Asparagine-84, a regulatory allosteric site residue, helps maintain the quaternary structure of Campylobacter jejuni dihydrodipicolinate synthase

• Published in: Journal of structural biology 2020-01, Vol.209 (1), p.107409, Article 107409
• Main Authors: Majdi Yazdi, Mohadeseh, Saran, Sagar, Mrozowich, Tyler, Lehnert, Cheyanne, Patel, Trushar R., Sanders, David A.R., Palmer, David R.J.
• Format: Article
• Language: English
• Subjects: Allosteric Regulation - genetics; Allostery; Asparagine - chemistry; Asparagine - genetics; Campylobacter jejuni - enzymology; Dihydrodipicolinate synthase; Hydro-Lyases - chemistry; Hydro-Lyases - genetics; Hydro-Lyases - ultrastructure; Protein Structure, Quaternary; Quaternary structure; SAXS; Site-directed mutagenesis; X-ray crystallography
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1016/j.jsb.2019.107409

[Display omitted] •We mutated Asn-84, found in the allosteric site of dihydrodipicolinate synthase (DHDPS).•N84A and N84D showed greatly reduced catalytic activity.•Loss of activity can be ascribed to disruption of the quaternary structure.•Mutant structures were studied by SAXS, MALS, and X-ray crystallography.•The quaternary structure of the mutants is concentration-dependent. Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.