Metallosphaera sedula is a thermoacidophilic archaeon that has carbon fixation ability using the 3-hydroxypropionate/4-hydroxybutyrate(3-HP/4-HB) cycle, and has an incomplete TCA cycle to produce necessary biosynthetic precursors. The citrate synthase from M. sedula (MsCS) is an enzyme involved in the first step of the incomplete TCA cycle, catalyzing the conversion of oxaloacetate and acetyl-CoA into citrate and coenzyme A. To investigate the molecular mechanism of MsCS, we determined its crystal structure at 1.8 angstrom resolution. As other known CSs, MsCS functions as a dimer, and each monomer consists of two domains, a large domain and a small domain. We also determined the structure of the complex with acetyl-CoA and revealed the acetyl-CoA binding mode of MsCS. Structural comparison of MsCS with another CS in complex with oxaloacetate enabled us to predict the oxaloacetate binding site. Moreover, we performed inhibitory kinetic analyses of MsCS, and showed that the protein is inhibited by citrate and ATP by competitive and non-competitive inhibition modes, respectively, but not by NADH. Based on these results, we suggest that MsCS belongs to the type-I CS with structural and biochemical properties similar to those of CSs involved in the conventional TCA cycle. (C) 2018 Elsevier Inc. All rights reserved.