Localization of C-S lyase activity in the root cells ofAlbizzia lophanta

Summary The distribution of C-S lyase activity in root cells ofAlbizzia lophanta Benth. plantlets was investigated histochemically. H₂S formed upon cleavage of exogenously applied L-cysteine was precipitated by Pb⁺⁺ in a capture reaction at the site of its formation. Enzyme activity was found to be localized at the root tip and in a layer of cortex cells adjacent to the endodermis throughout the whole length of the root. Distinct areas within the exodermis, distributed in a regular pattern on the root surface, also exhibited the specific reaction. In vivo roots ofAlbizzia lophanta actively excrete the strongly smelling methylene dithiol, formed by enzymatic cleavage of djenkolic acid, the natural substrate of C-S lyase inAlbizzia. The physiological meaning of this compound, as well as the localization and intracellular distribution of C-S lyase activity are discussed.     

耐药菌感染的噬菌体治疗专利技术

耐药菌,尤其是多重耐药菌的出现和持续进化给人类健康带来了巨大的威胁。在抗生素逐渐失去特效作用的情况下,科学界和医药界又把眼光重新投向了抗菌的天然生物-噬菌体,并在一些研究中证明了噬菌体可以作为新的武器去替代抗生素治疗耐药菌感染。通过对噬菌体治疗及衍生的裂解酶治疗的世界专利申请进行统计及分析,获得了专利发展趋势、申请人分布特点及主要专利申请人等信息,详细分析了噬菌体及裂解酶治疗的主要专利技术路线和研发热点。     

Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

Leishmania are auxotrophic for purines, and consequently purine acquisition from the host is a requisite nutritional function for the parasite. Both adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) have been identified as vital components of purine salvage in Leishmania donovani, and therefore Δadss and Δasl null mutants were constructed to test this hypothesis. Unlike wild type L. donovani, Δadss and Δasl parasites in culture exhibited a profoundly restricted growth phenotype in which the only permissive growth conditions were a 6-aminopurine source in the presence of 2′-deoxycoformycin, an inhibitor of adenine aminohydrolase activity. Although both knock-outs showed a diminished capacity to infect murine peritoneal macrophages, only the Δasl null mutant was profoundly incapacitated in its ability to infect mice. The enormous discrepancy in parasite loads observed in livers and spleens from mice infected with either Δadss or Δasl parasites can be explained by selective accumulation of adenylosuccinate in the Δasl knock-out and consequent starvation for guanylate nucleotides. Genetic complementation of a Δasl lesion in Escherichia coli implied that the L. donovani ASL could also recognize 5-aminoimidazole-(N-succinylocarboxamide) ribotide as a substrate, and purified recombinant ASL displayed an apparent K_m of ∼24 μm for adenylosuccinate. Unlike many components of the purine salvage pathway of L. donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania.     

Development of an assay and determination of kinetic parameters for chondroitin AC lyase using defined synthetic substrates

Many techniques have been developed for the assay of polysaccharide lyases; however, none have allowed the measurement of defined and reproducible k(cat) and K(m) values due to the inhomogeneous nature of the polymeric substrates. We have designed three different substrates for chondroitin AC lyase from Flavobacterium heparinum that can be monitored by three different techniques: UV/Vis spectroscopy, fluorescence spectroscopy, and use of a fluoride ion-selective electrode. Each is a continuous assay, free from interferences caused by other components present in crude enzyme preparations, and allows meaningful and reproducible kinetic parameters to be determined. The development of these defined synthetic substrates has opened up a wide variety of mechanistic studies that can be performed to elucidate the detailed catalytic mechanism of this, and other, polysaccharide lyases. The application of these techniques, which include kinetic isotope effects and linear free energy analyses, was not possible with the previous polymeric substrates and will allow this relatively poorly understood class of polysaccharide-degrading enzymes to be studied mechanistically.     

Characterization of a novel Agrobacterium tumefaciens galactarolactone cycloisomerase enzyme for direct conversion of D-galactarolactone to 3-deoxy-2-keto-L-threo-hexarate

Microorganisms use different pathways for D-galacturonate catabolism. In the known microbial oxidative pathway, D-galacturonate is oxidized to D-galactarolactone, the lactone hydrolyzed to galactarate, which is further converted to 3-deoxy-2-keto-hexarate and α-ketoglutarate. We have shown recently that Agrobacterium tumefaciens strain C58 contains an uronate dehydrogenase (At Udh) that oxidizes D-galacturonic acid to D-galactarolactone. Here we report identification of a novel enzyme from the same A. tumefaciens strain, which we named Galactarolactone cycloisomerase (At Gci) (E.C. 5.5.1.-), for the direct conversion of the D-galactarolactone to 3-deoxy-2-keto-hexarate. The At Gci enzyme is 378 amino acids long and belongs to the mandelate racemase subgroup in the enolase superfamily. At Gci was heterologously expressed in Escherichia coli, and the purified enzyme was found to exist as an octameric form. It is active both on D-galactarolactone and D-glucarolactone, but does not work on the corresponding linear hexaric acid forms. The details of the reaction mechanism were further studied by NMR and optical rotation demonstrating that the reaction product of At Gci from D-galactaro-1,4-lactone and D-glucaro-1,4-lactone conversion is in both cases the L-threo form of 3-deoxy-2-keto-hexarate.     

Bacterial versus human sphingosine-1-phosphate lyase (S1PL) in the design of potential S1PL inhibitors

A series of potential active-site sphingosine-1-phosphate lyase (S1PL) inhibitors have been designed from scaffolds 1 and 2, arising from virtual screening using the X-ray structures of the bacterial (StS1PL) and the human (hS1PL) enzymes. Both enzymes are very similar at the active site, as confirmed by the similar experimental kinetic constants shown by the fluorogenic substrate RBM13 in both cases. However, the docking scoring functions used probably overestimated the weight of electrostatic interactions between the ligands and key active-site residues in the protein environment, which may account for the modest activity found for the designed inhibitors. In addition, the possibility that the inhibitors do not reach the enzyme active site should not be overlooked. Finally, since both enzymes show remarkable structural differences at the access channel and in the proximity to the active site cavity, caution should be taken when designing inhibitors acting around that area, as evidenced by the much lower activity found in StS1PL for the potent hS1PL inhibitor D.     

Differences between human and rodent pancreatic islets: low pyruvate carboxylase, atp citrate lyase, and pyruvate carboxylation and high glucose-stimulated acetoacetate in human pancreatic islets

Anaplerosis, the net synthesis in mitochondria of citric acid cycle intermediates, and cataplerosis, their export to the cytosol, have been shown to be important for insulin secretion in rodent beta cells. However, human islets may be different. We observed that the enzyme activity, protein level, and relative mRNA level of the key anaplerotic enzyme pyruvate carboxylase (PC) were 80-90% lower in human pancreatic islets compared with islets of rats and mice and the rat insulinoma cell line INS-1 832/13. Activity and protein of ATP citrate lyase, which uses anaplerotic products in the cytosol, were 60-75% lower in human islets than in rodent islets or the cell line. In line with the lower PC, the percentage of glucose-derived pyruvate that entered mitochondrial metabolism via carboxylation in human islets was only 20-30% that in rat islets. This suggests human islets depend less on pyruvate carboxylation than rodent models that were used to establish the role of PC in insulin secretion. Human islets possessed high levels of succinyl-CoA:3-ketoacid-CoA transferase, an enzyme that forms acetoacetate in the mitochondria, and acetoacetyl-CoA synthetase, which uses acetoacetate to form acyl-CoAs in the cytosol. Glucose-stimulated human islets released insulin similarly to rat islets but formed much more acetoacetate. β-Hydroxybutyrate augmented insulin secretion in human islets. This information supports previous data that indicate beta cells can use a pathway involving succinyl-CoA:3-ketoacid-CoA transferase and acetoacetyl-CoA synthetase to synthesize and use acetoacetate and suggests human islets may use this pathway more than PC and citrate to form cytosolic acyl-CoAs.     

Insight into the Role of Substrate-binding Residues in Conferring Substrate Specificity for the Multifunctional Polysaccharide Lyase Smlt1473

Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling the degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously, we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a, which exhibited significant activity against hyaluronan (HA), poly-β-d-glucuronic acid (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly active and specific polysaccharide lyases.     

A Novel Eliminase from a Marine Bacterium That Degrades Hyaluronan and Chondroitin Sulfate

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ^4,5HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications.