Galactose metabolism in Rhizobium meliloti L5-30.

Data from previous studies of Rhizobium meliloti mutants have been consistent with the catabolism of hexoses via the Entner-Doudoroff pathway. However, galactose metabolism was not impaired in those mutants. We show here by enzymatic assay and by identification of a galactose mutant lacking 2-keto-3-deoxy-6-phosphogalactonate aldolase that the De Ley-Doudoroff pathway is used for galactose metabolism. Mutants in this pathway have not been previously reported for any organism.     

Crystal structure and stereochemical studies of KD(P)G aldolase from Thermoproteus tenax

Carbon-carbon bond forming enzymes offer great potential for organic biosynthesis. Hence there is an ongoing effort to improve their biocatalytic properties, regarding availability, activity, stability, and substrate specificity and selectivity. Aldolases belong to the class of C-C bond forming enzymes and play important roles in numerous cellular processes. In several hyperthermophilic Archaea the 2-keto-3-deoxy-(6-phospho)-gluconate (KD(P)G) aldolase was identified as a key player in the metabolic pathway. The carbohydrate metabolism of the hyperthermophilic Crenarchaeote Thermoproteus tenax, for example, has been found to employ a combination of a variant of the Embden-Meyerhof-Parnas pathway and an unusual branched Entner-Doudoroff pathway that harbors a nonphosphorylative and a semiphosphorylative branch. The KD(P)G aldolase catalyzes the reversible cleavage of 2-keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxygluconate (KDG) forming pyruvate and glyceraldehyde 3-phosphate or glyceraldehyde, respectively. In T. tenax initial studies revealed that the pathway is specific for glucose, whereas in the thermoacidophilic Crenarchaeote Sulfolobus solfataricus the pathway was shown to be promiscuous for glucose and galactose degradation. The KD(P)G aldolase of S. solfataricus lacks stereo control and displays additional activity with 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) and 2-keto-3-deoxygalactonate (KDGal), similar to the KD(P)G aldolase of Sulfolobus acidocaldarius. To address the stereo control of the T. tenax enzyme the formation of the two C4 epimers KDG and KDGal was analyzed via gas chromatography combined with mass spectroscopy. Furthermore, the crystal structure of the apoprotein was determined to a resolution of 2.0 A, and the crystal structure of the protein covalently linked to a pathway intermediate, namely pyruvate, was determined to 2.2 A. Interestingly, although the pathway seems to be specific for glucose in T. tenax the enzyme apparently also lacks stereo control, suggesting that the enzyme is a trade-off between required catabolic flexibility needed for the conversion of phosphorylated and nonphosphorylated substrates and required stereo control of cellular/physiological enzymatic reactions.     

Mutational Analysis of the Pentose Phosphate and Entner-Doudoroff Pathways in Gluconobacter oxydans Reveals Improved Growth of a {Delta}edd {Delta}eda Mutant on Mannitol

The obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H oxidizes sugars and sugar alcohols primarily in the periplasm, and only a small fraction is metabolized in the cytoplasm. The latter can occur either via the Entner-Doudoroff pathway (EDP) or via the pentose phosphate pathway (PPP). The Embden-Meyerhof pathway is nonfunctional, and a cyclic operation of the tricarboxylic acid cycle is prevented by the absence of succinate dehydrogenase. In this work, the cytoplasmic catabolism of fructose formed by oxidation of mannitol was analyzed with a Δgnd mutant lacking the oxidative PPP and a Δedd Δeda mutant devoid of the EDP. The growth characteristics of the two mutants under controlled conditions with mannitol as the carbon source and enzyme activities showed that the PPP is the main route for cytoplasmic fructose catabolism, whereas the EDP is dispensable and even unfavorable. The Δedd Δeda mutant (lacking 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) formed 24% more cell mass than the reference strain. In contrast, deletion of gnd (6-phosphogluconate dehydrogenase) severely inhibited growth and caused a strong selection pressure for secondary mutations inactivating glucose-6-phosphate dehydrogenase, thus preventing fructose catabolism via the EDP also. These Δgnd zwf* mutants (with a mutation in the zwf gene causing inactivation of the glucose-6-phosphate dehydrogenase) were almost totally disabled in fructose catabolism but still produced about 14% of the carbon dioxide of the reference strain, possibly by catabolizing substrates from the yeast extract. Overexpression of gnd in the reference strain improved biomass formation in a similar manner as deletion of edd and eda, further confirming the importance of the PPP for cytoplasmic fructose catabolism.     

Carbohydrate catabolism of Mima polymorpha. II. Abortive catabolism of glucose

Marus, Adrienne (University of Cincinnati, Cincinnati, Ohio), and Emily J. Bell. Carbohydrate catabolism of Mima polymorpha. II. Abortive catabolism of glucose. J. Bacteriol. 91:2229-2236. 1966.-Mima polymorpha, unable to grow in the presence of glucose as a sole carbon and energy source, is able to obtain supplemental, utilizable energy from the partial catabolism of this substrate. Various enzymes of hexose catabolism have been assayed in this organism and in M. polymorpha M, a mutant obtained by ultraviolet irradiation. The parent strain contains a functional glucose dehydrogenase, glucose-6-phosphate dehydrogenase, diphosphofructoaldolase, and a 2-keto-3-deoxy-6-phosphogluconate aldolase, but is lacking in glucokinase, gluconokinase, 2-ketogluconokinase, and 6-phosphogluconate dehydrogenase. The enzymes present indicate partially functioning hexose diphosphate and Entner-Doudoroff pathways. The absence of kinases explains the inability of the strain to grow on glucose and an absence of 6-phosphogluconate dehydrogenase would indicate the absence of the complete pentose pathway. The mutant strain, M. polymorpha M, possesses, in addition to those enzymes produced by the wild type, both gluconokinase and 6-phosphogluconate dehydrogenase. The presence of the former explains the mutant's ability to grow on glucose, and the presence of the latter indicates a more complete pentose shunt. The supplemental energy obtained from partial glucose catabolism (to gluconic acid) may be obtained from a cytochrome-linked reaction of the glucose dehydrogenase.     

Analysis of cloned structural and regulatory genes for carbohydrate utilization in Pseudomonas aeruginosa PAO.

Five of the genes required for phosphorylative catabolism of glucose in Pseudomonas aeruginosa were ordered on two different chromosomal fragments. Analysis of a previously isolated 6.0-kb EcoRI fragment containing three structural genes showed that the genes were present on a 4.6-kb fragment in the order glucose-binding protein (gltB)-glucokinase (glk)-6-phosphogluconate dehydratase (edd). Two genes, glucose-6-phosphate dehydrogenase (zwf) and 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), shown by transductional analysis to be linked to gltB and edd, were cloned on a separate 11-kb BamHI chromosomal DNA fragment and then subcloned and ordered on a 7-kb fragment. The 6.0-kb EcoRI fragment had been shown to complement a regulatory mutation, hexR, which caused noninducibility of four glucose catabolic enzymes. In this study, hexR was mapped coincident with edd. A second regulatory function, hexC, was cloned within a 0.6-kb fragment contiguous to the edd gene but containing none of the structural genes. The phenotypic effect of the hexC locus, when present on a multicopy plasmid, was elevated expression of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase activities in the absence of inducer.     

Improving upon nature: active site remodeling produces highly efficient aldolase activity toward hydrophobic electrophilic substrates

The substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm. We identified two active site mutations (T161S and S184L) that work additively to enhance the substrate specificity of this aldolase to include catalysis of retro-aldol cleavage of (4S)-2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (S-KHPB). These mutations improve the value of k(cat)/K(M)(S-KHPB) by >450-fold, resulting in a catalytic efficiency that is comparable to that of the wild-type enzyme with the natural substrate while retaining high stereoselectivity. Moreover, the value of k(cat)(S-KHPB) for this mutant enzyme, a parameter critical for biocatalytic applications, is 3-fold higher than the maximal value achieved by the natural aldolase with any substrate. This mutant also possesses high catalytic efficiency for the retro-aldol cleavage of the natural substrate, KDPG, and a >50-fold improved activity for cleavage of 2-keto-4-hydroxy-octonoate, a nonfunctionalized hydrophobic analogue. These data suggest a substrate binding mode that illuminates the origin of facial selectivity in aldol addition reactions catalyzed by KDPG and 2-keto-3-deoxy-6-phosphogalactonate aldolases. Furthermore, targeting mutations to the active site provides a marked improvement in substrate selectivity, demonstrating that structure-guided active site mutagenesis combined with selection techniques can efficiently identify proteins with characteristics that compare favorably to those of naturally occurring enzymes.     

Enzymes related to galactose utilization inPseudomonas cepacia

Pseudomonas cepacia produced a characteristic green sheen on EMB-galactose plates owing to production of galactonic acid by the constitutive membrane-associated glucose dehydrogenase of this bacterium. Mutants isolated as glucose dehydrogenase deficient (Gcd^–) also were deficient in membrane-associated galactose dehydrogenase. A strain that formed glucose dehydrogenase at 30°C but not at 40°C was also temperature sensitive with respect to formation of galactose dehydrogenase. The Gcd^– strains still utilized galactose. A second, NAD-specific, galactose dehydrogenase (not membrane associated) along with a transport system for galactose were induced during growth on galactose and constituted an alternative pathway of conversion of galactose to galactonate. Enzymes of the De Ley-Doudoroff pathway of conversion of galactonate to pyruvate and glyceraldehyde-3-phosphate were induced during growth on galactose. Unexpectedly, growth on galactose also elicited formation of enzymes of the Entner-Doudoroff (ED) route. Furthermore, mutants blocked in the ED pathway grew poorly on galactose. One interpretation of these findings is that glyceraldehyde-3-phosphate formed from galactose via the De Ley-Doudoroff route (by cleavage of 2-keto-3-deoxy-6-phosphogalaconate) is reconverted to hexose phosphate and metabolized via the ED pathway.     

Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase

The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.     

The Deficient Carbohydrate Metabolic Pathways and the Incomplete Tricarboxylic Acid Cycle in an Obligately Autotrophic Hydrogen-oxidizing Bacterium

The activities of enzymes of carbohydrate metabolism, enzymes of the tricarboxylic acid cycle and some related enzymes were measured in cell-free extracts of strain TK-6, an extremely thermophilic, obligately autotrophic, aerobic hydrogen-oxidizing bacterium. Activities of phosphofructokinase, aldolase, pyruvate kinase, 6-phosphogluconate dehydrase and 2-keto-3-deoxy-6-phosphogluconate aldolase, key enzymes of the Embden-Meyerhof and the Entner-Doudoroff pathways were not found in the extracts. All of the tricarboxylic acid cycle enzymes except α-ketoglutarate dehydrogenase, and reduced nicotinamide adenine dinucleotide oxidase were present. These metabolic defects are considered to be one of the reasons for the obligate autotrophy of strain TK-6.     

Utilization of gluconate by Escherichia coli. Induction of gluconate kinase and 6-phosphogluconate dehydratase activities

1. A mutant of Escherichia coli, devoid of phosphopyruvate synthetase, glucosephosphate isomerase and 6-phosphogluconate dehydrogenase activities, grew readily on gluconate and inducibly formed an uptake system for gluconate, gluconate kinase and 6-phosphogluconate dehydratase while doing so. 2. This mutant also grew on glucose 6-phosphate and inducibly formed 6-phosphogluconate dehydratase; however, the formation of the gluconate uptake system and gluconate kinase was not induced under these conditions. 3. The use of the Entner–Doudoroff pathway for the dissimilation of 6-phosphogluconate, derived from either gluconate or glucose 6-phosphate, by this mutant was also demonstrated by the accumulation of 2-keto-3-deoxy-6-phosphogluconate (3-deoxy-6-phospho-l-glycero-2-hexulosonate) from both these substrates in a similar mutant that also lacked phospho-2-keto-3-deoxygluconate aldolase activity. 4. Glucose 6-phosphate inhibits the continued utilization of fructose by cultures of the mutants growing on fructose, as it does in wild-type E. coli. 5. The mutants do not use glucose for growth. This is shown to be due to insufficiency of phosphopyruvate, which is required for glucose uptake.     

Glucose Catabolism in Strains of Acidophilic, Heterotrophic Bacteria

Pathways of glucose catabolism, potentially operational in six strains of obligately aerobic, acidophilic bacteria, including Acidiphilium cryptum strain Lhet2, were investigated by short-term radiorespirometry and enzyme assays. Short-term radiorespirometry was conducted at pH 3.0 with specifically labeled [¹⁴C]glucose. The high rate and yield of C-1 oxidized to CO₂ indicated that the Entner-Doudoroff, pentose phosphate, or both pathways were operational in all strains. Apparent nonequivalent yields of CO₂ from C-1 and estimated CO₂ from C-4 (C-1 > C-4) were suggestive of simultaneous glucose catabolism by both pathways in all strains tested. Variation in the relative contribution of the two pathways of glucose catabolism appears to account for observed strain differences. Calculation of the actual percent pathway participation was not feasible. Enzyme assays were completed with crude extracts of glucose-grown cells to substantiate the results obtained by radiorespirometry. The key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase) and the Entner-Doudoroff pathway (2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrase) were present in all strains examined (PW2, Lhet2, KLB, OP, and QBP). However, none of the strains exhibited detectable levels of the key enzyme of the Embden-Meyerhof-Parnas pathway, 6-phosphofructokinase. All strains contained glucose-6-phosphate dehydrogenase and fructose bisphosphate aldolase. The results of the enzyme study supported the contention that the pentose phosphate and Entner-Doudoroff pathways are operational for glucose catabolism in the acidophilic heterotrophs, and that the Embden-Meyerhof-Parnas pathway is apparently absent.     

Heterotrophic Metabolism of the Chemolithotroph Thiobacillus ferrooxidans

Glucose-6-phosphate dehydrogenase and the enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydrase and 2-keto-3-deoxy-6-phosphogluconate aldolase (assayed together), are induced during heterotrophic growth of Thiobacillus ferrooxidans on an iron-glucose-supplemented medium or on glucose alone. By contrast, autotrophic cells (iron-grown) contain low levels of these enzymes. Fructose 1, 6-diphosphate aldolase, an enzyme of the Embden-Meyerhof pathway, is present at low levels irrespective of the growth medium, suggesting that this enzyme is not involved in energy-yielding reactions but merely provides intermediates for biosynthesis. The Entner-Doudoroff and pentose-phosphate pathways are the principle means through which glucose is dissimilated and is presumed to be concerned with energy production. Isotopic studies showed that a high rate of CO(2) formation from specifically labeled glucose came from carbon atoms 1 and 4. An unexpectedly high rate of evolution of CO(2) also came from carbon 6, suggesting that the triose phosphate formed during glucose breakdown and specifically as a result of 2-keto-3-deoxy-6-phosphogluconate aldolase activity, was metabolized via some unorthodox metabolic route. Cells grown in the iron-supplemented and glucose-salts media have a complete tricarboxylic acid cycle, whereas autotrophically grown T. ferrooxidans lacked both alpha-ketoglutarate dehydrogenase and reduced nicotinamide adenine dinucleotide oxidase. Two isocitrate dehydrogenases [nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) specific] were present. NAD-linked enzyme was constitutive, whereas the NADP-linked enzyme was induced upon adaptation of autotrophic cells to heterotrophic growth.     

Cellobiose catabolism in the haloalkaliphilic hydrolytic bacterium <Emphasis Type="Italic">Alkaliflexus imshenetskii</Emphasis>

Cellobiose metabolism was studied in Alkaliflexus imshenetskii, a haloalkaliphilic hydrolytic bacterium capable of utilizing certain polymers of plant origin, as well as mono- and disaccharides. The major products of cellobiose fermentation by the bacterium were succinate and acetate, and formate was a minor product. Cellobiose could be split into glucose molecules by both β-glucosidase (hydrolytic pathway) and phosphorylase (phosphorolytic pathway); the activity of the former enzyme was two orders of magnitude higher (3600 nmol/(min mg) versus 36 nmol/(min mg)). In cell extracts of the bacterium, high activities of the Embden-Meyerhof-Parnas pathway enzymes—hexokinase, glucose-phosphate isomerase, and phosphofructokinase—were revealed, as well as the activities of glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and key enzymes of the Entner-Doudoroff pathway—6-phospho-gluconate dehydratase and 2-keto-3-deoxy-6-phospho-gluconate aldolase. Neither the activity of the key enzyme of the hexose-mono-phosphate pathway, 6-phospho-gluconate dehydrogenase, nor the activities of the key enzymes of the modified Entner-Doudoroff pathway, glucose dehydrogenase and 2-keto-3-deoxy-gluconate kinase, were revealed.     

A biochemical study of the intermediary carbon metabolism of Shewanella putrefaciens.

Cell extracts were used to determine the enzymes involved in the intermediary carbon metabolism of several strains of Shewanella putrefaciens. Enzymes of the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) were detected, but those of the Embden-Meyerhof-Parnas pathway were not. While several tricarboxylic acid cycle enzymes were present under both aerobic and anaerobic conditions, two key enzymes (2-oxoglutarate dehydrogenase and pyruvate dehydrogenase) were greatly diminished under anaerobic conditions. Extracts of cell grown anaerobically on formate as the sole source of carbon and energy were positive for hydroxypyruvate reductase, the key enzyme of the serine pathway in other methylotrophs, while no hexulose synthase activity was seen.     

Gluconate Regulation of Glucose Catabolism in Pseudomonas fluorescens

Induction of Entner-Doudoroff pathway enzymes in Pseudomonas fluorescens was investigated to study the role of gluconate as a possible inducer. Glucose oxidase-deficient mutants were isolated and characterized. One of these mutants, gox-7, was deficient in particulate glucose oxidase; another mutant, gox-17, was deficient in particulate glucose and gluconate oxidase activities. Gluconate, but not glucose, induced synthesis of gluconokinase and 6-phosphogluconate dehydratase in both mutants. High constitutive levels of 2-keto-3-deoxy-6-phosphogluconate aldolase were found when both mutants were grown on glucose. Growth of parent and both mutant strains on glycerol also resulted in high levels of Entner-Doudoroff pathway enzymes. It was concluded that glucose cannot serve as an inducer molecule for derepression of Entner-Doudoroff pathway enzymes in P. fluorescens. Evidence presented provides good support for gluconate being the true inducer of this pathway in P. fluorescens. A relationship is presented for explaining distribution of the Entner-Doudoroff pathway in certain groups of bacteria.     

Pathways of d-fructose and d-glucose catabolism in marine species of Alcaligenes,Pseudomonas marina,and Alteromonas communis

Cell-free extracts of d-fructose grown cells of marine species of Alcaligenes as well as Pseudomonas marina contained an activity which catalyzed a P-enolpyruvate-dependent phosphorylation of d-fructose in the 1-position as well as activities of the following enzymes: 1-P-fructokinase, fructose-1,6-P₂ aldolase, PPi-dependent 6-P-fructokinase, fructokinase, glucokinase, P-hexose isomerase, glucose-6-P dehydrogenase, 6-P-gluconate dehydrase, and 2-keto-3-deoxy-6-P-gluconate aldolase. The presence of these enzyme activities would allow d-fructose to be degraded by the Embden-Meyerhof pathway and/or the Entner-Doudoroff pathway. In cell-free extracts of d-glucose grown cells, the activity catalyzing a P-enolpyruvate-dependent phosphorylation of d-fructose as well as 1-P-fructokinase activity were reduced or absent while the remaining enzymes were present at levels similar to those found in d-fructose grown cells. Radiolabeling experiments suggested that both d-fructose and d-glucose were utilized primarily via the Entner-Doudoroff pathway. Alteromonas communis, a marine species lacking 1-P-fructokinase and the PPi-dependent 6-P-fructokinase, contained all the enzyme activities necessary for the catabolism of d-fructose and d-glucose by the Entner-Doudoroff pathway; the involvement of this pathway was also consistent with the results of the radiolabeling experiments.Non-Standard Abbreviations EDP Entner-Doudoroff pathway - EMP Embden-Meyerhof pathway - FDP fructose-1,6-P₂ - FDPase FDP phosphatase - F-1-P fructose-1-P - F-6-P fructose-6-P - FPTS PEP: d-fructose phosphotransferase system - PPi-6-PFK PPi dependent 6-PFK - G-6-P glucose-6-P - KDPG 2-keto-3-deoxy-6-P-gluconate - PEP P-enolpyruvate - 1-PFK 1-P-fructokinase - 6-PFK 6-P-fructokinase - 6-PGA 6-P-gluconate     

Pseudomonas cepacia mutants blocked in the Entner-Doudoroff pathway.

Pseudomonas cepacia mutants deficient in either 6-phosphogluconate (6PGA) dehydratase (Edd-) or 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (Eda-) failed to utilize glucose or gluconate despite the prominence of of 6-phosphogluconate dehydrogenase (6PGAD) ii this bacterium and the potential for utilizing the pentose shunt suggested by its growth on ribitol and xylose. The Eda- strains grew normally on glucuronic acid, indicating that in P. cepacia its degradation does not depend upon KDPG aldolase as it does in Escherichia coli. Both 6PGA dehydratase and KDPG aldolase were inducible enzymes, with 6PGA rather than gluconate the apparent inducer. Edd- as well as Eda- strains were sensitive to growth inhibition by glucose, gluconate, fructose, and related carbohydrates when these substrates were present in combination with alternate carbon sources such as citrate or phthalate, presumably as a consequence of accumulation and toxicity of 6PGA, KDPG, or both. Edd- mutants were somewhat less sensitive to such inhibition than were Eda- strains. Certain derivatives of the Edd- strains we examined were able to utilize gluconate despite their deficiency of 6PGA dehydratase. Such mutants formed higher levels of 6PGAD than did the wild type. It is likely that the elevated levels of 6PGAD in these strains prevents accumulation of toxic levels of 6PGA that would otherwise result from a block in he Entner-Doudoroff pathway. The results suggest that P. cepacia can mutate to grow slowly on gluconate utilizing only the pentose shunt.     

Fatty acid degradation in Caulobacter crescentus.

Fatty acid degradation was investigated in Caulobacter crescentus, a bacterium that exhibits membrane-mediated differentiation events. Two strains of C. crescentus were shown to utilize oleic acid as sole carbon source. Five enzymes of the fatty acid beta-oxidation pathway, acyl-coenzyme A (CoA) synthase, crotonase, thiolase, beta-hydroxyacyl-CoA dehydrogenase, and acyl-CoA dehydrogenase, were identified. The activities of these enzymes were significantly higher in C. crescentus than the fully induced levels observed in Escherichia coli. Growth in glucose or glucose plus oleic acid decreased fatty acid uptake and lowered the specific activity of the enzymes involved in beta-oxidation by 2- to 3-fold, in contrast to the 50-fold glucose repression found in E. coli. The mild glucose repression of the acyl-CoA synthase was reversed by exogenous dibutyryl cyclic AMP. Acyl-CoA synthase activity was shown to be the same in oleic acid-grown cells and in cells grown in the presence of succinate, a carbon source not affected by catabolite repression. Thus, fatty acid degradation by the beta-oxidation pathway is constitutive in C. crescentus and is only mildly affected by growth in the presence of glucose. Tn5 insertion mutants unable to form colonies when oleic acid was the sole carbon source were isolated. However, these mutants efficiently transported fatty acids and had beta-oxidation enzyme levels comparable with that of the wild type. Our inability to obtain fatty acid degradation mutants after a wide search, coupled with the high constitutive levels of the beta-oxidation enzymes, suggest that fatty acid turnover, as has proven to be the case fatty acid biosynthesis, might play an essential role in membrane biogenesis and cell cycle events in C. crescentus.     

Characterization and crystal structure of Escherichia coli KDPGal aldolase

2-Keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases catalyze an identical reaction differing in substrate specificity in only the configuration of a single stereocenter. However, the proteins show little sequence homology at the amino acid level. Here we investigate the determinants of substrate selectivity of these enzymes. The Escherichia coli KDPGal aldolase gene, cloned into a T7 expression vector and overexpressed in E. coli, catalyzes retro-aldol cleavage of the natural substrate, KDPGal, with values of k(cat)/K(M) and k(cat) of 1.9x10(4)M(-1)s(-1) and 4s(-1), respectively. In the synthetic direction, KDPGal aldolase efficiently catalyzes an aldol addition using a limited number of aldehyde substrates, including d-glyceraldehyde-3-phosphate (natural substrate), d-glyceraldehyde, glycolaldehyde, and 2-pyridinecarboxaldehyde. A preparative scale reaction between 2-pyridinecarboxaldehyde and pyruvate catalyzed by KDPGal aldolase produced the aldol adduct of the R stereochemistry in >99.7% ee, a result complementary to that observed using the related KDPG aldolase. The native crystal structure has been solved to a resolution of 2.4A and displays the same (alpha/beta)(8) topology, as KDPG aldolase. We have also determined a 2.1A structure of a Schiff base complex between the enzyme and its substrate. This model predicts that a single amino acid change, T161 in KDPG aldolase to V154 in KDPGal aldolase, plays an important role in determining the stereochemical course of enzyme catalysis and this prediction was borne out by site-directed mutagenesis studies. However, additional changes in the enzyme sequence are required to prepare an enzyme with both high catalytic efficiency and altered stereochemistry.     

Fermentation of pectin and glucose,and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bifidobacterium pseudolongum

AIMS: In a rabbit caecal bacterium Bifidobacterium pseudolongum, metabolites of pectin and glucose, and activities of enzymes involved in the degradation of pectin were assayed. Simultaneously, activities of these enzymes were assayed in a rumen pectinolytic strain of Streptococcus bovis. METHODS AND RESULTS: A strain B. pseudolongum P6 which grew best on pectin was selected among bifidobacteria isolated from the rabbit caecum. Cultures of B. pseudolongum P6 grown on pectin produced significantly less formate, lactate and ethanol, and more acetate and succinate than cultures grown on glucose. No CO2 production on pectin was observed. Pectin macromolecule was degraded by extracellular pectinase (EC 3.2.1.15). Cell extracts possessed the activity of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). Streptococcus bovis X4, possessed activity of exopectate lyase and pectinase, but not that of KDPG aldolase. CONCLUSIONS: Our results are consistent with the assumption that in B. pseudolongum P6 acidic products of pectin degradation are catabolized via a modified Entner-Doudoroff pathway, as shown previously in rumen pectin-utilizing bacteria. The missing KDPG aldolase activity in Strep. bovis X4 seems to be the reason for the absence of growth of this bacterium on pectin. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on polysaccharide metabolism in bifidobacteria is fragmentary. This study extends the knowledge on pectin metabolism in intestinal bacteria.