Document Type

Article

Language

eng

Format of Original

6 p.

Publication Date

1-2017

Publisher

Elsevier

Source Publication

Biochimica et Biophysica Acta: Proteins and Proteomics

Source ISSN

1878-1454

Original Item ID

DOI: 10.1016/j.bbapap.2016.09.013; PubMed Central: PMID: 27693250

Abstract

Nitrile hydratase (NHase), an industrially important enzyme that catalyzes the hydration of nitriles to their corresponding amides, has only been characterized from prokaryotic microbes. The putative NHase from the eukaryotic unicellular choanoflagellate organism Monosiga brevicollis (MbNHase) was heterologously expressed in Escherichia coli. The resulting enzyme expressed as a single polypeptide with fused α- and β-subunits linked by a seventeen-histidine region. Size-exclusion chromatography indicated that MbNHase exists primarily as an (αβ)2 homodimer in solution, analogous to the α2β2 homotetramer architecture observed for prokaryotic NHases. The NHase enzyme contained its full complement of Co(III) and was fully functional without the co-expression of an activator protein or E. coli GroES/EL molecular chaperones. The homology model of MbNHase was developed identifying Cys400, Cys403, and Cys405 as active site ligands. The results presented here provide the first experimental data for a mature and active eukaryotic NHase with fused subunits. Since this new member of the NHase family is expressed from a single gene without the requirement of an activator protein, it represents an alternative biocatalyst for industrial syntheses of important amide compounds.

Comments

Accepted version. Biochimica et Biophysica Acta: Proteins and Proteomics, Vol 1865, No. 1 (January 2017): pg. 107-112. DOI. © 2016 Elsevier B.V. Used with permission.

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