Biosynthesis of acetyl-CoA synthase and its derivatives /
Acetyl-CoA synthase (a.k.a. carbon monoxide dehydrogenase) from Clostridium thermoaceticum (ACS[Ct]/CODH[Ct]) is an α₂β₂ tetramer containing three metal-centers called the A-, B-, and C-clusters. This enzyme catalyzes the reversible reduction of CO₂ to CO at the C-cluster, and the synthesis of acety...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2001.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=726104581&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Acetyl-CoA synthase (a.k.a. carbon monoxide dehydrogenase) from Clostridium thermoaceticum (ACS[Ct]/CODH[Ct]) is an α₂β₂ tetramer containing three metal-centers called the A-, B-, and C-clusters. This enzyme catalyzes the reversible reduction of CO₂ to CO at the C-cluster, and the synthesis of acetyl-CoA at the A-cluster. Genes acsA and acsB encoding the β and α subunits were cloned into Escherichia coli JM109 and overexpressed at 37 C̊ under anaerobic conditions with Ni supplementation. The resulting recombinant His-tagged protein (AcsAB) exhibited CO oxidation activity and EPR signals with g-values and low spin intensities indistinguishable from those of the reduced states of the B- and C-clusters of ACS[Ct] from which Ni had been removed from the A-cluster. Upon overnight exposure to NiCl₂, the resulting recombinant enzyme (ACS[Ec]) developed CO exchange activity and exhibited an EPR signal indistinguishable from the NiFeC signal of Ni-replete ACS[Ct]. Two ORF's, acsF and acsG were identified in the acs operon. The predicted amino acid sequence of acsG has a high percentage of similarity to seven other proteins from archaea and Rhodospirillum rubrum, all of which contain ACS/CODH's. AcsG has a conserved "P-loop" which indicates ATP/GTP binding and is implicated in the C-cluster-Ni-insertion process through comparison to its homolog, CooC from R. rubrum. Furthermore, a fusion protein of glutathione S-transferase and AcsG that exhibited ATP hydrolysis was isolated. Three mini-metalloproteins of the acetyl-CoA synthase active site, a 737-, a 436-, and a 223-mer were similarly biosynthesized in E. coli as AcsAB. The 737- and 436-mers each contained a redox-active [Fe₄S₄]²⁺[/]¹⁺ cluster. The Ni-activated 737-mer exhibited an EPR signal and spin intensity indistinguishable from the NiFeC signal of ACS[Ct]. Unexpectedly, activated 737-mer also catalyzed the synthesis of acetyl-CoA, though at a rate ~ 10-fold slower than ACS[Ct]. The Ni-activated 436-mer exhibited an EPR signal indistinguishable from the pNiFeC signal of previously isolated α (Xia & Lindahl, 1996) but was catalytically inactive. The 223-mer contained a redox-inactive Fe₄S₄ cluster and was EPR and catalytically inactive. The characterizations of the three mini-proteins revealed proportional loss of physical properties that characterize the active site as the protein scaffold diminished in size. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xiii, 88 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 77-85). |