 1/5000 that for KDPG cleavage. TheKm for KDPGal is 0.2 mM, with aK _i of 0.85 mM. The E-KDPGal complex is reductively inactivated having aKd of 0.55 mM. TheV/K value for KDPG cleavage is 2.0×10⁸ sec^?1, while the value for KDPGal cleavage is 1220 sec^?1. The difference in first-order rate constants of 164,000-fold argues that a step in the cleavage of KDPGal mediated by the enzyme is uncatalyzed. The enzyme is reductively inactivated by trapping the E-pyruvate, E-KDPG, or E-KDPGal complex. The enzyme can also be inactivated by reductive trapping of a catalytically nonproductive E-glyceraldehyde-3P complex. This latter occurs with aKd for GaP of 20 mM and a rate constant equivalent to a limiting half-time of 1110 sec at 1 mM cyanoborohydride. Reductive inactivation half-times in the presence of high GaP/KDPG ratios were the sum of both E-GaP and E-KDPG trapping by cyanoborohydride so that the inactivation rate due to KDPG could be determined. It was found at 1 mM cyanoborohydride that the limiting half-time for the E-KDPG complex was 2382 sec. The corresponding value for the E-KDPGal complex was 215 sec. Consequently, the E-KDPGal complex is 11 times more sensitive to reductive derivativation than is the E-KDPG complex. This is interpreted to show that the enzyme binds the KDPGal in a “normal” step forming a ketimine. However, turnover to the eneamine with resultant C-C bond cleavage is uncatalyzed. For the case of KDPGal synthesized by KDPG aldolase, it is argued that the pyruvate eneamine is bound to the active site, which can be attacked by GaP with its aldehyde carbon in the catalytically nonproductive conformation as a side reaction, presumably forming a tertiary complex. Spontaneous protonation of the resultant alcoholate anion would generate KDPGal. The data are interpreted to support an argument that catalytic proton turnover at the OH of C-4 of KDPG is required for normal catalysis, and that this step, which catalytically interconverts ketimine/eneamine, imposes steric constraints controlling the overall stereochemistry of the reaction.     

Occurrence of 2-keto-3-deoxy-D-manno-octonic acid in lipopolysaccharides isolated from Vibrio parahaemolyticus.

The occurrence of 2-keto-3-deoxy-D-manno-octonic acid (KDO) in lipopolysaccharides (LPS) of Vibrio parahaemolyticus was demonstrated for the first time by gas chromatography-mass spectrometry after dephosphorylation, reduction, and methylation. KDO was virtually completely phosphorylated, since no KDO was detected by either gas chromatography or thiobarbituric acid assay before dephosphorylation. The level of KDO in all six strains of V. parahaemolyticus investigated ranged from 0.37 to 0.69%, which was considerably lower than that in enterobacterial LPS.     

Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase

Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 °C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 °C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40–60 °C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.     

Escherichia coli K-12 structural kdgT mutants exhibiting thermosensitive 2-keto-3-deoxy-D-gluconate uptake.

A specific method is described for selecting thermosensitive mutants of Escherichia coli K-12 able to grow on 2-keto-3-deoxy-D-gluconate (KDG) and D-glucuronate at 2, but not at 42 degrees C. The extensive analysis of one such mutant is consistent with the conclusion that the carrier molecule responsible for KDG and glucuronate uptake becomes thermolabile. (i) Growth on a variety of carbon sources is perfectly normal at 28 and 42 degrees C, whereas in the same temperature range it gradually diminishes on KDG and glucuronate. (ii) The apparent Km value for KDG is about twofold in the range 25 to 40 degrees C. In the same temperature range, the Vmax values for KDG influx are higher for the mutant compared with those of the wild-type strain, but the optimum temperature is 34 degrees C instead of 38 degrees C. On the contrary, the Vmax values for glucuronate influx are lower for the mutant than for the parental strain, and the optimum temperature for both strains is shifted beyond 40 degrees C. (iii) The activation energies for KDG and glucuronate uptake are about twofold higher in the mutant than in the wild-type strain. (iv) Kinetics of counterflow under deenergized conditions (overshoot) at different temperatures indicate that the defect is located in the translocation step rather than in the processes involved in energy coupling. (v) The first-order rate constants for thermal denaturation are, respectively, 2.5- and 5-fold higher at 40 and 30 degrees C in the mutant than in the wild-type strain, and the activation energy for thermal denaturation is lower. (vi) The carrier molecule in the mutant is also much more sensitive to denaturation by N-ethylmaleimide. (vii) Four independent thermosensitive mutations and one revertatn were located by transduction in or near the kdgT locus, defined previously as the site of nonconditional KDG transport-negative mutations. These results support the conclusion that kdgT represents the structural gene coding for the KDG transport system.     

Purification of 2-keto-3-deoxy-6-phosphohexonate aldolases of Pseudomonas saccharophila

Summary KDPG aldolase has been crystallized from extracts of sucrosegrown cells of Pseudomonas saccharophila. KDPGal aldolase, which is absent in sucrose-grown cells but is present together with KDPG aldolase in galactose-grown cells has been purified over 1000-fold. The two enzymes share many common features, but possess absolute substrate specificity and are immunochemically distinct. The equilibrium constants for the catalyzed reactions have been determined and found to favor the synthesis of the 2-keto-3-deoxy-6-phosphohexonic acids.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday. It was in his laboratory that Pseudomonas saccharophila was isolated and first studied some thirty years ago. In spite of a firm faith in enrichment culture techniques and incantations learned at Pacific Grove, repeated attempts to reisolate the organism from nature have been unsuccessful. One wonders whether it was the Knallgas atmosphere used for the original enrichment or that unique atmosphere of the master's laboratory, only occasionally tainted with a whiff of H₂S or mercury vapors, that nurtured this unique bacterium.This work was supported in part by a grant from the National Institutes of Health (AI-1808) and was conducted while the authors held tenure, respectively, as Postdoctoral Fellow and as Professor in the Miller Institute for Basic Research in Science at the University of California in Berkeley.