KatG

Catalase peroxidase KatG (PDB ID: 5L05) is a protein found in Burkholderia pseudomallei, a species of bacteria (1). Commonly found in bacteria, catalase peroxidases like KatG function as protection against oxidative stress (2). The protein breaks down hydrogen peroxide (H2O2) via two mechanisms: catalase, which breaks H₂O₂ down to water and oxygen, and peroxidase, which reacts H₂O₂ with an acid to form water and a deprotonated base. The removal of hydrogen peroxide from the organism prevents degradation of cells (3). KatG also has an enzymatic function in the prevention of Mycobacterium tuberculosis synthesis; KatG activates isoniazid (INH) and converts it to isonicotinoyl-NAD, which prevents M. tuberculosis from synthesizing or growing (4). Scientists are exploring the use of KatG in anti-tuberculosis drugs. Since M. tuberculosis is INH-resistant, INH would not be effective without activation from KatG (3).

KatG is a dimer composed primarily of random coils (two-thirds) and -helices and -sheets (one-third) (3). With an isoelectric point of 5.60, the protein contains more acidic residues and maintains a neutral charge at a pH of 5.60 (5). The protein has two subunits, each containing a heme group and binding site (3), with a total molecular weight of 158874.31 Da (5). Each subunit has 180-degree rotational symmetry, and the two are nearly identical; when superimposed, deviation is less than 0.8 angstroms (3). The two subunits are connected with a cross-linked disulfide bond between two cysteine residues (6). This connection holds the subunits together, but does not have any other significant function (3).

Due to a similar function via peroxidase reaction of H₂O₂, the structure of KatG resembles plant peroxidases, though it is nearly two times larger in size (2). Because of the larger subunit radius, active groups of KatG are more surrounded and less accessible than those in plants. Primary sequence deviations from the structure of plant peroxidases are predominantly found on the C-terminus of each subunit because this end of the peptide chain is less vital to the function of the protein. Fewer differences from plant peroxidase structure are found on the N-termini of each subunit, containing the functionally-important heme groups and binding sites (3). With structural changes to heme groups, a partial or entire loss of function may result.

To act as a defense against oxidative stress, KatG uses four functional ligands. The first is a peroxidized heme form 2 group, with one in each subunit. The group contains an iron atom at the center, bound to four surrounding nitrogen atoms. The remainder of the ligand is composed of salt bridges, hydrogen bonds, and hydrophobic interactions (1). The heme is the site where catalase and peroxidase reactions take place, starting with interaction of substrate with the iron cation to form a complex (3). This complex is oxidized to form “compound I.” Compound I is an intermediate, as both catalase and peroxidase reactions occur in two steps (7). The second functional ligand is a sodium ion, which is a binding site for the substrate. The remaining functional ligands include a main chain oxygen molecule and a (4S)-2-methyl-2,4-pentadienol group. Both can form hydrogen bonds with water and other surrounding molecules (3).

Some structural anomalies of KatG contribute to the function of the protein. On the distal side of the sodium ion is a funnel-shaped channel, narrow at Ser-324 and Asp-141 near the metal. His-55 and Glu-198 have major and minor conformations in this channel, adjusting the size of this pathway and allowing for selectivity in substrates entering the protein (3). Between the sodium ion and heme group is another adduct formed by Trp-111, Tyr-238, and Met-264. At the entrance of the adduct, Asp-141 controls selectivity by changing conformation, similar to His-55 and Glu-198 (2). Though the definite function of the adduct is not confirmed, scientists propose several potential functions. The adduct may act as structural reinforcement, a site for electron transfer, or prevention of Met-264 oxidation, which would happen more readily than hydroxide oxidation if it were not bound in the adduct (3). Met-264 may have an additional role as a vital residue to catalase and peroxidase reactions by acting as an electron donor (6). The combination of funnel and adduct surrounding the sodium ion collectively form a pathway for catalase-peroxidase reactions to take place. A possible second pathway is a polarized cleft that forms between the two domains of each subunit. Major conformations of Arg-426 and Thr-119 can adjust the depth of the space and encourage binding to the site (3).

Aside from contributing to structural pathways for the protein, residues of KatG also serve as binding sites. Arg-108, Trp-111, and His-112 of the heme group form an active site where the first step of catalase or peroxidase is initiated (3); here the substrate is reduced as the heme group is oxidized (7). It is possible that Arg-426 and Thr-119 form a second binding site. The residues are capable of changing conformation, indicating that they may have functional significance as they could conform to make binding easier (3). Additionally, Wiseman et al. found that activity of two separate pathways, as opposed to just one, was affected by the increase of oxidase, further supporting the speculation of multiple binding sites (7).

Ser-324 is a separate binding site for INH, unrelated to the catalase-peroxidase function. Following initial binding, Thr-232 and Ser-321 ionically bond and oxidize INH (3). The location of Ser-324 is unique in its selectivity; as it is located inside the adduct, the substrate must flow through it to bind with Ser-324. While INH can fit in the adduct, larger substrates will not fit, increasing substrate specificity of KatG (4).

Two bioinformatics servers allowed for comparison of KatG structure to other proteins. PSI-BLAST searches for proteins with similar primary structures, providing an E value to represent deviations in sequence; the smaller the E value, the more similar the sequence of the protein is to the KatG sequence. An E value greater than 0 and less than 0.05 indicates a significant similarity (8). Much like PSI-BLAST, the Dali Server finds proteins with similar tertiary structures, providing a Z-score for matches; a higher Z-score indicates more similarities. A Z-score greater than two indicates a significant similarity (9). A protein that fits these criteria in relation to KatG of B. pseudomallei is KatG in Escherichia coli (PDB ID: 1U2J), with an E value of 4x10^-133 and a Z-score of 43.3 (8, 9). KatG from E. coli, also a catalase peroxidase protein in bacteria, is known as hyperperoxidase I (HPI) (10). HPI removes hydrogen peroxide from the organism to prevent cell degradation, synonymous in function to KatG of B. pseudomallei but in a different species of bacteria (11).

Though it has a similar function, the structure of KatG in E. coli has differences. HPI also has two nearly identical subunits containing heme groups (12). Through the use of mass spectrometry, Hillar et al. discovered that each subunit contains four to eight heme groups, rather than just one (10). The heme groups remain the sites at which catalase and peroxidase reactions occur to break down hydrogen peroxide (12). However, the heme group contains a tryptophan in place of a phenylalanine, resulting in slower catalase activity compared with B. pseudomallei (3). Slower activity may be related to the absence of iron cation in some of the heme groups, making them unfit for reaction (11). Peroxidase activity is decreased as well, but to a smaller degree as it has little dependence on the tryptophan residue (10).

KatG of B. pseudomallei has the same functional groups as KatG of E. coli, and therefore, both carry out catalase and peroxidase reactions. With structural differences, reaction rates of each protein are altered (3). The similarity in structure and binding sites indicates that the proteins would function similarly (8, 9). KatG of B. pseudomallei, unlike in E. coli, is unique as an enzyme for INH conversion and is the focus in development of an anti-tuberculosis drug (4).