Mutagenesis of Key Residues in the Binding Center of l‐Aspartate‐β‐Semialdehyde Dehydrogenase from Escherichia coli Enhances Utilization of the Cofactor NAD(H)

Abstract

L-Aspartate-β-semialdehyde dehydrogenase (ASADH) is a key enzyme in the aspartate pathway. In bacteria, ASADH is highly specific for the cofactor NADP(+) rather than NAD(+). Limited information on cofactor utilization is available, and neither the wild-type protein nor the available mutants could utilize NAD(+) efficiently. In this study, we identified several residues crucial for cofactor utilization by Escherichia coli ASADH (ecASADH) by mutating residues within the cofactor binding center. Among the investigated mutants, ecASADH-Q350N and ecASADH-Q350N/H171A, which exhibited markedly improved NAD(+) utilization, were further investigated by various biochemical approaches and molecular modeling. Relative to the wild type, the two mutants showed approximately 44-fold and 66-fold increases, respectively, in the constant kcat /Km of NAD(+). As desired, they could also utilize NADH efficiently to synthesize l-homoserine in cascade reactions in vitro.

Structure of aspartate β-semialdehyde dehydrogenase fromFrancisella tularensis

“…The nonlinear regression analysis of the data yielded a K d value of 2.9 AE 0.9 mM for NADP + binding (n = 1.06 AE 0.04 per protein chain, ÁH = À8796 AE 482 cal mol À1 , ÁS = À4.2 cal mol À1 K À1 , ÁG = À7553 cal mol À1 ). Previously, a similar K d of 3.0 AE 0.2 mM was reported for the EcASADH double mutant (Q350N/H171A; Xu et al, 2016). However, wild-type EcASADH bound NADP + with a K d of 168 AE 22 mM.…”

NAD+‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

“…Until now, all sequences of 7β‐HSDHs reported in the literature are NADP + ‐dependent enzymes . However, protein engineering provides a straightforward method to change cofactor specificity; this has been the subject of many pieces of research in both academia and industry over the past few decades . Unfortunately, the resulting enzymes usually have a perfect cofactor switch, but in several cases the enzyme activity and/or substrate specificity decreases tremendously .…”

OsCERK1-Mediated Chitin Perception and Immune Signaling Requires Receptor-like Cytoplasmic Kinase 185 to Activate an MAPK Cascade in Rice

“…MBP-tag fused proteins were expressed in E. coli and purified according to a standard production instruction. His-tag fused proteins were purified according to a previous report (Xu et al, 2016). After elution, purified proteins were ultrafiltrated using Amicon Ultra-15 Centrifugal Filter Units (Merck Millipore) for buffer exchange with protein storage buffer containing 10 mM HEPES (pH 7.5) and 10% glycerol.…”