Abstract
Metallosphaera sedula is a thermoacidophilic archaeon and has an incomplete TCA/glyoxylate cycle that is used for production of biosynthetic precursors of essential metabolites. Citrate synthase from M. sedula (MsCS) is an enzyme involved in the first step of the incomplete TCA/glyoxylate cycle by converting oxaloacetate and acetyl-CoA into citrate and coenzyme A. To elucidate the inhibition properties of MsCS, we determined its crystal structure at 1.7 Å resolution. Like other Type-I CS, MsCS functions as a dimer and each monomer consists of two distinct domains, a large domain and a small domain. The oxaloacetate binding site locates at the cleft between the two domains, and the active site was more closed upon binding of the oxaloacetate substrate than binding of the citrate product. Interestingly, the inhibition kinetic analysis showed that, unlike other Type-I CSs, MsCS is non-competitively inhibited by NADH. Finally, amino acids and structural comparison of MsCS with other Type-II CSs, which were reported to be non-competitively inhibited by NADH, revealed that MsCS has quite unique NADH binding mode for non-competitive inhibition.
Highlights
Metallosphaera sedula belongs to the sulfolobaceae family
The gene coding for MsCS was amplified from chromosomal DNA of M. sedula by polymerase chain reaction (PCR)
The PCR products were digested by NdeI and XhoI restriction enzymes, and sub-cloned into the pET-30a expression vector, which contained a 6×His tag at the C-terminus of the target protein
Summary
Metallosphaera sedula belongs to the sulfolobaceae family. It is a thermoacidophilic archaea with optimum growth conditions of 73 ̊C and pH 2.0 [1,2,3]. The refined models of MsCS in complex with citrate and oxaloacetate were deposited in the Protein Data Bank [28] with PDB codes of 6ABX and 6ABY, respectively.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have