Functional Analysis, Overexpression, and Kinetic Characterization of Pyruvate Kinase from Methicillin-ResistantStaphylococcus aureus

Abstract

Novel antimicrobial targets are urgently needed to overcome rising antibiotic resistance of important human pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Here we report the essentiality and kinetic properties of MRSA pyruvate kinase (PK). Targetron-mediated gene disruption demonstrated PK is essential for S. aureus growth and survival, suggesting that this protein may be a potential drug target. The presence of the pfk (6-phosphofructokinase)-pyk operon in MRSA252, and the nonessential nature of PFK shown by targetron, further emphasized the essential role of PK in cell viability. The importance of PK in bacterial growth was confirmed by showing that its enzymatic activity peaked during the logarithmic phase of S. aureus growth. PK from Staphylococcus and several other species of bacteria have an extra C-terminal domain (CT) containing a phosphoenolpyruvate (PEP) binding motif. To elucidate the possible structure and function of this sequence, the quaternary structures and kinetic properties of the full-length MRSA PK and truncated MRSA PK lacking the CT domain were characterized. Our results showed that (1) MRSA PK is an allosteric enzyme with homotetramer architecture activated by AMP or ribose 5-phosphate (R5P), but not by fructose 1,6-bisphosphate (FBP), which suggests a different mode of allosteric regulation when compared with human isozymes, (2) the CT domain is not required for the tetramerization of the enzyme; homotetramerization occurred in a truncated PK lacking the domain, (3) truncated enzyme exhibited high affinity toward both PEP and ADP and exhibited hyperbolic kinetics toward PEP in the presence of activators (AMP and R5P) consistent with kinetic properties of full-length enzyme, indicating that the CT domain is not required for substrate binding or allosteric regulation observed in the holoenzyme, (4) the kinetic efficiency (k(cat)/S(0.5)) of truncated enzyme was decreased by 24- and 16-fold, in ligand-free state, toward PEP and ADP, respectively, but was restored by 3-fold in AMP-bound state, suggesting that the sequence containing the CT domain (Gly(473)-Leu(585)) plays a substantial role in enzyme activity and comformational stability, and (5) full-length MRSA PK activity was stimulated at low concentrations of ATP (e.g., 1 mM) and inhibited by inorganic phosphate and high concentrations of FBP (10 mM) and ATP (e.g., >2.5 mM), whereas for truncated enzyme, stimulation at low concentrations of ATP was lost. These findings suggest that the CT domain is involved in maintaining the specificity of allosteric regulation of MRSA PK by AMP, R5P, and ATP. The CT extension also encodes a protein domain with homology to enzyme I of the Escherichia coli sugar-PTS system, suggesting that MRSA PK may also exert an important regulatory role in sugar transport metabolism. These findings yield new insights into MRSA PK function and mode of allosteric regulation which may aid in the development of clinically important drugs targeting this enzyme and further define the role of the ext...

Identification of Pyruvate Kinase in Methicillin-Resistant Staphylococcus aureus as a Novel Antimicrobial Drug Target

“…As a result of this analysis, pyruvate kinase (PK), the product of a single-copy gene, was identified as a highly connected hub protein in MRSA. Furthermore, we also found that PK is absolutely essential for S. aureus viability based upon PK antisense and gene disruption experiments (44). The essential requirement for PK for bacterial growth was also reflected by its high enzymatic activity during the exponential phase of the S. aureus life cycle.…”

“…The essential requirement for PK for bacterial growth was also reflected by its high enzymatic activity during the exponential phase of the S. aureus life cycle. Taken together, these findings provide a clear rationale for selecting PK as a novel, candidate drug target (44).…”

“…1 and 2). MRSA PK is allosterically regulated by AMP and ribose 5-phosphate (R5P), which induces the conversion of the active R state of the enzyme from the inactive T state (44). In contrast, mammals possess four isoenzymes of PK, one (M1) of which is constitutively active and the other three (M2, L, and R) of which are allosterically regulated by fructose 1,6-bisphosphate (FBP) (23).…”

Methicillin-resistant Staphylococcus aureus (MRSA) Pyruvate Kinase as a Target for Bis-indole Alkaloids with Antibacterial Activities

“…Thus, identification of new targets for antibacterial development based upon novel scaffolds with unique mechanisms of action are critically needed (3). To this end, we recently identified pyruvate kinase (PK), an evolutionarily conserved, highly connected essential hub protein in MRSA, with structural features distinct from the mammalian orthologs, as a novel candidate drug target (1,4,5). Using structural modeling, virtual data base screening, and enzyme activity assays in combination with medicinal chemistry, we recently identified low micromolar selective inhibitors of MRSA PK exhibiting strong antibacterial activity against Staphylococci and a range of other Gram-positive bacteria such as Enterococcal and Streptococcal species with low micromolar minimal inhibitory concentration (MIC) values (5).…”

“…Sequence alignment between MRSA and human PKs revealed particular sequence divergence in domain C (e.g. sites for effector binding), which is involved in formation of the small interfaces between the C domains of adjacent PK subunits providing tetramer rigidity and efficient catalytic activity of the enzyme (2,4).…”