Recombinant Desulfovibrio vulgaris Shikimate kinase (aroK)

What structural features characterize bacterial shikimate kinases?

Bacterial shikimate kinases display a characteristic three-layer architecture consisting of a central β-sheet core flanked by α-helices . Crystal structures from various bacterial species reveal a conformationally elastic region (often called the LID region) that undergoes significant movement during substrate binding and catalysis. This region contains several conserved residues (corresponding to F48, R57, R116, and R132 in H. pylori SK) that interact directly with shikimate . The active site architecture includes distinct binding pockets for shikimate and ATP, with several conserved residues mediating substrate recognition and catalysis.

How do different bacterial shikimate kinases compare biochemically?

Biochemical properties of shikimate kinases vary significantly across bacterial species, as shown in the table below:

Some bacteria, like E. coli, possess two shikimate kinase isoenzymes with distinct kinetic properties and regulatory mechanisms, while others have only a single form .

What expression systems are most effective for recombinant shikimate kinase production?

For recombinant production of bacterial shikimate kinases, the T7 promoter system using pET vectors in E. coli has proven highly effective. This approach has successfully yielded large quantities of soluble enzyme for structural and biochemical studies . Based on documented success with S. aureus chorismate synthase (another shikimate pathway enzyme), expression from the T7 promoter can produce up to 100 mg of purified enzyme from 13 g of cells . For D. vulgaris shikimate kinase, similar expression strategies would likely be effective, with optimization of induction temperature, IPTG concentration, and host strain selection being critical factors.

What techniques are optimal for structural characterization of recombinant shikimate kinase?

X-ray crystallography has been the primary technique for determining the three-dimensional structures of shikimate kinases from various bacterial species. Crystal structures of shikimate kinases have been determined from E. coli, Erwinia chrysanthemi, Campylobacter jejuni, Aquifex aeolicus, Arabidopsis thaliana, M. tuberculosis, and H. pylori . For recombinant D. vulgaris shikimate kinase, a crystallization screening approach should include:

• Purification to >95% homogeneity using affinity chromatography followed by size-exclusion chromatography

• Screening in the presence of shikimate and ATP/ADP analogs to stabilize the enzyme conformation

• Crystal optimization with additives that promote crystal formation for kinases

• Data collection at synchrotron radiation sources for high-resolution structure determination

Additionally, small-angle X-ray scattering (SAXS) can provide valuable information about enzyme dynamics in solution, complementing the static crystal structures.

How should site-directed mutagenesis experiments be designed to probe catalytic mechanisms?

Based on studies with H. pylori shikimate kinase, several conserved residues are critical for substrate binding and catalysis . A comprehensive site-directed mutagenesis approach for D. vulgaris shikimate kinase should target:

• Residues corresponding to D33, F48, R57, R116, and R132 in H. pylori SK, which directly interact with shikimate

• Conserved residues in the ATP-binding P-loop motif

• Residues in the LID region that undergo conformational changes during catalysis

• Residues at the dimer interface if oligomerization is observed

Each mutant should be characterized through detailed kinetic analysis, thermal stability measurements, and where possible, structural determination to establish the precise role of each residue in enzyme function.

What kinetic assays provide the most reliable activity measurements for shikimate kinase?

Several complementary approaches can be used to accurately measure shikimate kinase activity:

• Coupled enzymatic assay: This approach links shikimate kinase activity to NADH oxidation via pyruvate kinase and lactate dehydrogenase, allowing continuous spectrophotometric monitoring at 340 nm

• Direct measurement of ADP formation using HPLC or mass spectrometry

• Malachite green assay for inorganic phosphate release in coupled reactions

• Isothermal titration calorimetry (ITC) for comprehensive thermodynamic characterization

For initial screening of enzyme variants or inhibitors, the coupled spectrophotometric assay offers the best combination of sensitivity and throughput, while more specialized techniques provide deeper mechanistic insights .

How does the shikimate binding pocket architecture influence substrate specificity?

The shikimate binding pocket in bacterial shikimate kinases contains a constellation of charged and polar residues that form specific interactions with the hydroxyl groups and carboxylate of shikimate. In H. pylori SK, residues D33, F48, R57, R116, and R132 create a network of hydrogen bonds and electrostatic interactions with the substrate . These interactions determine the high specificity of the enzyme for shikimate over similar metabolites.

For D. vulgaris shikimate kinase, comparative sequence analysis and homology modeling based on available bacterial SK structures would reveal conservation patterns in these critical residues. Substrate specificity studies with shikimate analogs and modified substrates could further delineate the structural constraints of the binding pocket, informing both mechanistic understanding and inhibitor design strategies.

What conformational changes occur during the catalytic cycle of shikimate kinase?

Crystal structures of shikimate kinases in different ligand-bound states reveal substantial conformational changes during catalysis . The most dramatic movements occur in the LID region, which closes over the active site upon substrate binding. This conformational change brings catalytic residues into optimal positions for facilitating phosphoryl transfer from ATP to shikimate.

For example, the crystal structure of H. pylori SK complexed with an inhibitor (162535) revealed a dramatic shift in the LID region, resulting in conformational locking into a distinctive form . This structural plasticity presents opportunities for designing inhibitors that target specific conformational states of the enzyme.

How does the ATP binding site architecture compare across bacterial shikimate kinases?

The ATP binding site in shikimate kinases contains the conserved P-loop motif characteristic of many kinases. While the core structure of this site is highly conserved, subtle differences in surrounding residues can influence nucleotide specificity and catalytic efficiency. Structural comparisons between shikimate kinases from different bacterial species reveal variations in residues that interact with the adenine and ribose moieties of ATP, potentially providing avenues for selective inhibitor design.

For D. vulgaris shikimate kinase, analysis of sequence conservation in the ATP binding region compared to pathogenic bacterial SKs would highlight unique features that might be exploited for developing species-selective inhibitors.

What strategies can be employed for selective inhibition of bacterial shikimate kinases?

The absence of the shikimate pathway in mammals makes shikimate kinase an attractive target for antimicrobial development without toxicity to human cells . Several approaches for designing selective inhibitors include:

• Shikimate-competitive inhibitors that mimic the transition state of the reaction

• ATP-competitive compounds with specificity for the bacterial enzyme

• Allosteric inhibitors that stabilize inactive conformations of the enzyme

• Covalent inhibitors targeting unique cysteine residues near the active site

The structure of an inhibitor complex, E114A- 162535, revealed a dramatic shift in the elastic LID region of H. pylori SK, resulting in conformational locking into a distinctive form . This suggests that inhibitors inducing specific conformational changes could be particularly effective.

How can recombinant D. vulgaris shikimate kinase serve as a model system for drug discovery?

While D. vulgaris is not a primary pathogen, its shikimate kinase can serve as a valuable model system for understanding the biochemical diversity of this enzyme family. Comparative studies between D. vulgaris SK and enzymes from pathogenic bacteria (such as M. tuberculosis, A. baumannii, and H. pylori) could:

• Identify conserved features essential for catalysis across all species

• Highlight structural differences that could be exploited for selective inhibition

• Provide insights into evolutionary adaptations of the enzyme in different bacterial metabolic contexts

• Serve as a non-pathogenic model for high-throughput screening of inhibitor libraries

The crystal structure of A. baumannii SK in complex with shikimate reveals the binding mode of the natural substrate and provides a template for structure-based inhibitor design .

What evolutionary relationships exist among bacterial shikimate kinases?

The distribution of shikimate kinase isoforms varies across bacterial species. In E. coli, two isoenzymes exist: shikimate kinase II (encoded by aroL) plays the dominant role in the pathway and is regulated by the TyrR and TrpR proteins with tyrosine and tryptophan as cofactors. Shikimate kinase I (encoded by aroK) is constitutively expressed and has a much lower affinity for shikimate .

Phylogenetic analysis of shikimate kinase sequences across diverse bacterial species would reveal evolutionary relationships and potential horizontal gene transfer events. For D. vulgaris shikimate kinase, determining its evolutionary position relative to the aroK/aroL divergence in other bacteria would provide context for interpreting its biochemical properties.

How does gene organization around aroK differ across bacterial species?

Gene organization in the shikimate pathway shows interesting variations across bacterial species. In S. aureus, the aroC gene (encoding chorismate synthase) likely forms the first gene in an operon that includes aroB (encoding dehydroquinate synthase) and aroA genes . The nucleoside diphosphate kinase gene (ndk) and gerCAgerCBgerCC genes are situated upstream from aroCaroB genes, a pattern also observed in B. subtilis .

Analyzing the genomic context of the aroK gene in D. vulgaris and comparing it with other bacteria would provide insights into potential co-regulation patterns and functional relationships with other metabolic pathways. This information could inform experimental approaches for investigating regulatory mechanisms and potential polar effects of gene disruption.