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

Abstract

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.

A fungal catalase reacts selectively with the 13S fatty acid hydroperoxide products of the adjacent lipoxygenase gene and exhibits 13S-hydroperoxide-dependent peroxidase activity

“…In comparison, the kcat/Km for Fg-cat in oxidizing ABTS using 13-HPOTE as co-substrate gave the slightly higher value of 22 μM −1 .s −1 (Figure 8). This kcat/Km for Fg-cat in the ABTS assay is about 200-fold higher than we determined previously for a small (33 kD) catalase-related peroxidase from Mycobacterium avium paratuberculosis [30]. …”

“…Peroxidase activity of Fg-cat was assayed as an increase in absorbance at 417 nm (green color) using a Lambda-35 UV-Vis spectrophotometer (Perkin-Elmer) of a Cary 60 UV-Vis spectrophotometer (Agilent) [28–30]. Reactions were conducted with 3 nM Fg-cat using 100 μM 9 S - or 13 S -HPOTE, 1 mM H 2 O 2 , 1 mM cumene hydroperoxide, and 1 mM t-butyl hydroperoxide substrates and ABTS (1 mM).…”

Catalase-Based Modified Graphite Electrode for Hydrogen Peroxide Detection in Different Beverages

“…To investigate CAT catalytic activity, it is important to study its capacity to perform direct electron transfer (DET) to the electrode surface. It is usually difficult to observe the DET because the heme groups are buried deeply inside in the large structure of the protein [61, 62]. Also, denaturation of the redox protein could occur on the sensor surface due to the immobilization method and to the matrix composition.…”

Oxidation of C18 Hydroxy‐Polyunsaturated Fatty Acids to Epoxide or Ketone by Catalase‐Related Hemoproteins Activated with Iodosylbenzene

“…paratuberculosis gene MAP_2744c, encoding a 33 kD mini-catalase [5] and human catalase was similarly expressed in E. coli [17]. The Pseudomonas fluorescens Pfl01 37.5 kD catalase (accession number WP_011333788.1) was cloned from the genomic DNA with an N-terminal His-tag (similarly to the other three catalases used in this study) and expressed in E. coli under the same conditions as used for Fg-cat [14].…”

“…The classic catalases generally have highly restricted access to the active site, which limits their reactivity to the metabolism of very small substrates, usually hydrogen peroxide [2, 3]. By contrast, the structure of the fatty acid peroxide-metabolizing catalase, while preserving the central “catalase fold”, allows access of substrates with C18 or C20 carbon chains [4, 5]. …”