Monosialogangliosides and Their Interaction with Cholera Toxin—Investigation by Molecular Modeling and Molecular Mechanics

Abstract

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface monosialogangliosides (GM3, GM2 and GM1) in aqueous environment. Water mediated hydrogen bonding network plays a significant role in the structural stabilization of GM3, GM2 and GM1. The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin reveals a limited allowed eulerian space of 2.4% with a much less allowed eulerian space (1.4%) for external galactose of GM1. The molecular mechanics of monosialoganglioside-cholera toxin complex reveals that cholera toxin can accommodate the monosialogangliosides in three different modes. Direct and water mediated hydrogen bonding interactions stabilize these binding modes and play an essential role in defining the order of specificity for different monosialogangliosides towards cholera toxin. This study identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.     

Enzymatic production of <Emphasis Type="SmallCaps">l</Emphasis>-amino acids from the corresponding <Emphasis Type="SmallCaps">dl</Emphasis>-5-substituted hydantoins by <Emphasis Type="Italic">Bacillus fordii</Emphasis> MH602

Bacillus fordii MH602 was newly screened from soil at 45 °C and exhibited high activities of hydantoinase and carbamoylase, efficiently yielding l-amino acids including phenylalanine, phenylglycine and tryptophan with the bioconversion yield of 60–100% from the corresponding dl-5-substituted hydantoins. Hydantoinase activity was found to be cell-associated and inducible. The optimal inducer was dl-5-methylhydantoin with concentration of 0.014 mol L⁻¹ and added to the fermentation medium in the exponential phase of growth. In the production of optically pure amino acids from dl-5-benylhydantoin, the optimal temperature and pH of this reaction were 45–50 °C and 7.5 respectively. The hydantoinase was non-stereoselective, while carmbamoylase was l-selective. The hydantoinase activity was not subject to substrate inhibition, or product inhibition by ammonia. In addition, The activities of both enzymes from crude extract of the strain were thermostable; the hydantoinase and carbamoylase retained about 90% and 60% activity after 6 h at 50 °C, respectively. Since reaction at higher temperature is advantageous for enhancement of solubility and for racemization of dl-5-substituted hydantoins, the relative paucity of l-selective hydantoinase systems, together with the high level of hydantoinase and carbamoylase activity and unusual substrate selectivity of the strain MH602, suggest that it has significant potential applications.     

In vitro transformation of paralytic shellfish toxins in the clams Mya arenaria and Protothaca staminea

Dissected tissues of two clam species, the Pacific littleneck, Protothaca staminea, and soft-shell, Mya arenaria, were evaluated for in vitro conversion of paralytic shellfish poisoning (PSP) toxins. Tissue homogenates were incubated with purified PSP toxins to determine the time-course of toxin conversion. The effects of boiling and addition of a natural reductant (glutathione) on toxin conversion were also assessed. For P. staminea, the digestive gland showed the greatest capacity for biotransformation, followed by gill, but mantle, adductor muscle, and siphon tissues exhibited very low conversion. In this species, the production of decarbamoyl derivatives was much greater from low potency N-sulfocarbamoyl toxins than from carbamate analogues. Decarbamolyation exhibited apparent specificity for α-epimers of all toxin substrates and this reaction was inhibited by boiling. Glutathione-mediated desulfation was tissue specific and had apparent specificity for β-epimers. These observations on P. staminea suggest that the above reactions are enzyme-mediated. In contrast, there was little toxin conversion in M. arenaria homogenates, but even this low activity was heat-labile and thus likely enzyme-mediated.     

The purification and characterization of a novel D(−)-specific carbamoylase enzyme from an Agrobacterium sp.

A carbamoylase enzyme was purified from a cell-free extract of Agrobacterium sp. with an overall yield of 81%. It was judged to be homogenous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight of 38,000 daltons. Further studies on the native enzyme suggested that the active enzyme was present as a dimer, with a pI of 5.5. It was able to cleave a variety of N-carbamoyl substrates, but was strictly D(−) specific. It was found to have a K_m of 0.82 m and a V_max of 31 U mg⁻¹ for D(−) N-carbamoyl hydroxyphenylglycine in the presence of 10 m dithiothreitol. It showed no metal ion requirements but was inhibited by iodoacetic acid and iodoacetamide, both thiol reagents. The N-terminal amino acid sequence of the enzyme was elucidated.     

Nitrile- and Amide-converting Microbial Enzymes: Stereo-, Regio- and Chemoselectivity

Biotransformations using microbial nitrile- and amide-converting enzymes have developed considerably in the recent years. Most processes profited from the stereo-, regio- and chemoselectivity of nitrile hydratases, nitrilases and amidases specific for primary or secondary amides. The aim of this review is to discuss the developments in this branch of biotransformations in the last ca. 5 years by taking highlights from research journals, patents and industrial applications.     

Nitrile- and Amide-converting Microbial Enzymes: Stereo-, Regio- and Chemoselectivity

Biotransformations using microbial nitrile- and amide-converting enzymes have developed considerably in the recent years. Most processes profited from the stereo-, regio- and chemoselectivity of nitrile hydratases, nitrilases and amidases specific for primary or secondary amides. The aim of this review is to discuss the developments in this branch of biotransformations in the last ca. 5 years by taking highlights from research journals, patents and industrial applications.