Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764
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...
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doaj-0300f90e7fea45e98ee0ea1d43d0c19d2021-02-11T04:22:20ZengElsevierBiochemistry and Biophysics Reports2405-58082021-03-0125100893Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764Lauren B. Jones0Xiaoqiang Wang1Jaya S. Gullapalli2Daniel A. Kunz3Division of Biochemistry and Molecular Biology, Department of Biological Sciences, University of North Texas, Denton, TX, 76203, USADivision of Biochemistry and Molecular Biology, Department of Biological Sciences, University of North Texas, Denton, TX, 76203, USA; BioDiscovery Institute, Department of Biological Sciences, University of North Texas, Denton, TX, 76203, USADivision of Biochemistry and Molecular Biology, Department of Biological Sciences, University of North Texas, Denton, TX, 76203, USADivision of Biochemistry and Molecular Biology, Department of Biological Sciences, University of North Texas, Denton, TX, 76203, USA; Corresponding author. Department of Biological Sciences, University of North Texas, 1155 Union Circle #305220, Denton, TX, 76203-5017, USA.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 situ 1H 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.http://www.sciencedirect.com/science/article/pii/S240558082030203XNitrilaseStructureEnzymePseudomonasNit1CCyanotroph |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Lauren B. Jones Xiaoqiang Wang Jaya S. Gullapalli Daniel A. Kunz |
spellingShingle |
Lauren B. Jones Xiaoqiang Wang Jaya S. Gullapalli Daniel A. Kunz Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764 Biochemistry and Biophysics Reports Nitrilase Structure Enzyme Pseudomonas Nit1C Cyanotroph |
author_facet |
Lauren B. Jones Xiaoqiang Wang Jaya S. Gullapalli Daniel A. Kunz |
author_sort |
Lauren B. Jones |
title |
Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764 |
title_short |
Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764 |
title_full |
Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764 |
title_fullStr |
Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764 |
title_full_unstemmed |
Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764 |
title_sort |
characterization of the nit6803 nitrilase homolog from the cyanotroph pseudomonas fluorescens ncimb 11764 |
publisher |
Elsevier |
series |
Biochemistry and Biophysics Reports |
issn |
2405-5808 |
publishDate |
2021-03-01 |
description |
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 situ 1H 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. |
topic |
Nitrilase Structure Enzyme Pseudomonas Nit1C Cyanotroph |
url |
http://www.sciencedirect.com/science/article/pii/S240558082030203X |
work_keys_str_mv |
AT laurenbjones characterizationofthenit6803nitrilasehomologfromthecyanotrophpseudomonasfluorescensncimb11764 AT xiaoqiangwang characterizationofthenit6803nitrilasehomologfromthecyanotrophpseudomonasfluorescensncimb11764 AT jayasgullapalli characterizationofthenit6803nitrilasehomologfromthecyanotrophpseudomonasfluorescensncimb11764 AT danielakunz characterizationofthenit6803nitrilasehomologfromthecyanotrophpseudomonasfluorescensncimb11764 |
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