Enzymatic characterization and molecular mechanism of a novel aspartokinase mutant M372I/T379W fromCorynebacterium pekinense

Abstract

A novel aspartokinase mutant M372I/T379W from Corynebacterium pekinense was constructed by using site-directed mutagenesis.

Mechanism of the feedback-inhibition resistance in aspartate kinase of Corynebacterium pekinense: from experiment to MD simulations

“…As reported, WT was inhibited by the synergistic feedback of threonine and lysine in a dose-dependent manner. [16][17][18][19] As can be seen from Table 1, the enzyme activity of mutant A380C was improved under different concentration of inhibitors, and even activated at high concentration of Lys, Lys + Thr, Lys + Met, Thr + Met, and Lys + Thr + Met, which was consistent with the decrease of the n value in the kinetic analysis. Compared with WT, 16 mutants had a more obvious disinhibition effect, and the activation effect of mutant is mainly contributed by Lys inhibitor.…”

“…In our previous studies, 17,18 the homology model for CpAK was obtained. Here we directly used the homology model, including the ligands (Asp and ATP) and inhibitor (Lys).…”

“…Elucidation of its allosteric regulatory mechanism is of great signicance for the construction of highly efficient microbial strains. [16][17][18] However, up to date, the detailed allosteric mechanism of CpAK is still unclear. In this work, we designed and expressed novel mutants with high-enzyme activity as well as feedback inhibition resistance on the basis of sites 379 and 380 by using site-directed mutagenesis and high-throughput screening techniques.…”

Discovery of an ʟ-amino acid ligase implicated in Staphylococcal sulfur amino acid metabolism

“…While most ʟ-Asp utilizing enzymes of S. aureus have not been characterized, a recent investigation of ʟ-Asp transcarbamoylase determined the ʟ-Asp KM as 4.5 mM (20). In multiple other bacterial species ʟ-Asp utilizing enzymes such as aspartokinase I -III have also been reported to possess similar KM values for ʟ-Asp, ranging between 1.9 and 21 mM (21)(22)(23)(24)(25). Investigations of Met limitation for S. aureus have also revealed that mutant strains with an inactivated de novo Met biosynthesis pathway require up to 100 µM ʟ-Met for a normal growth phenotype (5).…”