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Insights into the different catalytic activities of Clostridium neurotoxins.

ABSTRACT: The clostridial neurotoxins are among the most potent protein toxins for humans and are responsible for botulism, a flaccid paralysis elicited by the botulinum toxins (BoNT), and spastic paralysis elicited by tetanus toxin (TeNT). Seven serotypes of botulinum neurotoxins (A-G) and tetanus toxin showed different toxicities and cleave their substrates with different efficiencies. However, the molecular basis of their different catalytic activities with respect to their substrates is not clear. BoNT/B light chain (LC/B) and TeNT light chain (LC/T) cleave vesicle-associated membrane protein 2 (VAMP2) at the same scissile bond but possess different catalytic activities and substrate requirements, which make them the best candidates for studying the mechanisms of their different catalytic activities. The recognition of five major P sites of VAMP2 (P7, P6, P1, P1', and P2') and fine alignment of sites P2 and P3 and sites P2 and P4 by LC/B and LC/T, respectively, contributed to their substrate recognition and catalysis. Significantly, we found that the S1 pocket mutation LC/T(K(168)E) increased the rate of native VAMP2 cleavage so that it approached the rate of LC/B, which explains the molecular basis for the lower k(cat) that LC/T possesses for VAMP2 cleavage relative to that of LC/B. This analysis explains the molecular basis underlying the VAMP2 recognition and cleavage by LC/B and LC/T and provides insight that may extend the pharmacologic utility of these neurological reagents.

SUBMITTER: Chen S

PROVIDER: S-EPMC3542401 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Insights into the different catalytic activities of Clostridium neurotoxins.

Chen Sheng S Karalewitz Andrew P A AP Barbieri Joseph T JT

Biochemistry 20120424 18

The clostridial neurotoxins are among the most potent protein toxins for humans and are responsible for botulism, a flaccid paralysis elicited by the botulinum toxins (BoNT), and spastic paralysis elicited by tetanus toxin (TeNT). Seven serotypes of botulinum neurotoxins (A-G) and tetanus toxin showed different toxicities and cleave their substrates with different efficiencies. However, the molecular basis of their different catalytic activities with respect to their substrates is not clear. BoN ...[more]

PMID: 22510015

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Project description:BackgroundClostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to Clostridium and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families.ResultsThe C. botulinum strain A genome was examined for potential homologues of CNTs. A key link was identified between the neurotoxin and the flagellin gene (CBO0798) located immediately upstream of the BoNT/A neurotoxin gene cluster. This flagellin sequence displayed the strongest sequence similarity to the neurotoxin and NTNH homologue out of all proteins encoded within C. botulinum strain A. The CBO0798 gene contains a unique hypervariable region, which in closely related flagellins encodes a collagenase-like domain. Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity. Additional links to collagenase-related sequences and functions were detected by further analysis of CNTs and surrounding genes, including sequence similarities to collagen-adhesion domains and collagenases. Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains.ConclusionMultiple lines of evidence suggest that the neurotoxin and adjacent genes evolved from an ancestral collagenase-like gene cluster, linking CNTs to another major family of clostridial proteolytic toxins. Duplication, reshuffling and assembly of neighboring genes within the BoNT/A neurotoxin gene cluster may have lead to the neurotoxin's unique architecture. This work provides new insights into the evolution of C. botulinum neurotoxins and the evolutionary mechanisms underlying the origins of virulent genes.

Project description:BackgroundEfficient biomass bioconversion is a promising solution to alternative energy resources and environmental issues associated with lignocellulosic wastes. The Trichoderma species of cellulolytic fungi have strong cellulose-degrading capability, and their cellulase systems have been extensively studied. Currently, a major limitation of Trichoderma strains is their low production of β-glucosidases.ResultsWe isolated two Trichoderma hamatum strains YYH13 and YYH16 with drastically different cellulose degrading efficiencies. YYH13 has higher cellobiose-hydrolyzing efficiency. To understand mechanisms underlying such differences, we sequenced the genomes of YYH13 and YYH16, which are essentially identical (38.93 and 38.92 Mb, respectively) and are similar to that of the T. hamatum strain GD12. Using GeneMark-ES, we annotated 11,316 and 11,755 protein-coding genes in YYH13 and YYH16, respectively. Comparative analysis identified 13 functionally important genes in YYH13 under positive selection. Through examining orthologous relationships, we identified 172,655, and 320 genome-specific genes in YYH13, YYH16, and GD12, respectively. We found 15 protease families that show differences between YYH13 and YYH16. Enzymatic tests showed that exoglucanase, endoglucanase, and β-glucosidase activities were higher in YYH13 than YYH16. Additionally, YYH13 contains 10 families of carbohydrate-active enzymes, including GH1, GH3, GH18, GH35, and GH55 families of chitinases, glucosidases, galactosidases, and glucanases, which are subject to stronger positive selection pressure. Furthermore, we found that the β-glucosidase gene (YYH1311079) and pGEX-KG/YYH1311079 bacterial expression vector may provide valuable insight for designing β-glucosidase with higher cellobiose-hydrolyzing efficiencies.ConclusionsThis study suggests that the YYH13 strain of T. hamatum has the potential to serve as a model organism for producing cellulase because of its strong ability to efficiently degrade cellulosic biomass. The genome sequences of YYH13 and YYH16 represents a valuable resource for studying efficient production of biofuels.

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Insights into the different catalytic activities of Clostridium neurotoxins.

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Insights into the different catalytic activities of Clostridium neurotoxins.

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