Structural Basis for a Bispecific NADP+ and CoA Binding Site in an Archaeal Malonyl-Coenzyme A Reductase

• Published in: The Journal of biological chemistry Vol. 288; no. 9; pp. 6363 - 6370
• Main Authors: Demmer, Ulrike, Warkentin, Eberhard, Srivastava, Ankita, Kockelkorn, Daniel, Pötter, Markus, Marx, Achim, Fuchs, Georg, Ermler, Ulrich
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Archaeal Proteins - chemistry; Binding Sites; Biotechnology; Coenzyme A; Coenzyme A - chemistry; Crystal Structure; Enzyme Mechanisms; Enzymology; NADP - chemistry; Nicotinamide; Oxidoreductases - chemistry; Structure-Activity Relationship; Sulfolobus - enzymology; Enzyme Mechanisms; Biotechnology; Crystal Structure; Coenzyme A; Nicotinamide
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.421263

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. Background: Malonyl-CoA reductase of the CO2 assimilating 3-hydroxypropionate/4-hydroxybutyrate cycle reduces malonyl-CoA to malonic semialdehyde. Results: Malonyl-CoA reductase complexed with CoA and NADP+ was structurally characterized. Conclusion: The protein acts as rigid template to press CoA and NADP+ into similar S-shaped, superimposable forms. Significance: The data indicate how to construct a bispecific cofactor binding site and to engineer a malonyl- into methyl-malonyl-CoA reductase for polyester building block production.