Stereospecificity in meta-fission catabolic pathways

The stereospecificities of 2-keto-4-pentenoate hydratase and 4-hydroxy-2-ketovalerate aldolase were studied in Escherichia coli, Pseudomonas putida, and Acinetobacter sp. Hydration was stereospecific in all three; however, only P. putida and Acinetobacter sp. showed stereospecificity in their aldolase reactions.     

The earliest catabolic pathways

Summary Anaerobic catabolism of amino acids may have provided the main source of energy for primitive microorganisms. Examples are given of amino acid catabolic reactions coupled to substrate level phosphorylations occurring in present-day anaerobes which may be biochemical fossils from a very early stage of the evolution of procaryotes.     

Evolution of chlorocatechol catabolic pathways

The aerobic bacterial degradation of chloroaromatic compounds often involves chlorosubstituted catechols as central intermediates. They are converted to 3-oxoadipate in a series of reactions similar to that for catechol catabolism and therefore designated as modifiedortho-cleavage pathway. Among the enzymes of this catabolic route, the chlorocatechol 1,2-dioxygenases are known to have a relaxed substrate specificity. In contrast, several chloromuconate cycloisomerases are more specific, and the dienelactone hydrolases of chlorocatechol catabolic pathways do not even convert the corresponding intermediate of catechol degradation, 3-oxoadipate enol-lactone. While the sequences of chlorocatechol 1,2-dioxygenases and chloromuconate cycloisomerases are very similar to those of catechol 1,2-dioxygenases and muconate cycloisomerases, respectively, the relationship between dienelactone hydrolases and 3-oxoadipate enol-lactone hydrolases is more distant. They seem to share an / hydrolase fold, but the sequences comprising the fold are quite dissimilar. Therefore, for chlorocatechol catabolism, dienelactone hydrolases might have been recruited from some other, preexisting pathway. Their relationship to dienelactone (hydrolases identified in 4-fluorobenzoate utilizing strains ofAlcaligenes andBurkholderia (Pseudomonas) cepacia is investigated). Sequence evidence suggests that the chlorocatechol catabolic operons of the plasmids pJP4, pAC27, and pP51 have been derived from a common precursor. The latter seems to have evolved for the purpose of halocatechol catabolism, and may be considerably older than the chemical industry.     

Development of catabolic pathways in insect flight muscles. A comparative study

Activities of enzymes representative of glycolytic and β-oxidative pathways and citric acid and glycerophosphate cycles were measured in the developing flight muscles of three species: Calliphora erythrocephala, Locusta migratoria, and Philosamia cynthia. The activities were measured in vitro under optimal conditions.The enzyme pattern of young flight muscles is quite different from the adult pattern. In the second half of the developmental period final differentiation towards the adult metabolic pattern takes place, in Calliphora leading to exclusively carbohydrate-oxidizing capacities, in Locusta to properties enabling both aerobic glycolytic and β-oxidative processes, whereas Philosamia becomes oriented to fatty acid oxidation. This differentiation starts after a temporary rise of lactate dehydrogenase activity, a phenomenon that seems to be connected with invagination of tracheoblasts into the muscle fibres. This tracheolization might be necessary for differentiation towards the species specific metabolic properties of the adult flight muscle.Theoretical aspects of the enzyme activities, as they were measured in the in vitro assays, are discussed and related to the physiological qualities of the flight muscles of the three species investigated.     

Laboratory evolution of catabolic enzymes and pathways

The laboratory evolution of environmentally relevant enzymes and proteins has resulted in the generation of optimized and stabilized enzymes, as well as enzymes with activity against new substrates. Numerous methods, including random mutagenesis, site-directed mutagenesis and DNA shuffling, have been widely used to generate variants of existing enzymes. These evolved catabolic enzymes have application for improving biodegradation pathways, generating engineered pathways for the degradation of particularly recalcitrant compounds, and for the development of biocatalytic processes to produce useful compounds. Regulatory proteins associated with catabolic pathways have been utilized to generate biosensors for the detection of bioavailable concentrations of environmentally relevant chemicals.     

Enzyme induction and repression in anabolic and catabolic pathways

Summary Microorganisms have evolved enzymes which catalyze a large number of reactions in the sequences to form essential cellular constituents and liberate energy and carbon for cellular processes. Regulation of the use of energy and of the monomeric cellular precursors to the synthesis of those enzymes required under changing environmental conditions depends on the one hand on the level of end products of a reaction sequence and on the other upon the presence of the first, or early members of a reaction sequence. These cases in turn represent product repression and substrate, or substrate like, induction of enzyme formation. Though the repression system has generally been considered to operate in anabolic and the induction system in catabolic processes, the experiments presented demonstrate a role for both types of control in formation of biosynthetic and peripheral pathway enzymes. The induction of biosynthetic enzymes is shown in Pseudomonas putida, and organism with three clusters of genes for the tryptophan pathway. The repression of degradative enzymes is shown in an extended pathway of peripheral oxidation of terpenoid compounds. The enzymes for steps following conversion of neutral to non-essential acidic products are repressed as well as enzymes beyond convergence with isobutyrate formation and conversion to the succinyl and propionyl intermediates.Dedicated to C. B. van Niel on the occasion of his 70th birthday. Supported in part by grant G24037 from the National Science Foundation.     

On the catabolic pathways of sugars in green algae

Respiratory metabolism of the cultures of algaeChlorella pyrenoidosa (82) andScenedesmus obliquus (125) was investigated. One part of algae were cultivated on a mineral nutrient solution, another two parts on the solution with glucose and on the solution with glucose and yeast decoction. Individual steps of respiratory metabolism—endogenous as well as in the presence of exogenous sugars—were estimated according to the response to the following inhibitors: monoiodacetate, NaF, NaN₃, and 2,4-DNP. In the last two cases, fructose, glucose-6-phosphate and fructose-1,6-diphosphate were applied in parallel for comparison. Na-monoiodacetate was found to inhibit the respiration of both endogenous and exogenous (with glucose) substrates, NaF (concentrations up to 2.5.10^?2 m) stimulated the O₂ uptake. The effect of sodium azide and 2,4-DNP depended in both strains on previous cultivation. On the basis of the results obtained, the presence of particular respiration pathways in the dissimilation of glucose is discussed. The following catabolic processes are to be considered: a) direct oxidation (with both autotrophically cultivated strains and with theChlorella cultivated on glucosecontaining medium), b) the process similar to glycolysis, which, however, does not necessarily involve the enolase (it is not inhibited by NaF) c) pentosephosphate cycle (Chlorella), and d) glycolysis, in which both algae can operate when sugars previously phosphorylated are applied.     

Anabolic and catabolic pathways of pyrimidine metabolism in rat liver

• 1.1. Use of pyrimidine nucleotide precursors labelled in various positions on the ring shows that in rat liver, pyrimidine nucleotides formed from orotate follow anabolic pathways almost exclusively, whereas trace quantities of uridine are mostly degraded to β-alanine and its metabolites.
• 2.2. Annomalies in the ratios of [¹⁴C] and [³H] in various common nucleotide products of orotate and uridine can be accounted for on the basis of metabolic compartmentation and recycling of CO₂.

     

Genomic islands and the evolution of catabolic pathways in bacteria

Genes for the degradation of organic pollutants have usually been allocated to plasmid DNAs in bacteria or considered non-mobile when detected in the chromosome. New discoveries have shown that catabolic genes can also be part of so-called integrative and conjugative elements (ICElands), a group of mobile DNA elements also known as genomic islands and conjugative transposons. One such ICEland is the clc element for chlorobenzoate and chlorocatechol degradation in Pseudomonas sp. strain B13. Genome comparisons and genetic data on integrase functioning reveal that the clc element and several other unclassified ICElands belong to a group of elements with conserved features. The clc element is unique among them in carrying the genetic information for several degradation pathways, whereas the others give evidence for pathogenicity functions. Many more such elements may exist, bridging the gap between pathogenicity and degradation functions.     

Filling gaps in bacterial catabolic pathways with computation and high-throughput genetics

To discover novel catabolic enzymes and transporters, we combined high-throughput genetic data from 29 bacteria with an automated tool to find gaps in their catabolic pathways. GapMind for carbon sources automatically annotates the uptake and catabolism of 62 compounds in bacterial and archaeal genomes. For the compounds that are utilized by the 29 bacteria, we systematically examined the gaps in GapMind’s predicted pathways, and we used the mutant fitness data to find additional genes that were involved in their utilization. We identified novel pathways or enzymes for the utilization of glucosamine, citrulline, myo-inositol, lactose, and phenylacetate, and we annotated 299 diverged enzymes and transporters. We also curated 125 proteins from published reports. For the 29 bacteria with genetic data, GapMind finds high-confidence paths for 85% of utilized carbon sources. In diverse bacteria and archaea, 38% of utilized carbon sources have high-confidence paths, which was improved from 27% by incorporating the fitness-based annotations and our curation. GapMind for carbon sources is available as a web server (http://papers.genomics.lbl.gov/carbon) and takes just 30 seconds for the typical genome.     

Parallel metabotropic pathways in the heart of the toad, Bufo marinus

This study examined the transduction pathways activated by epinephrine in the pacemaker region of the toad heart. Recordings of membrane potential, force, and intracellular Ca(2+) concentration ([Ca(2+)](i)) were made from arrested toad sinus venosus. Sympathetic nerve stimulation activated non-alpha-, non-beta-adrenoceptors to evoke a membrane depolarization and a transient increase in [Ca(2+)](i). In contrast, the beta-adrenoceptor agonist isoprenaline (10 microM) caused membrane hyperpolarization and decreased [Ca(2+)](i). The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM) mimicked the isoprenaline-evoked membrane hyperpolarization. Epinephrine (10-50 microM) caused an initial membrane depolarization and an increase in [Ca(2+)](i) followed by membrane hyperpolarization and decreased [Ca(2+)](i). The membrane depolarizations evoked by sympathetic nerve stimulation or epinephrine were abolished either by the phospholipase C inhibitor U-73122 (20 microM) or by the blocker of D-myo-inositol 1,4,5,-trisphosphate-induced Ca(2+) release, 2-aminoethoxydiphenyl borate (2-APB, 60 microM). Neither U-73122 nor 2-APB had an affect on the membrane hyperpolarization evoked by beta-adrenoceptor activation. These results suggest that in the toad sinus venosus, two distinct transduction pathways can be activated by epinephrine to cause an increase in heart rate.