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Crystal structure of the Streptococcus pneumoniae phosphomevalonate kinase, a member of the GHMP kinase superfamily

Introduction. S. pneumoniae phosphomevalonate kinase (PMK, PMVK, MvaK2; EC 2.7.4.2; 335 residues; predicted molecular mass 37026.5 Da) is a cytoplasmic enzyme in the mevalonate-dependent isoprenoid biosynthesis pathway. The enzyme catalyzes transfer of the -phosphoryl group of ATP to the phosphate oxygen of (R)-5-phosphomevalonate to form (R)-5-pyrophosphomevalonate. PMK is a member of the GHMP kinase superfamily [named for galactokinase (GK), homoserine kinase (HSK), mevalonate kinase (MK), and PMK], which includes at least 19 30%-identical clusters of proteins with various enzymatic activities. Previously determined structures of two GHMP kinases, Methanococcus jannaschii HSK (PDB ID 1FWK) and Saccharomyces cerevisiae mevalonate-5-diphosphate decarboxylase (MDD; PDB ID 1FI4), produced low-accuracy homology models for a number of PMKs. Phylogenetic analyses of nearly 200 superfamily members suggested that an experimental structure of at least one PMK would be required to produce more accurate models for members of this GHMP kinase subfamily. We cloned and expressed two PMKs: one from Staphylococcus aureus and one from S. pneumoniae. S. aureus PMK proved to be largely insoluble under standard purification conditions, whereas the S. pneumoniae homolog emerged as the better structure determination target. PMK, like HSK and MDD, is an / protein (dimensions 59 Å 53 Å 48 Å) composed of two domains. The linear arrangement of the secondary structural elements is 1, 310, 2, 3, A, 4, B, C, D, E, 5, F, G, 6, 7, H, I, J, K, 8, 9, L, and 10 (Fig. 1). The N-terminal domain contains a six-stranded -sheet of mixed polarity (antiparallel 65214 followed by 3 parallel to 4) arching over an -helical region ( C, E, A, D, G, and F) (Fig. 2). 1 (11 residues) and 2 (17 residues containing a -corner) further subdivide the six-stranded -sheet into two four-stranded -sheets: one antiparallel ( 653N-terminal portion of 23C-terminal portion of 1) and the other of mixed polarity (antiparallel N-terminal portion of 13C-terminal portion of 23 4 followed by 3 parallel to 4). The C-terminal domain is composed of an antiparallel four-stranded -sheet ( 89710) flanked by J, K, and L. Visual inspection of the surface of PMK revealed a deep cleft between two -sheets ( 6521 and 89710). This feature almost certainly represents the enzyme active site because it contains three conserved GHMP kinase motifs (I, II, and III). Previously, the New York Structural Genomics Research Consortium (NYSGRC) reported homology modeling of various PMKs. With the X-ray structure of S. pneumoniae PMK, we can evaluate the accuracy of these modeling attempts (PMK versus an HSK-derived model gave a root-mean-square deviation (RMSD) 2.8 Å; PMK versus an MDD-derived model gave an RMSD 3.6 Å). It is not surprising that the PMK models proved to be of relatively low accuracy because they were both calculated with distantly related templates (sequence identity between PMK and HSK is 16%, between PMK and MDD 8%). In both cases, modeling was compromised by errors in the alignment of the modeled sequence with the template and by the presence of insertions and deletions. Automated homology modeling with MODPIPE using PMK as a template yielded 250 protein models (model score 0.7, model length 170 residues) reflecting conservation of the GHMP kinase fold. Highor mediumaccuracy models in the 50–100% and 30–50% sequence identity ranges were obtained for prokaryotic PMKs. Lower-accuracy models in the 20–30% sequence identity range represent other PMKs and some proteins annotated as MKs. Models with 20% sequence identity include fungal PMKs, various MKs, HSKs, GKs, and MDDs, plus proteins of unknown function. The X-ray structure of PMK provided a useful test of the GHMP kinase homology modeling. In addition, modeling