Molecular architecture of KedS8, a sugar N‐methyltransferase from Streptoalloteichus sp. ATCC 53650

Kedarcidin, produced by Streptoalloteichus sp. ATCC 53650, is a fascinating chromoprotein of 114 amino acid residues that displays both antibiotic and anticancer activity. The chromophore responsible for its chemotherapeutic activity is an ansa‐bridged enediyne with two attached sugars, l ‐mycarose, and l ‐kedarosamine. The biosynthesis of l ‐kedarosamine, a highly unusual trideoxysugar, is beginning to be revealed through bioinformatics approaches. One of the enzymes putatively involved in the production of this carbohydrate is referred to as KedS8. It has been proposed that KedS8 is an N‐methyltransferase that utilizes S‐adenosylmethionine as the methyl donor and a dTDP‐linked C‐4′ amino sugar as the substrate. Here we describe the three‐dimensional architecture of KedS8 in complex with S‐adenosylhomocysteine. The structure was solved to 2.0 Å resolution and refined to an overall R‐factor of 17.1%. Unlike that observed for other sugar N‐methyltransferases, KedS8 adopts a novel tetrameric quaternary structure due to the swapping of β‐strands at the N‐termini of its subunits. The structure presented here represents the first example of an N‐methyltransferase that functions on C‐4′ rather than C‐3′ amino sugars.     

The Mycobacterium tuberculosis complex has a pathway for the biosynthesis of 4‐formamido‐4,6‐dideoxy‐d‐glucose

Recent studies have demonstrated that the O‐antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N‐formylated sugars (3‐formamido‐3,6‐dideoxy‐d ‐glucose or 4‐formamido‐4,6‐dideoxy‐d ‐glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6‐dehydratase, a pyridoxal 5'‐phosphate or PLP‐dependent aminotransferase, and an N‐formyltransferase. To date, there have been no published reports of N‐formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N‐formyltransferase. Given that M. tuberculosis produces l ‐rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6‐dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N‐formylated sugar in M. tuberculosis, namely a PLP‐dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined.     

Investigation of a sugar N‐formyltransferase from the plant pathogen Pantoea ananatis

Pantoea ananatis is a Gram‐negative bacterium first recognized in 1928 as the causative agent of pineapple rot in the Philippines. Since then various strains of the organism have been implicated in the devastation of agriculturally important crops. Some strains, however, have been shown to function as non‐pathogenic plant growth promoting organisms. To date, the factors that determine pathogenicity or lack thereof between the various strains are not well understood. All P. ananatis strains contain lipopolysaccharides, which differ with respect to the identities of their associated sugars. Given our research interest on the presence of the unusual sugar, 4‐formamido‐4,6‐dideoxy‐d ‐glucose, found on the lipopolysaccharides of Campylobacter jejuni and Francisella tularensis, we were curious as to whether other bacteria have the appropriate biosynthetic machinery to produce these unique carbohydrates. Four enzymes are typically required for their biosynthesis: a thymidylyltransferase, a 4,6‐dehydratase, an aminotransferase, and an N‐formyltransferase. Here, we report that the gene SAMN03097714_1080 from the P. ananatis strain NFR11 does, indeed, encode for an N‐formyltransferase, hereafter referred to as PA1080c. Our kinetic analysis demonstrates that PA1080c displays classical Michaelis–Menten kinetics with dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose as the substrate and N¹⁰‐formyltetrahydrofolate as the carbon source. In addition, the X‐ray structure of PA1080c, determined to 1.7 Å resolution, shows that the enzyme adopts the molecular architecture observed for other sugar N‐formyltransferases. Analysis of the P. ananatis NFR11 genome suggests that the three other enzymes necessary for N‐formylated sugar biosynthesis are also present. Intriguingly, those strains of P. ananatis that are non‐pathogenic apparently do not contain these genes.     

Structural investigation on WlaRG from Campylobacter jejuni: A sugar aminotransferase

Campylobacter jejuni is a Gram‐negative bacterium that represents a leading cause of human gastroenteritis worldwide. Of particular concern is the link between C. jejuni infections and the subsequent development of Guillain‐Barré syndrome, an acquired autoimmune disorder leading to paralysis. All Gram‐negative bacteria contain complex glycoconjugates anchored to their outer membranes, but in most strains of C. jejuni, this lipoglycan lacks the O‐antigen repeating units. Recent mass spectrometry analyses indicate that the C. jejuni 81116 (Penner serotype HS:6) lipoglycan contains two dideoxyhexosamine residues, and enzymological assay data show that this bacterial strain can synthesize both dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐glucose and dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐galactose. The focus of this investigation is on WlaRG from C. jejuni, which plays a key role in the production of these unusual sugars by functioning as a pyridoxal 5′‐phosphate dependent aminotransferase. Here, we describe the first three‐dimensional structures of the enzyme in various complexes determined to resolutions of 1.7 Å or higher. Of particular significance are the external aldimine structures of WlaRG solved in the presence of either dTDP‐3‐amino‐3,6‐dideoxy‐d ‐galactose or dTDP‐3‐amino‐3,6‐dideoxy‐d ‐glucose. These models highlight the manner in which WlaRG can accommodate sugars with differing stereochemistries about their C‐4′ carbon positions. In addition, we present a corrected structure of WbpE, a related sugar aminotransferase from Pseudomonas aeruginosa, solved to 1.3 Å resolution.     

Probing the catalytic mechanism of a C-3'-methyltransferase involved in the biosynthesis of D-tetronitrose

D ‐Tetronitrose is a nitro‐containing tetradeoxysugar found attached to the antitumor and antibacterial agent tetrocarcin A. The biosynthesis of this highly unusual sugar in Micromonospora chalcea requires 10 enzymes. The fifth step in the pathway involves the transfer of a methyl group from S‐adenosyl‐L ‐methionine (SAM) to the C‐3′ carbon of dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐D ‐glucose. The enzyme responsible for this transformation is referred to as TcaB9. It is a monomeric enzyme with a molecular architecture based around three domains. The N‐terminal motif contains a binding site for a structural zinc ion. The middle‐ and C‐terminal domains serve to anchor the SAM and dTDP–sugar ligands, respectively, to the protein, and the active site of TcaB9 is wedged between these two regions. For this investigation, the roles of Tyr 76, His 181, Tyr 222, Glu 224, and His 225, which form the active site of TcaB9, were probed by site‐directed mutagenesis, kinetic analyses, and X‐ray structural studies. In addition, two ternary complexes of the enzyme with bound S‐adenosyl‐L ‐homocysteine and either dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐D ‐glucose or dTDP‐3‐amino‐2,3,6‐trideoxy‐D ‐galactose were determined to 1.5 or 1.6 Å resolution, respectively. Taken together, these investigations highlight the important role of His 225 in methyl transfer. In addition, the structural data suggest that the methylation reaction occurs via retention of configuration about the C‐3′ carbon of the sugar.     

Structural studies on KijD1, a sugar C‐3′‐methyltransferase

Kijanimicin is an antitumor antibiotic isolated from Actinomadura kijaniata. It is composed of three distinct moieties: a pentacyclic core, a monosaccharide referred to as d ‐kijanose, and a tetrasaccharide chain composed of l ‐digitoxose units. d ‐Kijanose is a highly unusual nitro‐containing tetradeoxysugar, which requires at least ten enzymes for its production. Here we describe a structural analysis of one of these enzymes, namely KijD1, which functions as a C‐3′‐methyltransferase using S‐adenosylmethionine as its cofactor. For this investigation, two ternary complexes of KijD1, determined in the presence of S‐adenosylhomocysteine (SAH) and dTDP or SAH and dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐3‐methyl‐d ‐glucose, were solved to 1.7 or 1.6 Å resolution, respectively. Unexpectedly, these structures, as well as additional biochemical analyses, demonstrated that the quaternary structure of KijD1 is a dimer. Indeed, this is in sharp contrast to that previously observed for the sugar C‐3′‐methyltransferase isolated from Micromonospora chalcea. By the judicious use of site‐directed mutagenesis, it was possible to convert the dimeric form of KijD1 into a monomeric version. The quaternary structure of KijD1 could not have been deduced based solely on bioinformatics approaches, and thus this investigation highlights the continuing need for experimental validation.     

Misannotations of the genes encoding sugar N‐formyltransferases

Tens of thousands of bacterial genome sequences are now known due to the development of rapid and inexpensive sequencing technologies. An important key in utilizing these vast amounts of data in a biologically meaningful way is to infer the function of the proteins encoded in the genomes via bioinformatics techniques. Whereas these approaches are absolutely critical to the annotation of gene function, there are still issues of misidentifications, which must be experimentally corrected. For example, many of the bacterial DNA sequences encoding sugar N‐formyltransferases have been annotated as l ‐methionyl‐tRNA transferases in the databases. These mistakes may be due in part to the fact that until recently the structures and functions of these enzymes were not well known. Herein we describe the misannotation of two genes, WP_088211966.1 and WP_096244125.1, from Shewanella spp. and Pseudomonas congelans, respectively. Although the proteins encoded by these genes were originally suggested to function as l ‐methionyl‐tRNA transferases, we demonstrate that they actually catalyze the conversion of dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose to dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose utilizing N¹⁰‐formyltetrahydrofolate as the carbon source. For this analysis, the genes encoding these enzymes were cloned and the corresponding proteins purified. X‐ray structures of the two proteins were determined to high resolution and kinetic analyses were conducted. Both enzymes display classical Michaelis–Menten kinetics and adopt the characteristic three‐dimensional structural fold previously observed for other sugar N‐formyltransferases. The results presented herein will aid in the future annotation of these fascinating enzymes.     

C‐terminal tail derived from the neighboring subunit is critical for the activity of Thermoplasma acidophilumD‐aldohexose dehydrogenase

The D ‐aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a homotetrameric enzyme that catalyzes the oxidation of several D ‐aldohexoses, especially D ‐mannose. AldT comprises a unique C‐terminal tail motif (residues 247–255) that shuts the active‐site pocket of the neighboring subunit. The functional role of the C‐terminal tail of AldT has been investigated using mutational and crystallographic analyses. A total of four C‐terminal deletion mutants (Δ254, Δ253, Δ252, and Δ249) and two site‐specific mutants (Y86G and P254G) were expressed by Escherichia coli and purified. Enzymatic characterization of these mutants revealed that the C‐terminal tail is a requisite and that the interaction between Tyr86 and Pro254 is critical for enzyme activity. The crystal structure of the Δ249 mutant was also determined. The structure showed that the active‐site loops undergo a significant conformational change, which leads to the structural deformation of the substrate‐binding pocket. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Quaternary variations in the structural assembly of N‐acetylglucosamine‐6‐phosphate deacetylase from Pasteurella multocida

N‐acetylglucosamine 6‐phosphate deacetylase (NagA) catalyzes the conversion of N‐acetylglucosamine‐6‐phosphate to glucosamine‐6‐phosphate in amino sugar catabolism. This conversion is an essential step in the catabolism of sialic acid in several pathogenic bacteria, including Pasteurella multocida, and thus NagA is identified as a potential drug target. Here, we report the unique structural features of NagA from P. multocida (PmNagA) resolved to 1.95 Å. PmNagA displays an altered quaternary architecture with unique interface interactions compared to its close homolog, the Escherichia coli NagA (EcNagA). We confirmed that the altered quaternary structure is not a crystallographic artifact using single particle electron cryo‐microscopy. Analysis of the determined crystal structure reveals a set of hot‐spot residues involved in novel interactions at the dimer‐dimer interface. PmNagA binds to one Zn²⁺ ion in the active site and demonstrates kinetic parameters comparable to other bacterial homologs. Kinetic studies reveal that at high substrate concentrations (~10‐fold the K_M), the tetrameric PmNagA displays hysteresis similar to its distant neighbor, the dimeric Staphylococcus aureus NagA (SaNagA). Our findings provide key information on structural and functional properties of NagA in P. multocida that could be utilized to design novel antibacterials.     

Structure of the Escherichia coli ArnA N‐formyltransferase domain in complex with N5‐formyltetrahydrofolate and UDP‐Ara4N

ArnA from Escherichia coli is a key enzyme involved in the formation of 4‐amino‐4‐deoxy‐l ‐arabinose. The addition of this sugar to the lipid A moiety of the lipopolysaccharide of pathogenic Gram‐negative bacteria allows these organisms to evade the cationic antimicrobial peptides of the host immune system. Indeed, it is thought that such modifications may be responsible for the repeated infections of cystic fibrosis patients with Pseudomonas aeruginosa. ArnA is a bifunctional enzyme with the N‐ and C‐terminal domains catalyzing formylation and oxidative decarboxylation reactions, respectively. The catalytically competent cofactor for the formylation reaction is N¹⁰‐formyltetrahydrofolate. Here we describe the structure of the isolated N‐terminal domain of ArnA in complex with its UDP‐sugar substrate and N⁵‐formyltetrahydrofolate. The model presented herein may prove valuable in the development of new antimicrobial therapeutics.     

Arg‐85 and Thr‐430 in murine 5‐aminolevulinate synthase coordinate acyl‐CoA‐binding and contribute to substrate specificity

5‐Aminolevulinate synthase (ALAS) controls the rate‐limiting step of heme biosynthesis in mammals by catalyzing the condensation of succinyl‐coenzyme A and glycine to produce 5‐aminolevulinate, coenzyme‐A (CoA), and carbon dioxide. ALAS is a member of the α‐oxoamine synthase family of pyridoxal 5′‐phosphate (PLP)‐dependent enzymes and shares high degree of structural similarity and reaction mechanism with the other members of the family. The X‐ray crystal structure of ALAS from Rhodobacter capsulatus reveals that the alkanoate component of succinyl‐CoA is coordinated by a conserved arginine and a threonine. The functions of the corresponding acyl‐CoA‐binding residues in murine erthyroid ALAS (R85 and T430) in relation to acyl‐CoA binding and substrate discrimination were examined using site‐directed mutagenesis and a series of CoA‐derivatives. The catalytic efficiency of the R85L variant with octanoyl‐CoA was 66‐fold higher than that of the wild‐type protein, supporting the proposal of this residue as key in discriminating substrate binding. Substitution of the acyl‐CoA‐binding residues with hydrophobic amino acids caused a ligand‐induced negative dichroic band at 420 nm in the CD spectra, suggesting that these residues affect substrate‐mediated changes to the PLP microenvironment. Transient kinetic analyses of the R85K variant‐catalyzed reactions confirm that this substitution decreases microscopic rates associated with formation and decay of a key reaction intermediate and show that the nature of the acyl‐CoA tail seriously affect product binding. These results show that the bifurcate interaction of the carboxylate moiety of succinyl‐CoA with R85 and T430 is an important determinant in ALAS function and may play a role in substrate specificity.     

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Biosynthesis of the nucleotide sugar precursor dTDP‐L‐rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP‐L‐rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP‐L‐rhamnose biosynthesis through their action as dTDP‐glucose‐4,6‐dehydratase and dTDP‐4‐keto‐6‐deoxyglucose‐3,5‐epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio‐layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP‐rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose‐dependent streptococcal pathogens as well as M. tuberculosis with an IC₅₀ of 120–410 µM. Importantly, we confirmed that Ri03 inhibited dTDP‐L‐rhamnose formation in a concentration‐dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP‐L‐rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP‐rhamnose biosynthesis in pathogenic bacteria.     

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

The sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A Streptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium Salmonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gacA in a S. mutans rmlD knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gacA as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.     

A novel Fe(II)/α‐ketoglutarate‐dependent dioxygenase from Burkholderia ambifaria has β‐hydroxylating activity of N‐succinyl l‐leucine

An Fe(II)/α‐ketoglutarate‐dependent dioxygenase, SadA, was obtained from Burkholderia ambifaria AMMD and heterologously expressed in Escherichia coli. Purified recombinant SadA had catalytic activity towards several N‐substituted l‐amino acids, which was especially strong with N‐succinyl l‐leucine. With the NMR and LC‐MS analysis, SadA converted N‐succinyl l‐leucine into N‐succinyl l‐threo‐β‐hydroxyleucine with >99% diastereoselectivity. SadA is the first enzyme catalysing β‐hydroxylation of aliphatic amino acid‐related substances and a potent biocatalyst for the preparation of optically active β‐hydroxy amino acids.     

New m‐calpain substrate‐based azapeptide inhibitors

Calpains are intracellular cysteine proteases with several important physiological functions. Calpain inhibitors may be promising tools in the analysis of the function of the enzyme in diseases caused by overexpression/activation. Here, we report on the synthesis, solution conformation, and characterization of novel group of azapeptides whose sequences originate from an efficient m‐calpain substrate, TPLKSPPPSPR, described by us earlier and possess varying levels of calpain inhibition. The Lys residue at P₁ position was replaced with azaglycine (NH₂‐NH‐COOH) and further changes were made as follows: the N‐terminal or/and C‐terminal were truncated, amino acids were also changed at P₃, P₂, P′₁, or P′₂ positions. Our results indicate that the identity of amino acid moieties between P₄ and P′₅ positions is essential for the inhibitory activity. Only changes at position P₃ (Pro) are tolerated. Azapeptide analogs, described in this communication could be considered as useful set of compounds for elucidation of the enzyme interaction at P and P′ sites. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

A pseudo steady‐state model for galacto‐oligosaccharides synthesis with β‐galactosidase from Aspergillus oryzae

A pseudo steady‐state model for the kinetically controlled synthesis of galacto‐oligosaccharides (GOS) with Aspergillus oryzae β‐galactosidase is presented. The model accounts for the dynamics of lactose consumption and production of galactose, glucose, di, tri, tetra, and penta‐oligosaccharides during the synthesis, being able to describe the total GOS content in the reaction medium at the experimental conditions evaluated. Experimental results show that the formation of GOS containing only galactose residues is significant at high conversions of substrate, which was taken into account in the model. The formation of enzyme transition complexes was considered and reasonable assumptions were made to reduce the number of parameters to be determined. The model developed has 8 parameters; 2 of them were experimentally determined and the other 6 were estimated by fitting to the experimental data using multiresponse regression. Temperature effect on kinetic and affinity constants was determined in the range from 40 to 55°C, and the data were fitted to Arrhenius type equation. Parameters of the proposed model are independent from the enzyme load in the reaction medium and, differently from previously reported models, they have a clear biochemical meaning. The magnitude of the kinetic and affinity constants of the enzyme suggests that the liberation of galactose from the galactosyl–enzyme complex is a very slow reaction and such complex is driven into GOS formation. It also suggests that the affinity for sugars of the galactosyl–enzyme complex is higher than that of the free enzyme. Biotechnol. Bioeng. 2011;108: 2270–2279. © 2011 Wiley Periodicals, Inc.     

Deletion of fucose residues in plant N‐glycans by repression of the GDP‐mannose 4,6‐dehydratase gene using virus‐induced gene silencing and RNA interference

Production of pharmaceutical glycoproteins in plants has many advantages in terms of safety and reduced costs. However, plant‐produced glycoproteins have N‐glycans with plant‐specific sugar residues (core β‐1,2‐xylose and α‐1,3‐fucose) and a Lewis a (Le^a) epitope, i.e., Galβ(1‐3)[Fucα(1‐4)]GlcNAc. Because these sugar residues and glycan structures seemed to be immunogenic, several attempts have been made to delete them by repressing their respective glycosyltransferase genes. However, until date, such deletions have not been successful in completely eliminating the fucose residues. In this study, we simultaneously reduced the plant‐specific core α‐1,3‐fucose and α‐1,4‐fucose residues in the Le^a epitopes by repressing the Guanosine 5′‐diphosphate (GDP)‐D‐mannose 4,6‐dehydratase (GMD) gene, which is associated with GDP‐L‐fucose biosynthesis, in Nicotiana benthamiana plants. Repression of GMD was achieved using virus‐induced gene silencing (VIGS) and RNA interference (RNAi). The proportion of fucose‐free N‐glycans found in total soluble protein from GMD gene‐repressed plants increased by 80% and 95% following VIGS and RNAi, respectively, compared to wild‐type plants. A small amount of putative galactose substitution in N‐glycans from the NbGMD gene‐repressed plants was observed, similar to what has been previously reported GMD‐knockout Arabidopsis mutant. On the other hand, the recombinant mouse granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) with fucose‐deleted N‐glycans was successfully produced in NbGMD‐RNAi transgenic N. benthamiana plants. Thus, repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants.     

The structure and peroxidase activity of a 33‐kDa catalase‐related protein from Mycobacterium avium ssp. paratuberculosis

True catalases are tyrosine‐liganded, usually tetrameric, hemoproteins with subunit sizes of ～55–84 kDa. Recently characterized hemoproteins with a catalase‐related structure, yet lacking in catalatic activity, include the 40–43 kDa allene oxide synthases of marine invertebrates and cyanobacteria. Herein, we describe the 1.8 Å X‐ray crystal structure of a 33 kDa subunit hemoprotein from Mycobacterium avium ssp. paratuberculosis (annotated as MAP‐2744c), that retains the core elements of the catalase fold and exhibits an organic peroxide‐dependent peroxidase activity. MAP‐2744c exhibits negligible catalatic activity, weak peroxidatic activity using hydrogen peroxide (20/s) and strong peroxidase activity (～300/s) using organic hydroperoxides as co‐substrate. Key amino acid differences significantly impact prosthetic group conformation and placement and confer a distinct activity to this prototypical member of a group of conserved bacterial “minicatalases”. Its structural features and the result of the enzyme assays support a role for MAP‐2744c and its close homologues in mitigating challenge by a variety of reactive oxygen species.