Probing C-terminal interactions of the Pseudomonas stutzeri cyanide-degrading CynD protein

Abstract

The cyanide dihydratases from Bacillus pumilus and Pseudomonas stutzeri share high amino acid sequence similarity throughout except for their highly divergent C-termini. However, deletion or exchange of the C-termini had different effects upon each enzyme. Here we extended previous studies and investigated how the C-terminus affects the activity and stability of three nitrilases, the cyanide dihydratases from B. pumilus (CynDpum) and P. stutzeri (CynDstut) and the cyanide hydratase from Neurospora crassa. Enzymes in which the C-terminal residues were deleted decreased in both activity and thermostability with increasing deletion lengths. However, CynDstut was more sensitive to such truncation than the other two enzymes. A domain of the P. stutzeri CynDstut C-terminus not found in the other enzymes, 306GERDST311, was shown to be necessary for functionality and explains the inactivity of the previously described CynDstut-pum hybrid. This suggests that the B. pumilus C-terminus, which lacks this motif, may have specific interactions elsewhere in the protein, preventing it from acting in trans on a heterologous CynD protein. We identify the dimerization interface A-surface region 195-206 (A2) from CynDpum as this interaction site. However, this A2 region did not rescue activity in C-terminally truncated CynDstutΔ302 or enhance the activity of full-length CynDstut and therefore does not act as a general stability motif.

Systems Approaches to Predict the Functions of Glycoside Hydrolases during the Life Cycle of Aspergillus niger Using Developmental Mutants ∆brlA and ∆flbA

“…As described previously [ 5 ], cfcA was one of the genes with the highest increase in transcription after one day of carbon starvation in the wild-type strain ( Table 1A ). Cfca hydrolyzes cell wall chitin during carbon starvation and is required for fragmentation [ 39 ]. Expression of the homologs of this chitinase in A .…”

“…Chitinase CfcA hydrolyzed chitin in the cell wall and strongly contributed to cell wall fragmentation in submerged fermentations where the pH control was released at the start of carbon starvation. These results show that under higher pH values, CfcA is present in the fungal culture, the enzyme has higher activity then at low pH values, and its effect on the cell wall can readily be measured and observed [ 39 ].…”

Bacillus pumilus Cyanide Dihydratase Mutants with Higher Catalytic Activity

“…Studying the effects of various C-terminal truncations has led to clues on the role and location of this extension. 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 ).…”

From sequence to function: a new workflow for nitrilase identification

“…Sequences for aromatic nitrilases were obtained from Rhodococcus rhodochrous J1 (D11425; Kobayashi et al 1992a), R. rhodochrous NCIMB 11216 (AX235749; Ress-Löschke et al 2001), and Aeribacillus pallidus RAPc8 (DQ826045; Williamson et al 2010). References for cyanide dihydratases were taken from Pseudomonas stutzeri AK61 (KM459551; Crum et al 2015), Bacillus pumilus C1 (AF492815; Jandhyala et al 2003), and B. pumilus 8A3 (AF492814; Jandhyala et al 2003). β-Cyano-L-alanine nitrilases originated from Pseudomonas protegens Pf-5 (CP000076; Paulsen et al 2005;, P. fluorescens Pf0-1 (CP000094; Silby et al 2009), and Pseudomonas pseudoalcaligenes CECT 5344 (HG916826; Wibberg et al 2014;Acera et al 2017).…”