**3.2 KARI sequence alignment**

The alignment of KARI enzyme was carried out using BLAST and Clustal W. Multiple-sequence alignment of *Ng* KARI and KARIs from human pathogens,

Neisseria gonorrhoeae *Ketol-Acid Reductoisomerase Is a Potential Therapeutic Target DOI: http://dx.doi.org/10.5772/intechopen.107993*

## **Figure 1.**

*Phylogenetic tree based on KARI sequence between N. gonorrhoeae and ESKAPE pathogens (a) and other human pathogens (b). Accession numbers are as follows: (a) A. baumannii (EHU1490884), P. aeruginosa CIG1 (EJY 59157), E. faecium (KXH23108), S. aureus (MBU4945389), E. coli K12 (AKD89606), and K. pneumoniae (MBC4258974). (b) S. dysenteriae (WP 134806311.1), S. flexneri (SRM99818.1), S. sonnei (SRM99818.1), Y. pseudotuberculosis (WP 207968213), Y. enterocolitica (CQH59307.1), F. tularensis (MWY11435.1), S. pneumoniae (CJL31550.1), N. meningitidis ATCC 13091 (EFM04987.1), S. enterica (EBV8528786.1), M. tuberculosis (9WKJ7.2), B. cereus (CUB23876.1), and B. anthracis (PFE27432.1).*

revealed that residues constituting NADP(H) and Mg2+ binding sites are well conserved, while the overall length of each KARI is different. The tested bacteria share different residues, especially in the active pocket, and cofactors binding sites. Indeed, different KARIs have almost identical active site structures.

### **Figure 2.**

*Phylogenetic tree based on KARI sequence between N. gonorrhoeae and some members of the gut microbiota. Accession numbers are as follows: (a) F. nucleatum (PZA04987.1), B. bifidum (ERI84126), Lactobacillus (RRG12499), A. muciniphila (QWP74035), P. dentalis (AGB28157), and B. fragilis (KXU42727). The tree was constructed using neighbor-joining analysis based on KARI protein sequences. The scale bar represents 0.5 substitutions per nucleotide position.*

Analysis of residues contacting NADP(H) and Mg2+ identified five amino acid residues, in *Sa* KARI, as contacting ones: Arg-47, Asp-81, Ser-51, Asp-189, and Glu-193 (**Figure 3b**). KARIs harbor a GxGxxG motif, which is part of the nucleotidebinding site by phosphate-bridging interaction (**Figure 3c**), and Mg2+ is required for NADP(H) binding. The study of residue mutations' effect on NADPH binding to the KARI's structure show that residues A71, R76, and S78 are in the loop connecting the β2 sheet and the αB helix, referred to as the β2αB loop. R76, and S78 establish direct contact with the 2′-phosphate of NADPH. Sequence alignment of KARI show a variable length of the β2αB loop among tested bacteria (**Figure 4**). This loop is crucial for the cofactor specificity [24].

Upon the binding of cofactors, NADP(H) and Mg2+, the N-terminal domain of KARI undergoes large local conformational changes, only in the NADP(H) binding site. Four Mg2+-binding residues are also identified (D190, E194, E226, and E230) [19]. The side chains of these residues rotate upon metal ion binding. Previously, the mutation of R47 and D81 induce rotameric changes in other bulky residues (His-31, Lys-52, Phe-54, and His-135), resulting in the NADP(H) binding pocket broadening, and then a weak binding to the structure [25].

Other conserved residues are identified. According to available KARI's structure analysis, these amino acids interact with inhibitors. Notably, KARI binds different ligands other than metal ions and NADP(H), such as IpOHA, cyclopropane-1, 1 dicarboxylic acid (CPD) and 2-(dimethyl phosphoryl)-2-hydroxyacetic acid

Neisseria gonorrhoeae *Ketol-Acid Reductoisomerase Is a Potential Therapeutic Target DOI: http://dx.doi.org/10.5772/intechopen.107993*

### **Figure 3.**

*Surface representation of the crystal structure of KARI from Staphylococcus aureus (Sa KARI) (PDBID: 5w3k) (Sa KARI), the NADP(H) binding site is shown as a red cavity. (b) Stereo view of the binding mode of NADP(H). NADP(H) is shown as yellow sticks, and metals are shown as green spheres. Polar contacts with residues within 5 of the NADPH are shown in magenta in dashed lines (c) surface representation of the NADP(H) binding site pocket of Sa KARI. NADP(H) is shown as yellow sticks.*


## **Figure 4.**

*Multiple alignments of KARI partial sequence from members of the ESKAPE pathogens groups. Clustal W was used to align KARI sequences from six members of the ESKAPE (Escherichia coli strain K12, Staphylococcus aureus strain MOS225, Klebsiella pneumoniae strain K783, Acinobacter baumannii strain MRSN7301, Pseudomonas aeruginosa strain CIG1, and Entercoccus faecium strain VRE-1503646) against the orthologue from N. gonorrhoeae. The alignments were used to identify regions possessing the greatest similarities. The conservation of residues is indicated above the alignments as follows: asterisk, complete identity; colon, conservation of a strong group; period, conservation of a weak group. GxGxxG motif is shown in red line, while β2αB loop is shown in blue line.*


## **Table 1.**

*Summarization of ligands residues for KARI's inhibitors.*

(Hoe704). These compounds are the most extensively characterized KARI inhibitors investigated to date. They are transition state analogs. Residues involved in these inhibitors binding are conserved among bacteria, as described in **Table 1**. These inhibitors could be tested with KARI from *N. gonorrhoeae*, to assess its effect on bacterial growth rate, its viability, and antimicrobial resistance.

In the presence of Mg2+ ions, the active site of KARI becomes open and accessible to solvent, while the NADP(H) binding reduces the space between the domains, and the active site adopt a closed conformation. Hence, the active site changes its surface structure to become appropriate for substrate binding. The open-close transition state has been thought to facilitate substrate binding and catalysis. This feature provides then possibilities for the development of inhibitors able to bind to both of structural conformations of KARI (i.e. ± NADPH) [6].
