Novel Dual-Coenzyme Specificity and Thermostability of Malate Dehydrogenase Identified in the Cyanobacterium Microcystis aeruginosa PCC7806

TL;DR

This study identifies a unique malate dehydrogenase in a cyanobacterium that works with both NAD+ and NADP+ and is highly stable at high temperatures.

Contribution

The discovery of a dual-coenzyme specific and thermostable MDH in Microcystis aeruginosa provides new insights into the evolution of coenzyme specificity.

Findings

MaMDH shows dual-coenzyme specificity with a slight preference for NAD+.

The enzyme retains over 90% activity after 20 minutes at 70°C.

Structure-guided mutagenesis shifted cofactor preference from NAD+ to NADP+.

Abstract

Malate dehydrogenase (MDH) is a key energy metabolic enzyme with distinct coenzyme specificity for either NAD+ or NADP+ in all domains of life. Here, we characterize a novel MDH from the bloom-forming cyanobacterium Microcystis aeruginosa PCC7806 (MaMDH), which displays dual-coenzyme specificity with comparable efficiency for both NAD+ and NADP+, albeit with a slight preference for NAD+. MaMDH exists as a 72.1 kDa homodimer with a subunit mass of 36.2 kDa in solution. Kinetic measurements yielded Km values of 33.140 μM for NAD+ and 113.200 μM for NADP+, with a kcat ratio (NAD⁺/NADP⁺) of 3.64. The enzyme exhibited optimal activity at pH 8.0 and 40 °C, along with notable thermostability, retaining over 90% activity after incubation at 70 °C for 20 min. Through structure-guided mutagenesis of the predicted coenzyme-binding motif, we shifted MaMDH cofactor preference from NAD+ toward NADP+, supporting the hypothesis that dual-specificity MDHs may represent evolutionary intermediates in the emergence of NADP+-dependent chloroplast MDHs. This study provides new insights into the molecular evolution mechanisms of coenzyme specificity within the MDH family.

Figures