The role of substrate-binding groups in the mechanism of aspartate-β-semialdehyde dehydrogenase

Abstract

The reversible dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde (ASA) in the aspartate biosynthetic pathway is catalyzed by aspartate-beta-semialdehyde dehydrogenase (ASADH). The product of this reaction is a key intermediate in the biosynthesis of diaminopimelic acid, an integral component of bacterial cell walls and a metabolic precursor of lysine and also a precursor in the biosynthesis of threonine, isoleucine and methionine. The structures of selected Haemophilus influenzae ASADH mutants were determined in order to evaluate the residues that are proposed to interact with the substrates ASA or phosphate. The substrate Km values are not altered by replacement of either an active-site arginine (Arg270) with a lysine or a putative phosphate-binding group (Lys246) with an arginine. However, the interaction of phosphate with the enzyme is adversely affected by replacement of Arg103 with lysine and is significantly altered when a neutral leucine is substituted at this position. A conservative Glu243 to aspartate mutant does not alter either ASA or phosphate binding, but instead results in an eightfold increase in the Km for the coenzyme NADP. Each of the mutations is shown to cause specific subtle active-site structural alterations and each of these changes results in decreases in catalytic efficiency ranging from significant (approximately 3% native activity) to substantial (<0.1% native activity).

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

“…This Arg103 is engaged in an electrostatic interaction with the phosphate ion in the reverse reaction. 19 Given the similarity in the reactions catalyzed by MtbAGPR and HiASADH, the alignment of their structures (Figure 7) suggests that the catalytic roles of Cys158 and Arg114 in MtbAGPR are analogous to those of Cys136 and Arg103 in HiASADH.…”

“…To study a conservation of residues located close to this area the sequence alignment was carried out among AGPRs with both known and unknown structure. This alignment resulted in nine residues, Asp108, Arg114, Tyr211, His217, His219, Glu222, Asp319, Asn320, and Leu321, as well as six glycine residues (numbers 16,19,157,192,249,324) from MtbAGPR to be fully conserved. There are also several other residues with a high level of conservation.…”

“…Its catalytic mechanism was analyzed using the structures of the protein in complexes with the cofactor NADP + , with the substrate ASA, and with different substrate analogs as well as the structures of the protein with mutated critical catalytic residues. 10,18,19 We shall use the structures of HiASADH complexed with NADP + and ASA 10,18 in discussing the MtbAGPR catalytic mechanism.…”

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.…”

“…Based on the selection criteria for these compounds and their competitive inhibition versus ASA, it is reasonable to assume that each can bind by engaging the two highly conserved active site arginines (Arg 99 and 245 for spASADH or Arg 101 and 267 for vcASADH) that have been shown to bind phosphate and the ASA carboxyl group, respectively. 16 The proposed alignment to bridge these active site binding groups is highlighted in bold for each of these compounds (Fig. 5).…”