Structural basis for the catalytic mechanism of homoserine dehydrogenase

Abstract

Homoserine dehydrogenase (HSD) is an oxidoreductase in the aspartic acid pathway. This enzyme coordinates a critical branch point of the metabolic pathway that leads to the synthesis of bacterial cell-wall components such as L-lysine and m-DAP in addition to other amino acids such as L-threonine, L-methionine and L-isoleucine. Here, a structural rationale for the hydride-transfer step in the reaction mechanism of HSD is reported. The structure of Staphylococcus aureus HSD was determined at different pH conditions to understand the basis for the enhanced enzymatic activity at basic pH. An analysis of the crystal structure revealed that Lys105, which is located at the interface of the catalytic and cofactor-binding sites, could mediate the hydride-transfer step of the reaction mechanism. The role of Lys105 was subsequently confirmed by mutational analysis. Put together, these studies reveal the role of conserved water molecules and a lysine residue in hydride transfer between the substrate and the cofactor.

Biochemical Characterization of Homoserine Dehydrogenase from Pseudomonas aeruginosa

“…In addition, we also carried out size‐exclusion chromatography, circular dichroism spectroscopy, and thermal denaturation test. In particular, the size‐exclusion chromatography demonstrated that PaHSD exists as a tetramer in solution, which is in contrast to the previously reported HSDs from other species that commonly form dimers …”

“…The content of the secondary structure of PaHSD was calculated by BeStSel, and it showed 16.5% for helices, 22.5% for strands, 14.8% for turns, and 46.1% for others. The crystal structures of HSDs reported so far from other organisms have shown that they take on a common α/β structure containing the α/β Rossman folding domain, which is in good agreement with the above CD spectra analysis of PaHSD . In addition, this analysis suggests that the PaHSD purified in this study maintains its structural integrity in a stable manner.…”

“…The peak of PaHSD appeared at the elution volume of 11.8 mL, and its molecular mass was estimated from the standard curve to be 235 kDa, which is 4.9 times larger than the 47.1 kDa of a single chain PaHSD. It was previously shown that HSD from Staphylococcus aureus forms a dimer in solution, as well as in a crystal lattice . In addition, HSDs from Saccharomyces cerevisiae , Thermus thermophiles , Pyrococcus horikoshii , and Sulfolobus tokodaii also show the same dimeric assembly in the crystal lattice .…”

“…4 Structural studies on HSDs from other microorganisms have revealed that HSD consists of three domains: the nucleotide-binding domain with a characteristic α/β Rossmann fold, the substrate-binding domain, and the dimerization domain. [4][5][6][7][8] Even though structural and enzymatic characterization studies have been reported for various microorganisms, studies on HSD from Pseudomonas aeruginosa, which is a ubiquitous environmental bacterium causing one of the top three opportunistic human infections, have not yet been reported. 9 The HSD from P. aeruginosa (PaHSD) consists of 434 amino acid residues, and its molecular mass is 46 224 Da.…”

“…In particular, the sizeexclusion chromatography demonstrated that PaHSD exists as a tetramer in solution, which is in contrast to the previously reported HSDs from other species that commonly form dimers. [4][5][6][7][8]…”