Genetic Construction of Truncated and Chimeric Metalloproteins Derived from the Alpha Subunit of Acetyl-CoA Synthase from Clostridium thermoaceticum

Journal Article · Fri Jun 28 04:00:00 EDT 2002 · Journal of the American Chemical Society

DOI:https://doi.org/10.1021/ja025924w· OSTI ID:826199

Loke, Huay-Keng ^[1]; Tan, Xiangshi; Lindahl, Paul A

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1. Texas A&M University

In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha2beta2 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 7 metal-sulfur clusters of 4 different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kda beta 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 alpha 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 alpha subunit was removed, as long as CO, rather than CO2 and a low-potential reductant, was used as a substrate. Truncating an {approx} 30 kda region from the N-terminus of the alpha 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 kda and 82 kda fragments of the alpha 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 multi-centered metalloenzymes and to generate new complex metalloenzymes with interesting properties.

• Research Organization: Texas A& M University, College Station, TX (US)
• Sponsoring Organization: USDOE Office of Energy Research (ER) (US)
• DOE Contract Number: FG03-01ER15177
• OSTI ID: 826199
• Report Number(s): DOE/ER/15177-1
• Journal Information: Journal of the American Chemical Society, Journal Name: Journal of the American Chemical Society Journal Issue: 29 Vol. 124
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ACETYL-COENZYME A SYNTHASE
CARBON MONOXIDE DEHYDROGENASE
CLOSTRIDIUM
CONSTRUCTION
EPR SPECTROSCOPY
FERREDOXIN
GENES
GENETICS
IRON-SULFUR CLUSTERS
METALLOPROTEINS
NICKEL
PROTEINS
SPECTRA
SULFUR
SYNTHESIS