GK

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Introduction

In most bacteria, glycerol uptake is catalyzed in an energy-independent manner by a membrane channel protein, the glycerol facilitator (GlpF). The motive force of glycerol diffusion stems from the imbalance of intra- and extra-cellular glycerol concentration caused by glycerol metabolism. Typically, glycerol kinase (GlpK) converts triols to glycerol-3-P (G3P), and because G3P is not recognized as substrate by GlpF, GlpK plays an integral role in intracellular capture of glycerol. In Firmicutes (Gram-positive) and enterobacteriaceae (Gram-negative), GlpK synthesis is controlled by carbon catabolite inhibition and enzymatic activity is regulated by the sugar phosphotransferase system (PTS). PTS is a carbohydrate transport and phosphorylation system in which four of its five proteins (or domains) use phosphoenolpyruvate (PEP) as a phosphate donor to form a phosphorylation cascade. In in vivo studies, the presence of PTS sugars resulted in poor phosphorylation of PTS proteins and also significantly reduced the extent of GlpK phosphorylation and activation in glycerol.

Surprisingly, although these enzymes typically exhibit 50% to 60% sequence identity, the mechanisms controlling GlpK activity are quite different in firmicutes and enterobacteriaceae. In enterobacteriaceae such as E. coli, unphosphorylated EIIA^Glc interacts with GlpK and inhibits its activity, thereby preventing the biosynthesis of the inducer G3P. The interaction of EIIA^Glc with GlpK occurs in the C-terminal domain approximately 30 Å from the glycerol binding site and is stimulated by the presence of Zn²⁺ ions. In firmicutes GlpK, the activation loop is also located away from the active site, as shown in the crystal structure. Several spontaneous mutations in Bacillus subtilis lead to glycerol utilization in the absence of functional PTS. GlpK of B. subtilis is phosphorylated at His-230. These findings suggest that the aforementioned mutations and GlpK phosphorylation cause similar structural changes leading to activation of the enzyme. Mutant GlpK purified from E. casseliflavus and B. subtilis exhibited 7- to 19-fold higher activity than the unphosphorylated wild-type (WT) enzyme.

Figure 1. Overview of the Enterococcus casseliflavus GlpK O-X Dimer (Yeh, J.I.; et al. 2009)

Catalytic Site

The binding of glycerol to GlpK is an ordered reaction, with glycerol binding first, followed by ATP. Although most studies were performed on the E. coli GlpK enzyme, sequence conservation of catalytic cleft residues was found across organisms, whereas greater sequence diversity was found at regulatory regions of the GlpK. Therefore, the active site configuration and catalytic mechanism of various GlpK enzymes may be similar.

Glycerol is bound deep within the catalytic cleft through hydrogen bonds to multiple residues. The strong electron density evident in Fo-Fc maps of the glycerol-bound catalytic cleft supports the presence of a single glycerol molecule per subunit. The His232Arg, His232Glu, and His232Ala active sites share strong similarities around the glycerol binding site, and the hydrogen bonding distances between glycerol and amino acids in the active sites differ by less than 0.2 Å. At the active site, with the assistance of Glu85, Arg84, Asp 246 and Tyr136 are responsible for anchoring and aligning the bound glycerol. Residues Trp104 and Phe271 provide hydrophobic surfaces to interact with the glycerol carbons, further aligning the glycerol. The similarity between the orientations of active site residues in all GlpK structures suggests that differences in activity are not due to direct changes in glycerol binding at the catalytic cleft.

The binding of GlpK to the substrate results in the closure of the catalytic cleft, thus bringing domains I and II closer together. Glycerol binds deep within the catalytic cleft, while the putative ATP binding site is located at the entrance to the catalytic cleft. While glycerol is readily identified at the active site, additional significant positive residual electron densities were found in the His232Arg, His232Arg-B, and His232Glu differential density maps indicating that the tetragonal molecule is close to glycerol, at the position bridging the glycerol and ATP binding sites. Based on the crystallization conditions of each protein, a phosphate ion was modeled as His232Arg density, while sulfate ions were placed in His232Glu and His232Arg-B. After refinement, the phosphate group is 4.4 Å away from the glycerol O3 atom, while the sulfate is 4.8 Å (His232Glu) and 3.5 Å (His232Arg-B).

Figure 2. Glycerol Binding Site in His232Arg and His232Glu GlpK Mutants (Yeh, J.I.; et al. 2009)