Genetic Construction of Truncated and Chimeric Metalloproteins Derived from the α Subunit of Acetyl-CoA Synthase from Clostridium t hermoaceticum

In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an α2β2 tetrameric 310 kDa bifunctional enzyme. ACS catalyzes the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA from CO (or CO2 in the presence of low-pote...

Full description

Saved in:
Bibliographic Details
Published in:Journal of the American Chemical Society 2002-07, Vol.124 (29), p.8667-8672
Main Authors: Loke, Huay-Keng, Tan, Xiangshi, Lindahl, Paul A
Format: Article
Language:English
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an α2β2 tetrameric 310 kDa bifunctional enzyme. ACS catalyzes the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA from CO (or CO2 in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains seven metal−sulfur clusters of four different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kDa β subunit, while the A-cluster, a Ni−X−Fe4S4 cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kDa α subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire β subunit was removed, as long as CO, rather than CO2 and a low-potential reductant, was used as a substrate. Truncating an ∼30 kDa region from the N-terminus of the α subunit yielded a 49 kDa protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO-binding properties. Further truncation afforded a 23 kDa protein that lacked recognizable A-cluster properties except for UV−vis spectra typical of [Fe4S4]2+ clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 and 82 kDa fragments of the α subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multicentered metalloenzymes and to generate new complex metalloenzymes with interesting properties.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja025924w