Sequence Determinants of Substrate Ambiguity in a HAD Phosphosugar Phosphatase of Arabidopsis Thaliana

Abstract

The Arabidopsis thaliana broad-range sugar phosphate phosphatase AtSgpp (NP_565895.1, locus AT2G38740) and the specific DL-glycerol-3-phosphatase AtGpp (NP_568858.1, locus AT5G57440) are members of the wide family of magnesium-dependent acid phosphatases subfamily I with the C1-type cap domain haloacid dehalogenase-like hydrolase proteins (HAD). Although both AtSgpp and AtGpp have a superimporsable α/β Rossmann core active site, they differ with respect to the loop-5 of the cap domain, accounting for the differences in substrate specificity. Recent functional studies have demonstrated the essential but not sufficient role of the signature sequence within the motif-5 in substrate discrimination. To better understand the mechanism underlying the control of specificity, we explored additional sequence determinants underpinning the divergent evolutionary selection exerted on the substrate affinity of both enzymes. The most evident difference was found in the loop preceding the loop-5 of the cap domain, which is extended in three additional residues in AtSgpp. To determine if the shortening of this loop would constrain the substrate ambiguity of AtSgpp, we deleted these three aminoacids. The kinetic analyses of the resulting mutant protein AtSgpp3Δ (ΔF53, ΔN54, ΔN55) indicate that promiscuity is compromised. AtSgpp3Δ displays highest level of discrimination for D-ribose-5-phosphate compared to the rest of phosphate ester metabolites tested, which may suggest a proper orientation of D-ribose-5-phosphate in the AtSgpp3Δ active site.

A phosphatase gene is linked to nectar dihydroxyacetone accumulation in mānuka (Leptospermum scoparium)

“…S4). All the predicted mānuka proteins have a similar loop sequence to AtSGPP that is involved in the protein recognising multiple substrates (Caparrós‐Martín et al ., 2019) (Fig. 3b).…”

Genome-wide analysis of haloacid dehalogenase genes reveals their function in phosphate starvation responses in rice

“…The cap structure has a great influence on the substrate specificity of the enzyme. Mutation of the amino acids (△F53, △N54, △N55) in the cap structure will reduce the kinetics of AtHAD15 [ 51 ]. The substrate specificity of HAD proteins with C1 and C2 cap structures is lower than that of C0, which may be related to the specific enzyme residues of caps that can bind to different substrates [ 50 ].…”