HutW from Vibrio cholerae Is an Anaerobic Heme-Degrading Enzyme with Unique Functional Properties

Abstract

Increasing antibiotic resistance, and a growing recognition of the importance of the human microbiome, demand that new therapeutic targets be identified. Characterization of metabolic pathways that are unique to enteric pathogens represents a promising approach. Iron is often the rate-limiting factor for growth, and Vibrio cholerae, the causative agent of cholera, has been shown to contain numerous genes that function in the acquisition of iron from the environment. Included in this arsenal of genes are operons dedicated to obtaining iron from heme and heme-containing proteins. Given the persistence of cholera, an important outstanding question is whether V. cholerae is capable of anaerobic heme degradation as was recently reported for enterohemorrhagic Escherichia coli O157:H7. In this work, we demonstrate that HutW from V. cholerae is a radical S-adenosylmethionine methyl transferase involved in the anaerobic opening of the porphyrin ring of heme. However, in contrast to the enzyme ChuW, found in enterohemorrhagic E. coli O157:H7, there are notable differences in the mechanism and products of the HutW reaction. Of particular interest are data that demonstrate HutW will catalyze ring opening as well as tetrapyrrole reduction and can utilize reduced nicotinamide adenine dinucleotide phosphate as an electron source. The biochemical and biophysical properties of HutW are presented, and the evolutionary implications are discussed.

Radical SAM‐Dependent Demetallation of Heme

“…To test this possibility, we performed quantification analysis and compared the reaction efficiencies on the two substrates, hemin and PPIX. Because accurate quantification of anaerobilin is impractical owing to its instability, [ 33 ] dAdoH and SAH were used as indicators of the reaction efficiency. A detailed time course analysis of ChuW reaction showed that hemin is a much better substrate than the metal‐free PPIX, as the yields of both dAdoH and SAH in the reaction with hemin are generally more than 2‐folds compared to those in the reaction with PPIX (Figure 3B).…”

Class C Radical SAM Methyltransferases Involved in Anaerobic Heme Degradation

“…In the case of the latter, the methylene radical is added to a double bond, resulting in a methyl transfer reaction and, in many cases, further radical-catalyzed chemical rearrangements . For both ChuW and HutW, this includes the β-scission of a carbon–carbon bond, resulting in opening of the porphyrin ring along with the liberation of the iron atom. , …”

“…Similarly, V. cholera also expresses an enzyme, HutW (39% sequence identity with ChuW), that is capable of opening the porphyrin ring under anaerobic conditions . However, in contrast, V.…”

“…As Figure shows, the iron atom does not participate in the reaction, and in fact, several metal-free porphyrins have been shown to be acceptable substrates for ChuW as well as HutW. , It is also important to point out that the electron source as well as the tetrapyrrole product produced by ChuW is different from what has been observed for HutW. ChuW utilizes the E.…”

Nutritional immunity: the battle for nutrient metals at the host–pathogen interface

“…Subsequently, Fe must be liberated from the tetrapyrrole ring of haem via the activity of haem oxygenases in the bacterial cytosol 30 . Haem oxygenases are classified into five different enzyme families including the HO-1 family, the IsdG family, ChuZ, ChuW and HutW 31 , 32 .…”