C-terminal hybrid mutant of <i>Bacillus pumilus</i>
 cyanide dihydratase dramatically enhances thermal stability and pH tolerance by reinforcing oligomerization

Crum, M.A.; Park, Jason M.; Sewell, B. Trevor; Benedik, Michael J.

doi:10.1111/jam.12754

Cited by 16 publications

(13 citation statements)

References 18 publications

(48 reference statements)

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“…The cellular conditions may be allowing CynD R67C to partially oligomerize in an active form, which is disrupted upon lysis, although the protein does not appear to degrade more rapidly than wild type. The R67C defect has also been shown to be suppressed by stabilizing changes at the c-terminus of the protein, supporting the conclusion that only oligomer formation is perturbed ( Crum et al, 2015 ).…”

Section: Discussionmentioning

confidence: 57%

Probing an Interfacial Surface in the Cyanide Dihydratase from Bacillus pumilus, A Spiral Forming Nitrilase

et al. 2016

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Nitrilases are of significant interest both due to their potential for industrial production of valuable products as well as degradation of hazardous nitrile-containing wastes. All known functional members of the nitrilase superfamily have an underlying dimer structure. The true nitrilases expand upon this basic dimer and form large spiral or helical homo-oligomers. The formation of this larger structure is linked to both the activity and substrate specificity of these nitrilases. The sequences of the spiral nitrilases differ from the non-spiral forming homologs by the presence of two insertion regions. Homology modeling suggests that these regions are responsible for associating the nitrilase dimers into the oligomer. Here we used cysteine scanning across these two regions, in the spiral forming nitrilase cyanide dihydratase from Bacillus pumilus (CynD), to identify residues altering the oligomeric state or activity of the nitrilase. Several mutations were found to cause changes to the size of the oligomer as well as reduction in activity. Additionally one mutation, R67C, caused a partial defect in oligomerization with the accumulation of smaller oligomer variants. These results support the hypothesis that these insertion regions contribute to the unique quaternary structure of the spiral microbial nitrilases.

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Section: Discussionmentioning

confidence: 57%

Probing an Interfacial Surface in the Cyanide Dihydratase from Bacillus pumilus, A Spiral Forming Nitrilase

et al. 2016

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“…2). These interactions stabilize the C- and A-interfaces, explaining why C-terminal truncation decreases thermostability 40 and why the C-terminal tail is an important factor in oligomerization 3,41,42 . Interactions across the helical groove (D- or F-interfaces) were not observed (Fig.…”

Section: Discussionmentioning

confidence: 99%

Cryo-EM and directed evolution reveal how Arabidopsis nitrilase specificity is influenced by its quaternary structure

2019

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Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced nitrilases with altered specificity. Plant nitrilase substrate size and specificity correlate with helical twist, but molecular details of this relationship are lacking. Here we determine, to our knowledge, the first close-to-atomic resolution (3.4 Å) cryo-EM structure of an active helical nitrilase, the nitrilase 4 from Arabidopsis thaliana . We apply site-saturation mutagenesis directed evolution to three residues (R95, S224, and L169) and generate a mutant with an altered helical twist that accepts substrates not catalyzed by known plant nitrilases. We reveal that a loop between α2 and α3 limits the length of the binding pocket and propose that it shifts position as a function of helical twist. These insights will allow us to start designing nitrilases for chemoenzymatic synthesis.

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“…Deletion of more than 28 amino acids from the B. pumilus CynD C-terminus disrupted its activity but shorter deletions remained active in vivo ( Sewell et al, 2005 ; Crum et al, 2015a ). Exchanging C-termini between the CynD enzymes from B. pumilus and P. stutzeri led to a dramatic increase in stability as well ( Crum et al, 2015b ).…”

Section: Discussionmentioning

confidence: 99%

Bacillus pumilus Cyanide Dihydratase Mutants with Higher Catalytic Activity

2016

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Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied. K93R and D172N increased the enzyme’s thermostability whereas E327K mutation had a less pronounced effect on stability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 but lowered its kcat. However, the A202T mutation, located in the dimerization or the A surface, destabilized the protein and abolished its activity. No significant effect on activity at alkaline pH was observed for any of the purified mutants. These mutations help confirm the model of CynD and are discussed in the context of the protein–protein interfaces leading to the protein quaternary structure.

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C-terminal hybrid mutant of Bacillus pumilus cyanide dihydratase dramatically enhances thermal stability and pH tolerance by reinforcing oligomerization

Abstract: The study characterized a chimeric protein with remarkable thermal stability and tolerance to alkaline conditions, features essential for practical application as industrial cyanide solutions are maintained as highly alkaline solutions to prevent formation of hydrogen cyanide gas.

Cited by 16 publications

References 18 publications

Probing an Interfacial Surface in the Cyanide Dihydratase from Bacillus pumilus, A Spiral Forming Nitrilase

Probing an Interfacial Surface in the Cyanide Dihydratase from Bacillus pumilus, A Spiral Forming Nitrilase

Cryo-EM and directed evolution reveal how Arabidopsis nitrilase specificity is influenced by its quaternary structure

Bacillus pumilus Cyanide Dihydratase Mutants with Higher Catalytic Activity

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