Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764

• Published in: Biochemistry and biophysics reports 2021-03, Vol.25, p.100893-100893, Article 100893
• Main Authors: Jones, Lauren B., Wang, Xiaoqiang, Gullapalli, Jaya S., Kunz, Daniel A.
• Format: Article
• Language: English
• Subjects: Cyanotroph; Enzyme; Nit1C; Nitrilase; Pseudomonas; Structure
• ISSN: 2405-5808; 2405-5808
• DOI: 10.1016/j.bbrep.2020.100893

We report the purification and characterization of a nitrilase (E.C. 3.5.5.1) (Nit11764) essential for the assimilation of cyanide as the sole nitrogen source by the cyanotroph, Pseudomonas fluorescens NCIMB 11764. Nit11764, is a member of a family of homologous proteins (nitrile_sll0784) for which the genes typically reside in a conserved seven-gene cluster known as Nit1C. The physical properties and substrate specificity of Nit11764 resemble those of Nit6803, the current reference protein for the family, and the only true nitrilase that has been crystallized. The substrate binding pocket of the two enzymes places the substrate in direct proximity to the active site nucleophile (C160) and conserved catalytic triad (Glu44, Lys126). The two enzymes exhibit a similar substrate profile, however, for Nit11764, cinnamonitrile, was found to be an even better substrate than fumaronitrile the best substrate previously identified for Nit6803. A higher affinity for cinnamonitrile (Km 1.27 mM) compared to fumaronitrile (Km 8.57 mM) is consistent with docking studies predicting a more favorable interaction with hydrophobic residues lining the binding pocket. By comparison, 3,4-dimethoxycinnamonitrile was a poorer substrate the substituted methoxyl groups apparently hindering entry into the binding pocket. in situ1H NMR studies revealed that only one of the two nitrile substituents in the dinitrile, fumaronitrile, was attacked yielding trans-3-cyanoacrylate (plus ammonia) as a product. The essentiality of Nit11764 for cyanotrophy remains uncertain given that cyanide itself is a poor substrate and the catalytic efficiencies for even the best of nitrile substrates (~5 × 103 M−1 s−1) is less than stellar. •The nitrilase, Nit11764, shares properties with Nit6803 both derived from a conserved gene cluster in cyanotrophic bacteria.•The best substrate for Nit117674 was cinnamonitrile consistent with favorable docking studies.•The dinitrile, fumaronitrile, was the second best substrate for which 3-cyanoacrylate was identified by 1NMR.•While annotated as an aliphatic nitrilase the physiological role of Nit11764 remains unclear.