Substrate specificity and evolutionary implications of a NifDK enzyme carrying NifB-co at its active site

Soboh, Basem; Boyd, Eric S.; Zhao, Dehua; Peters, John W.; Rubio, Luis M.

doi:10.1016/j.febslet.2010.02.064

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Volume 584, Issue 8 , Pages 1487-1492, 16 April 2010

Substrate specificity and evolutionary implications of a NifDK enzyme carrying NifB-co at its active site

Edited by Stuart Ferguson

Basem Soboh
- Institute of Biology/Microbiology, Martin-Luther University Halle-Wittenberg, Halle 06120, Germany
- Both authors equally contributed to this study.
Eric S. Boyd
- Department of Chemistry and Biochemistry and Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman 59717, USA
- Both authors equally contributed to this study.
Dehua Zhao
- Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158, USA
John W. Peters
- Department of Chemistry and Biochemistry and Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman 59717, USA
- Corresponding authors. Fax: +1 406 994 7212 (J.W. Peters), +34 91 715 7721 (L.M. Rubio).
Luis M. Rubio
- Instituto Madrileño de Estudios Avanzados (IMDEA) Energía, Madrid 28023, Spain
- Corresponding authors. Fax: +1 406 994 7212 (J.W. Peters), +34 91 715 7721 (L.M. Rubio).

Received 8 January 2010; received in revised form 4 February 2010; accepted 23 February 2010. published online 26 February 2010.

Abstract

The in vitro reconstitution of molybdenum nitrogenase was manipulated to generate a chimeric enzyme in which the active site iron–molybdenum cofactor (FeMo-co) is replaced by NifB-co. The NifDK/NifB-co enzyme was unable to reduce N₂ to NH₃, while exhibiting residual C₂H₄ and considerable H₂ production activities. Production of H₂ by NifDK/NifB-co was stimulated by N₂ and was dependent on NifH and ATP hydrolysis. Thus, NifDK/NifB-co is a useful tool to gain insights into the catalytic mechanism of nitrogenase. Furthermore, phylogenetic analysis of D and K homologs indicates that several early emerging lineages, which contain NifB, NifH and NifDK encoding genes but which lack other genes required for processing NifB-co into FeMo-co, might encode an enzyme with similar catalytic properties to NifDK/NifB-co.

Abbreviations: FeMo-co, iron–molybdenum cofactor, ICP-OES, inductively coupled plasma-optical emission spectrometry, EPR, electron paramagnetic resonance, DTH, sodium dithionite, Sarkosyl, n-lauroyl sarcosine, MOPS, (N-morpholino)propanesulfonic acid