Structural and functional analysis of the yeast N -acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism

Abstract

Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog L-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the L-proline and L-arginine metabolism by acetylating L-Δ 1 -pyrroline-5-carboxylate, leading to the L-arginine-dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-L-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA-binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent k cat value. The replacement of Asn135 led to a remarkable increase in the apparent K m value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the L-Δ 1 -pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs. cyclic amine N-acetyltransferase | X-ray crystallography | reaction mechanism | antioxidant enzyme

Metabolomic analysis of acid stress response in Saccharomyces cerevisiae

“…Proline might protect cells from acid-induced oxidative stress by the following contribution. Overexpressed Mpr1 (encoding N-acetyltransferase which detoxifies proline-analogue compound) may lead to decreased ROS and increased cell viability (67,68). Proline is also suggested to act as a scavenger of more reactive oxidative stress compounds produced under acid stress conditions, such as hydroxy radicals ( OH) (69), which are caused by lactate induction (anion) via Fenton reaction (70,71).…”

Structure-based molecular design for thermostabilization ofN-acetyltransferase Mpr1 involved in a novel pathway of l-arginine synthesis in yeast

“…Our previous study revealed that Mpr1 forms a dimer in solution (13). In the crystal structure, Asn203 is located at the interface of two molecules of Mpr1, which seem to be a pair of dimers.…”

“…A recent structural analysis clarified the mechanism by which the substitution of Leu for Phe65 increases the thermostability of Mpr1, and also provided clues to the design of novel mutations for improvement of the enzymatic functions (13). Here, we successfully constructed new stable Mpr1 variants by structure-based molecular design, and indicated that some of them had increased abilities to contribute to L-arginine biosynthesis.…”

“…The Mpr1 protein was overexpressed as previously reported (13). Briefly, the E. coli strain BL21(DE3) harboring pQE2-MPR1(SacIHindIII) was cultured at 37 C in M9+CA medium.…”

“…Recently, we determined the three-dimensional structure of Mpr1 by crystallographic analysis (13), and showed that the folding pattern of Mpr1 is similar to those of proteins in the Gcn5-related N-acetyltransferase (GNAT) superfamily, to which Mpr1 also belongs. The crystal structure of Mpr1 helped us to understand the mechanism by which Phe65Leu substitution increased its thermostability.…”