A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.     

Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16

ReBktB is a β-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3 Å, and the overall structure has a similar fold to that of type II biosynthetic thiolases, such as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBktB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that K_m, V_max, and K_cat values of 11.58 μM, 1.5 μmol/min, and 102.18 s⁻¹, respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.     

Up-regulation of the peroxisomal β-oxidation system occurs in butyrate-grown Candida tropicalis following disruption of the gene encoding peroxisomal 3-ketoacyl-CoA thiolase

In the yeast Candida tropicalis, two thiolase isozymes, peroxisomal acetoacetyl-CoA thiolase and peroxisomal 3-ketoacyl-CoA thiolase, participate in the peroxisomal fatty acid β-oxidation system. Their individual contributions have been demonstrated in cells grown on butyrate, with C. tropicalis able to grow in the absence of either one. In the present study, a lack of peroxisomal 3-ketoacyl-CoA thiolase protein resulted in increased expression (up-regulation) of acetoacetyl-CoA thiolase and other peroxisomal proteins, whereas a lack of peroxisomal acetoacetyl-CoA thiolase produced no corresponding effect. Overexpression of the acetoacetyl-CoA thiolase gene did not suppress the up-regulation or the growth retardation on butyrate in cells without peroxisomal 3-ketoacyl-CoA thiolase, even though large amounts of the overexpressed acetoacetyl-CoA thiolase were detected in most of the peroxisomes of butyrate-grown cells. These results provide important evidence of the greater contribution of 3-ketoacyl-CoA thiolase to the peroxisomal β-oxidation system than acetoacetyl-CoA thiolase in C. tropicalis and a novel insight into the regulation of the peroxisomal β-oxidation system.     

Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos

Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.     

Understanding the function of bacterial and eukaryotic thiolases II by integrating evolutionary and functional approaches

Acetoacetyl-CoA thiolase (EC 2.3.1.9), commonly named thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA and CoA. This enzyme acts in anabolic processes as the first step in the biosynthesis of isoprenoids and polyhydroxybutyrate in eukaryotes and bacteria, respectively. We have recently reported the evolutionary and functional equivalence of these enzymes, suggesting that thiolase II could be the rate limiting enzyme in these pathways and presented evidence indicating that this enzyme modulates the availability of reducing equivalents during abiotic stress adaptation in bacteria and plants. However, these results are not sufficient to clarify why thiolase II was evolutionary selected as a critical enzyme in the production of antioxidant compounds. Regarding this intriguing topic, we propose that thiolase II could sense changes in the acetyl-CoA/CoA ratio induced by the inhibition of the tricarboxylic acid cycle under abiotic stress. Thus, the high level of evolutionary and functional constraint of thiolase II may be due to the connection of this enzyme with an ancient and conserved metabolic route.     

Interactive effects of season and temperature on enzyme activities,tissue and whole animal respiration in roach,Rutilus rutilus

Synopsis This paper reviews investigations on the ecophysiology of a population of roach, Rutilus rutilus, from a subalpine oligotrophic lake in the Austrian Tirol. Metabolic responses to season and temperature were studied in whole animals, tissues and selected enzymes. The exponent of the relationship between body mass and three levels of the metabolic rate of acclimated fish was 0.82 ± 0.02, 0.60 ± 0.15, and 0.75 ± 0.01 at 4, 12, and 20° C respectively. Various combinations of long-term acclimation to constant or seasonally fluctuating temperatures and long-term (up to 14 days) monitoring of O₂ at the acclimation temperature led to the conclusion that the aerobic power of fish swimming in the routine mode does not show any sign of being temperature compensated. On the other hand, there are several indications that the energy expenditure of spontaneously swimming fish is adjusted to the seasonal pattern of environmental change and that these responses of metabolism and behaviour are controlled by both endogenous and exogenous factors. The rate of oxygen consumption of gill and muscle tissue brei from fish caught during a seasonal cycle and measured at 15° C appears to follow closely the reproductive and gonadal cycle of the living fish. The same holds for the activities of phosphofructokinase, acetoacetyl-CoA thiolase, and cytochrome oxidase. On the other hand, the Na⁺, K⁺-ATPase of the kidney shows near perfect temperature compensation when fish acclimated to 5 and 25° C are compared, whereas an equally pronounced case of inverse temperature acclimation has been reported for the activity of digestive enzymes in the gut. Summarizing these data it is pointed out that the temperature relationship of a poikilothermic organism is the sum of often very diverse temperature relationships of specific metabolic and behavioural functions. In the case of the roach, strong effects of acclimation temperature on the molecular level, sometimes in the opposite direction, combine with seasonal effects on enzyme activities and tissue respiration. However, on the whole animal level the fish behave as strictly non-compensating poikilotherms, the reproductive cycle being the only detectable influence capable of modulating the basic temperature relationship of energy expenditure.     

High resolution crystal structures of human cytosolic thiolase (CT): a comparison of the active sites of human CT, bacterial thiolase, and bacterial KAS I

Thiolases belong to a superfamily of condensing enzymes that includes also beta-ketoacyl acyl carrier protein synthases (KAS enzymes), involved in fatty acid synthesis. Here, we describe the high resolution structure of human cytosolic acetoacetyl-CoA thiolase (CT), both unliganded (at 2.3 angstroms resolution) and in complex with CoA (at 1.6 angstroms resolution). CT catalyses the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA, which is the first reaction of the metabolic pathway leading to the synthesis of cholesterol. CT is a homotetramer of exact 222 symmetry. There is an excess of positively charged residues at the interdimer surface leading towards the CoA-binding pocket, possibly important for the efficient capture of substrates. The geometry of the catalytic site, including the three catalytic residues Cys92, His 353, Cys383, and the two oxyanion holes, is highly conserved between the human and bacterial Zoogloea ramigera thiolase. In human CT, the first oxyanion hole is formed by Wat38 (stabilised by Asn321) and NE2(His353), and the second by N(Cys92) and N(Gly385). The active site of this superfamily is constructed on top of four active site loops, near Cys92, Asn321, His353, and Cys383, respectively. These loops were used for the superpositioning of CT on the bacterial thiolase and on the Escherichia coli KAS I. This comparison indicates that the two thiolase oxyanion holes also exist in KAS I at topologically equivalent positions. Interestingly, the hydrogen bonding interactions at the first oxyanion hole are different in thiolase and KAS I. In KAS I, the hydrogen bonding partners are two histidine NE2 atoms, instead of a water and a NE2 side-chain atom in thiolase. The second oxyanion hole is in both structures shaped by corresponding main chain peptide NH-groups. The possible importance of bound water molecules at the catalytic site of thiolase for the reaction mechanism is discussed.     

PTS2 protein import into mammalian peroxisomes

Peroxisome targeting signal (PTS)2 directs proteins from their site of synthesis in the cytosol to the lumen of the peroxisome. Unlike PTS1 which is present in the great majority of peroxisomal matrix proteins and whose import mechanics have been dissected in considerable detail, PTS2 is a relatively rare topogenic signal whose import mechanisms are far less well understood. However, as is the case for PTS1 proteins, an inability to import PTS2 proteins leads to human disease. In this report, we describe the biochemical characterization of mammalian PTS2 protein import using a semi-permeabilized cell system. We show that a PTS2-containing reporter molecule is taken up by peroxisomes in a reaction that is time-, temperature-, ATP-, and cytosol-dependent. Furthermore, the import process is specific, saturable, and requires action of the chaperone Hsc70, the cochaperone Hsp40, and the peroxins Pex5p and Pex14p. We also demonstrate peroxisomal translocation of PTS2 reporter/antibody complexes confirming the import competence of higher order structures. Importantly, cultured fibroblasts from patients with the rhizomelic form of chondrodysplasia punctata (RCDP) which are deficient for the PTS2 receptor protein, Pex7p, are unable to import the PTS2 reporter in this assay. The ability to monitor PTS2 import in vitro will permit, for the first time, a detailed comparison of the biochemical properties of PTS1 and PTS2 protein import.