A Novel ADP-forming Succinyl-CoA Synthetase in Thermococcus kodakaraensis Structurally Related to the Archaeal Nucleoside Diphosphate-forming Acetyl-CoA Synthetases

2. March 2008 by HealingMindN.

Originally published In Press as doi:10.1074/jbc.M702694200 on July 19, 2007 J. Biol. Chem., Vol. 282, Issue 37, 26963-26970, September 14, 2007

Kenichi Shikata, Toshiaki Fukui, Haruyuki Atomi, and Tadayuki Imanaka¹

From the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

We have identified and characterized a structurally novel succinyl-CoA synthetase (SCS) from the hyperthermophilic Archaea Thermococcus kodakaraensis. The presence of an SCS completes the metabolic pathway from glutamate to succinate in Thermococcales, which had not been clarified because of the absence of classical SCS homologs on their genomes.

The SCS from T. kodakaraensis (SCS_Tk) is a heteromeric enzyme (₂₂) encoded by TK1880 (-subunit) and TK0943 (-subunit). Although both SCS_Tk and classical SCSs harbor the five domains present in enzymes of the acyl-CoA synthetase (nucleoside diphosphate-forming) superfamily, the domain order and distribution among subunits in SCS_Tk (-subunit, domains 1-2-5; -subunit, domains 3-4) are distinct from those of classical SCSs (-subunit, domains 1-2; -subunit, domains 3-4-5) and instead resemble the acetyl-CoA synthetases from Pyrococcus furiosus (ACSs I_Pf and II_Pf).

Comparison of the four Thermococcales genomes revealed that each strain harbors five - and two -subunit homologs. Sequence similarity suggests that the -subunit of SCS_Tk is also a component of the presumed ACS II from T. kodakaraensis (ACS II_Tk).

We coexpressed the /-genes of SCS_Tk (TK1880/TK0943) and of ACS II_Tk (TK0139/TK0943). ACS II_Tk recognizes a broad range of hydrophobic/aromatic acid compounds, as is the case with ACS II_Pf, whereas SCS_Tk displays a distinct and relatively strict substrate specificity for several acids, including succinate. This indicates that the -subunits are responsible for the distinct substrate specificities of SCS_Tk and ACS II_Tk.

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Received for publication, March 29, 2007 , and in revised form, July 19, 2007. ^* This work was supported by Grant-in-aid for Scientific Research 14103011 (to T. I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement“ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¹ To whom correspondence should be addressed. Tel.: 81-75-383-2777; Fax: 81-75-383-2778

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