Crystal structure and biochemical properties of msed_0281, the citrate synthase from Metallosphaera sedula

Highlights

• The crystal structure of archaeal citrate synthase from Metallosphaera sedula (MsCS) was determined.

• The cofactor binding mode of MsCS was confirmed and inhibition properties of MsCS was elucidated.

• MsCS belongs to type I with structural and biochemical properties similar to those of CSs involved in the TCA cycle.

Abstract

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 Å 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.

Introduction

Metallosphaera sedula was isolated from a volcanic field in Italy [1,2]. This microorganism belongs to the Sulfolobaceae family and is an extremely thermoacidophilic archaea, with an optimum growth temperature of 73 °C, at pH 2.0 [3,4]. M. sedula is also highly tolerant to heavy metals and grows chemolithoautotrophically using metal sulfides or molecular hydrogen [5]. M. sedula has attracted attention due to its carbon fixation ability using the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle [[5], [6], [7], [8]]. However, because of the lack of 2-oxoglutarate dehydrogenase, which is an enzyme converting 2-oxoglutarate to succinyl-CoA, the TCA/glyoxylate cycle is incomplete in M. sedula [9]. The accumulated 2-oxoglutarate is used as a biosynthetic precursor for amino acids, such as glutamate, proline, glutamine, and arginine (Fig. 1A) [9]. The lack of reducing power, which occurs as the result of an incomplete TCA/glyoxylate cycle, is overcome by supplies through metal sulfide oxidation and iron oxidation [4].

Citrate synthase (CS, EC 2.3.3.1) is a common enzyme found in most organisms from bacteria to mammalians. This enzyme catalyzes the irreversible conversion of oxaloacetate and acetyl-CoA into citrate (Fig. 1A) [[10], [11], [12], [13]]. This reaction has citryl-CoA as an intermediate, and a conformational change occurs when the first substrate (oxaloacetate) binds to CS, producing a suitable binding pocket for the second substrate (acetyl-CoA) [12,14,15]. So far many CS structures were reported, and CS enzymes are categorized into two distinct types: type-I and type-II CSs [15,[17], [18], [19], [20], [21]]. Type-I CSs function as homo-dimers and are found in gram-positive bacteria, archaea, and eukaryotes [16]. Type-II CSs are mostly found in gram-negative bacteria and usually exist as hexamers with extra β-strands at the N-terminus [17,[22], [23], [24]]. Since CS catalyzes an irreversible reaction, several inhibitors are known to regulate its activity. CS inhibitors are divided into allosteric inhibitors, such as NADH and ATP, and competitive inhibitors, such as citrate and succinyl-CoA [22,25,26].

Although CSs have been studied extensively, no structural or biochemical comparisons have been made between CSs involved in the incomplete TCA/glyoxylate cycle and those involved in the conventional TCA cycle. Herein, we report the crystal structures of MsCS in the apo-form and in complex with acetyl-CoA. We also report kinetic and inhibition analyses on MsCS.