Critical catalytic functional groups in the mechanism of aspartate-β-semialdehyde dehydrogenase

Abstract

Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes the reductive dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde in the aspartate biosynthetic pathway. This pathway is not found in humans or other eukaryotic organisms, yet is required for the production of threonine, isoleucine, methionine and lysine in most microorganisms. The mechanism of this enzyme has been examined through the structures of two active-site mutants of ASADH from Haemophilus influenzae. Replacement of the enzyme active-site cysteine with serine (C136S) leads to a dramatic loss of catalytic activity caused by the expected decrease in nucleophilicity, but also by a change in the orientation of the serine hydroxyl group relative to the cysteine thiolate. In contrast, in the H277N active-site mutant the introduced amide is oriented in virtually the same position as that of the histidine imidazole ring. However, a shift in the position of the bound reaction intermediate to accommodate this shorter asparagine side chain, coupled with the inability of this introduced amide to serve as a proton acceptor, results in a 100-fold decrease in the catalytic efficiency of H277N relative to the native enzyme. These mutant enzymes have the same overall fold and high structural identity to native ASADH. However, small perturbations in the positioning of essential catalytic groups or reactive intermediates have dramatic effects on catalytic efficiency.

Crystal Structure of N-acetyl-γ-glutamyl-phosphate Reductase from Mycobacterium tuberculosis in Complex with NADP+

“…The crystal structures of the ASADHs indicate that their catalytic residues are located on the surface of this cleft. 18 One might expect the same for MtbAGPR given its cofactor position. The enzyme HiASADH has been studied in most detail.…”

Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene inP. putidaCA-3

“…Previous characterization of TodS domains 5 reveal certain overlapping similarities with the workings of StyS, e.g., the critical nature of the C-terminal HK2. The localization of kinase activity to the carboxy terminus has been well documented among other sensor kinase proteins also, including EnvZ, 12 VirA, 16 and FixL.…”

“…These findings are in keeping with previous work on the autophosphorylation capacities of truncated forms of the TodS sensor kinase. 5 In contrast, constructs harboring a deletion of input 2 (SOE4 Δ568-730), or histidine kinase 1 (SOE1 Δ192-399), respectively, achieved reporter system activities comparable with the StySR wild type construct, suggesting the non-essential nature of these domains in response to extracellular styrene presence (Fig. 1B).…”

“…8 In tandem with the contribution of bioinformatic inputs, functional characterisations of TCSs also have an important role to play, particularly with TCS systems involving multiple or duplicate domains, such as the TodS system, which has been well characterized in recent years. 5,36 Amino acid sequence alignment of StyS from P. putida CA-3 with the TodS sensor kinase revealed Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains.…”

1 Molecular Docking and Enzymatic Evaluation to Identify Selective Inhibitors of Aspartate Semialdehyde Dehydrogenase

“…11 The catalytic mechanism of ASADH is supported by kinetic studies, 12,13 mutagenesis studies [14][15][16] and structural characterization of several key catalytic intermediates. 8,9 Our molecular level understanding of the structural and mechanism of ASADH is being used to guide the identification of selective enzyme inhibitors.…”