Crystallization and X-ray analysis ofD-threonine aldolase fromChlamydomonas reinhardtii

Abstract

D-Threonine aldolase from the green alga Chlamydomonas reinhardtii (CrDTA) catalyzes the interconversion of several β-hydroxy-D-amino acids (e.g. D-threonine) and glycine plus the corresponding aldehydes. Recombinant CrDTA was overexpressed in Escherichia coli and purified to homogeneity; it was subsequently crystallized using the hanging-drop vapour-diffusion method at 295 K. Data were collected and processed at 1.85 Å resolution. Analysis of the diffraction pattern showed that the crystal belonged to space group P1, with unit-cell parameters a = 64.79, b = 74.10, c = 89.94 Å, α = 77.07, β = 69.34, γ = 71.93°. The asymmetric unit contained four molecules of CrDTA. The Matthews coefficient was calculated to be 2.12 Å Da and the solvent content was 41.9%.

Structure of pyridoxal 5′-phosphate-bound D-threonine aldolase from Chlamydomonas reinhardtii

“…We have previously described the crystallization and preliminary X-ray analysis of CrDTA (Hirato, Goto et al, 2017). The crystal of CrDTA was obtained by the hanging-drop vapor-diffusion method.…”

“…However, the highest resolution data could only be processed in the triclinic space group P1. The highest resolution diffraction data were processed with XDS (Kabsch, 2010), POINTLESS (Evans, 2011) and SCALA (Evans, 2006) within the CCP4 package (Winn et al, 2011) as described previously (Hirato, Goto et al, 2017).The analyzed diffraction pattern demonstrated that the crystal belonged to space group P1, with unit-cell parameters a = 64.79, b = 74.10, c = 89.94 A ˚, = 77.07, = 69.34, = 71.93 . The crystal contained four protein molecules in the asymmetric unit.…”

“…The solvation-free energy released upon dimer formation by CrDTA was calculated to be À26 kcal mol À1 using PRODIGY (Vangone & Bonvin, 2015;Xue et al, 2016), which indicates that the CrDTA crystal structure is stable as a dimer. We had previously suggested that CrDTA exists in a monomer-dimer equilibrium because the molecular mass of CrDTA determined via gel filtration was 60 kDa, although that of the CrDTA monomer is approximately 45 kDa on SDS-PAGE (Hirato, Goto et al, 2017;. The crystal structure was shown to be a dimer.…”