Abstract
β-Phosphoglucomutase (βPGM) is a magnesium-dependent phosphoryl transfer enzyme that catalyses the reversible isomerisation of β-glucose 1-phosphate and glucose 6-phosphate, via two phosphoryl transfer steps and a β-glucose 1,6-bisphosphate intermediate. Substrate-free βPGM is an essential component of the catalytic cycle and an understanding of its dynamics would present significant insights into βPGM functionality, and enzyme catalysed phosphoryl transfer in general. Previously, 30 residues around the active site of substrate-free βPGMWT were identified as undergoing extensive millisecond dynamics and were unassignable. Here we report 1H, 15N and 13C backbone resonance assignments of the P146A variant (βPGMP146A) in its substrate-free form, where the K145–A146 peptide bond adopts a trans conformation in contrast to all crystal structures of βPGMWT, where the K145–P146 peptide bond is cis. In βPGMP146A millisecond dynamics are suppressed for all but 17 residues, allowing 92% of backbone resonances to be assigned. Secondary structure predictions using TALOS-N reflect βPGM crystal structures, and a chemical shift comparison between substrate-free βPGMP146A and βPGMWT confirms that the solution conformations are very similar, except for the D137–A147 loop. Hence, the isomerisation state of the 145–146 peptide bond has little effect on structure but the cis conformation triggers millisecond dynamics in the hinge (V12–T16), the nucleophile (D8) and residues that coordinate the transferring phosphate group (D8 and S114–S116), and the D137–A147 loop (V141–A142 and K145). These millisecond dynamics occur in addition to those for residues involved in coordinating the catalytic MgII ion and the L44–L53 loop responsible for substrate discrimination.
Highlights
Protein expression and purificationSite-directed mutagenesis (QuikChange II Site-Directed Mutagenesis Kit, Agilent Technologies) of the pgmB from Lactococcus lactis cloned in the pET-22b(+) expression plasmid was employed to generate βPGMP146A using primers with single-site base changes
Biological context β-Phosphoglucomutase from Lactococcus lactis is a magnesium-dependent phosphoryl transfer enzyme of the haloacid dehalogenase superfamily (Lahiri et al 2002a; Allen and Dunaway-Mariano 2004; Dai et al 2009)
Structural analysis coupled with metalfluoride ground state and transition state analogue (TSA) complexes have allowed the atomic resolution description of several discrete species found in the catalytic cycle i.e. substrate-free βPGMWT (PDB: 2WHE; Baxter et al 2010), a ground state βPGMWP T analogue, two ground state βPGMPWT:glucose 6-phosphate (G6P) complexes, two βPGMD10N:βG16BP complexes (PDB: 5OK1; PDB: 5OK0; Johnson et al 2018), a βPGMPWT:G6P TSA complex and a βPGMPWT:β-glucose 1-phosphate (βG1P) TSA complex
Summary
Site-directed mutagenesis (QuikChange II Site-Directed Mutagenesis Kit, Agilent Technologies) of the pgmB from Lactococcus lactis cloned in the pET-22b(+) expression plasmid was employed to generate βPGMP146A using primers with single-site base changes. Proteins bound to the DEAE-Sepharose column were eluted with a gradient of 0 to 50% standard working buffer containing 1 M NaCl. Fractions were checked for the presence of βPGMP146A by SDS-PAGE, pooled together and concentrated by Vivaspin (10 kDa MWCO, Sartorius). The protein sample was loaded onto a prepacked Hiload 26/60 Superdex 75 size-exclusion column connected to an ÄKTA purification system previously washed with 1 column volume of 1 M NaOH and equilibrated with 1.5 column volumes of standard working buffer containing 1 M NaCl. Fractions containing βPGMP146A were checked for purity by SDS-PAGE, pooled together and buffer exchanged into standard working buffer and concentrated to ~ 1.6 mM by Vivaspin (10 kDa MWCO) for storage as 1 mL aliquots at − 20 °C. All reagents were of analytical grade and were purchased from Sigma-Aldrich (UK), except for the stable isotopicallylabelled compounds 15NH4Cl (99%), 13C, 2H7-d-Glucose (U–13C6, 99%; 1,2,3,4,5,6,6-d7 97–98%) and 2H2O (99.8%), which were purchased from CortecNet (France) and used as received
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