Structural basis for a bispecific NADP+ and CoA binding site in an archaeal malonyl-coenzyme A reductase

Abstract

Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO2 into cell material. The product of the initial acetyl-CoA carboxylation with CO2, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP(+) and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP(+) and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3'- and 2'-ribose phosphate group of CoA and NADP(+), respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with the α-phosphate of NADP(+). Evolution from an NADP(+) to a bispecific NADP(+) and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP(+) binding. Based on the paralogous aspartate-β-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis.

FIGURE 1.

Reactions catalyzed by MCR (A) and ASD (B).

FIGURE 2.

Structure of MCR from S. todokaii. The MCR homotetramer is arranged as a dimer of dimers (colored in magenta/blue and gray/green). Each monomer is built up of a dinucleotide binding (dark green) and a dimerization domain (light green). The active site cleft lies between them and is occupied in the MCR_CoA structure by CoA (drawn as a stick model). Two dimers form an extended interface that includes both cover loops (black arrow) being involved in substrate binding.

FIGURE 3.

Cofactor binding to MCR. A, NADP⁺ binding in stereo. NADP⁺ binds in a characteristic S-shaped conformation and is sandwiched between the loops of the C-terminal end of the β-sheet (light gray) and the cover loop (dark gray). Only residues forming hydrogen bonds to the polypeptide are shown. NADP⁺ is firmly anchored to the polypeptide at the 2′-ribose phosphate binding site. Proton donors comprise Lys⁴¹ NH, Gly⁴² NH, Ser⁴³ NH, Ser⁴³ O_γH, and Thr¹⁶ O_γ1H as well as via a solvent molecule Ala³⁹ O, Thr¹⁶ NH, and Ala¹⁵ NH. The 2F_obs − F_calc electron density (cyan) is drawn at a contour level of 1.3σ. B, CoA binding in stereo. CoA is arranged in an S-shaped, rather compressed conformation (distance between α-phosphate O2A and β-cysteamine N4P, 8.7 Å) and embedded into the same binding site as NADP⁺. A metal ion seems to be positioned between the α- and β-phosphate and the Tyr¹⁸⁷ carbonyl oxygens. The 2F_obs − F_calc electron density (cyan) is drawn at a contour level of 1.5σ. C, superposition of NADP⁺ and CoA. The carbons of NADP⁺ are colored in yellow, and CoA carbons are in green. The distances between the β-phosphate of CoA and the α-phosphate of NADP⁺ and between their 3′- and 2′-ribose phosphates are ∼1.4 Å.

FIGURE 4.

Binding of the enzyme-malonyl thioacyl adduct. The Cys¹⁵³-malonyl adduct (malonyl carbons in green) of MCR is modeled on the basis of the corresponding cysteine-aspartyl template of ASD. Although the thioacyl and the carboxylate surroundings of malonyl and aspartyl are highly conserved, the absence of the ammonium group tracks with an exchange of a glutamate and an asparagine in ASD with a tyrosine (Tyr²⁰⁶) and a leucine (Leu¹⁵²) in MCR, respectively.

FIGURE 5.

Proposed enzymatic mechanism. A, malonyl-CoA is bound and subsequently attacked by the Cys¹⁵³ thiolate forming a tetrahedral intermediate. The negative charge of the oxyanion is stabilized by basic residues and by the positively charged N-terminal end of helix 152:168. B, the tetrahedral intermediate is converted to a thioacyl adduct, CoA is released, and NADPH is bound. CoA and NADPH use an identical binding site. C, NADPH transfers a hydride from the B-side to the thioacyl carbon forming a hemithioacetal intermediate, which is converted to the product malonic semialdehyde, thereby restoring Cys¹⁵³ (D).