Significance of Arg3, Arg54, and Tyr58 of l-aspartate α-decarboxylase from Corynebacterium glutamicum in the process of self-cleavage

Abstract

We have elucidated the significance of three key amino acid residues of L-aspartate α-decarboxylase that act remotely from its cleavage site for its functional self-cleavage as well as for its catalytic activity. These results provide useful fundamental information for engineering L-aspartate α-decarboxylase. L-Aspartate α-decarboxylase (ADC) from Corynebacterium glutamicum, and encoded by panD, was cloned and expressed in Escherichia coli and then purified. Three amino acid residues were found to be related to ADC self-cleavage. Mutating R3 to either A, Q, N, L, D, or E produced only the unprocessed pro-enzyme. Although mutating R54 and Y58 into A or K and A or T, respectively, partly influenced ADC self-cleavage, the specific activity of each of the four ßmutants decreased to 3.5, 4, 2.4, and 2.6 U mg(-1), respectively, compared with a specific activity of 690 U mg(-1) for the wild-type enzyme. Thus, R3 triggers ADC self-cleavage and completes the modification of the active site with assistance by R54 and Y58. These results will help to engineer ADC for improved industrial applications.

Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress

“…These modifications allow organisms to rapidly respond and adapt to changing environmental conditions, forgoing the need to transcribe and translate new proteins by simply modifying the function of existing proteins. Examples of PTMs include acetylation (2), glycosylation (3), lipidation (4), methylation (5), S-nitrosylation (6), phosphorylation (7,8), succinylation (9), ubiquitinylation (10), adenylylation and phosphocholinylation (11), ADP-ribosylation (12), serine/threonine O-acetylation (13), proteolysis (14)(15)(16), and others.…”

Discovery and Engineering of a Novel Bacterial L-Aspartate α-Decarboxylase for Efficient Bioconversion

“…The results revealed that the L-aspartate α-decarboxylase probe, encoded by the panD gene of C. glutamicum, was in a different evolutionary lineage from that of B. subtilis, despite both enzymes belonging to the pyruvoyl-dependent enzyme class. However, both enzymes shared the G24-S25 conserved motif, involved in cleaving [1,11], and T57-Y58, involved in catalysis [11,33]. Given that the ADCs from these two sources displayed different enzymatic activities, we speculated that the enzymatic properties were not closely related to the evolutionary relationship shown on the phylogenetic tree.…”

“…Within this region, 22 residues were identified, varying degrees of conservation (Figure 4c). Highly conserved residues, such as R54, T57, and Y58, were excluded due to their importance in catalysis [1,11,33]. Finally, eight residues (H11, C26, I49, L55, T56, N72, I88, Y90) were chosen using Rosetta_ddg for virtual mutagenesis analysis.…”

“…Previous studies have shown that R3 residue might be involved in the self-processing of the enzyme from C. glutamicum, although it is far from the residues around the active center [33]. However, the contribution of R3 to catalytic activity has remained unknown.…”

Enhancement of β-Alanine Biosynthesis in Escherichia coli Based on Multivariate Modular Metabolic Engineering

“…Various sources of L-aspartate-α-decarboxylase, such as E. coli [32], C. glutamicum [4,33], Bacillus subtilis [15], Helicobacter pylori [34], Mycobacterium tuberculosis [35], and Tribolium castaneum [34,35], have been reported, among which the L-aspartate-α-decarboxylase from T. castaneum (TcpanD) exhibited the highest activity [36,37]. As the rate-limiting step of β-alanine generation is the reaction from L-aspartate to β-alanine [17], to further increase β-alanine generation, the CgpanD used in B0016-06 was substituted by TcpanD, generating the B0016-07 strain, which harbored the plasmids pETpL-TcpanD-aspA and pCDFPL-ppc-gldA-dhaKLM.…”