Engineering Malic Enzyme CO₂ Fixation Activity via a Structure–Sequence–SCANNER (3S) Co-Evolution Strategy

Enzymatic CO₂ fixation offers great potential for the sustainable synthesis of value-added compounds. Malic enzyme (ME) catalyzes the reverse carboxylation of pyruvate to malate, enabling direct CO₂ conversion into C₄ compounds with broad biosynthetic applications. However, the reverse carboxylation activity of wild-type ME is insufficient, and conventional enzyme engineering strategies remain limited by the complexity of identifying distal functional sites. Here, we present a Structure–Sequence–SCANNER (3S) co-evolution strategy that integrates protein structural analysis, sequence conservation profiling, and co-evolutionary network analysis to enable systematic identification of functionally relevant hotspot residues. Using this approach, we engineered Escherichia coli ME (EcME) variants with enhanced CO₂ fixation activities. In total, 106 single-point variants were constructed and screened. Among these, variants A464S and D97E exhibited significantly improved reverse carboxylation activities, with 1.7-fold and 1.6-fold increases in catalytic activity and 1.5-fold and 1.8-fold improvements in catalytic efficiency (k_cat/K_m), respectively, compared to wild-type EcME. Their catalytic efficiencies (k_cat/K_m) improved by 1.5-fold and 1.8-fold, increasing from 80 mM⁻¹·min⁻¹ for the wild-type enzyme to 120 and 130 mM⁻¹·min⁻¹, respectively. Mechanistic analyses revealed that A464S introduces a stabilizing hydrogen bond with N462, enhancing NADPH binding, while D97E forms a new salt bridge network with K513, resulting in contraction of the substrate pocket entrance and increased pyruvate affinity. These findings demonstrate the effectiveness of the 3S strategy in reprogramming enzyme functions and highlight its potential for constructing efficient artificial CO₂ fixation systems.