Unexpected Diversity of Chlorite Dismutases: a Catalytically Efficient Dimeric Enzyme from Nitrobacter winogradskyi

Abstract

Chlorite dismutase (Cld) is a unique heme enzyme catalyzing the conversion of ClO 2؊ to Cl ؊ and O 2 . Cld is usually found in perchlorate-or chlorate-reducing bacteria but was also recently identified in a nitriteoxidizing bacterium of the genus Nitrospira. Here we characterized a novel Cld-like protein from the chemolithoautotrophic nitrite oxidizer Nitrobacter winogradskyi which is significantly smaller than all previously known chlorite dismutases. Its three-dimensional (3D) crystal structure revealed a dimer of two identical subunits, which sharply contrasts with the penta-or hexameric structures of other chlorite dismutases. Despite a truncated N-terminal domain in each subunit, this novel enzyme turned out to be a highly efficient chlorite dismutase (K m ‫؍‬ 90 M; k cat ‫؍‬ 190 s ؊1 ; k cat /K m ‫؍‬ 2.1 ؋ 10 6 M ؊1 s ؊1 ), demonstrating a greater structural and phylogenetic diversity of these enzymes than was previously known. Based on comparative analyses of Cld sequences and 3D structures, signature amino acid residues that can be employed to assess whether uncharacterized Cld-like proteins may have a high chlorite-dismutating activity were identified. Interestingly, proteins that contain all these signatures and are phylogenetically closely related to the novel-type Cld of N. winogradskyi exist in a large number of other microbes, including other nitrite oxidizers.

A structural and functional perspective of DyP-type peroxidase family

“…292 Therefore, the important histidine residue is located near the cen-293 ter of the long a-helix. The amino acid residues that form the long 294 a-helix are well conserved, suggesting that the histidine residue proteins that do not form a dimeric a + b barrel structure because 353 of a truncation in the N-terminus been discovered [42]. These 354 observations suggest that the structural superfamily that includes 355 DyP-type peroxidases needs to be reassessed.…”

From chlorite dismutase towards HemQ–the role of the proximal H-bonding network in haeme binding

“…However, in most haeme proteins the Fe-proximal His represents the only covalent bond between the haeme chromophore and the protein, and stable incorporation of the prosthetic group occurs via several non-covalent interactions. These include H-bonding networks at the proximal side [54] as shown for haeme b-containing Clds [12,20,30,55].…”

“…This includes Clds from perchlorate reducing bacteria (Azospira oryzae GR-1, Ideonella dechloratans, Dechloromonas aromatica, Pseudomonas chloritidismutans, Magnetospirillum sp.) [13][14][15][16][17][18], nitrite-oxidizing bacteria ('Candidatus Nitrospira defluvii', Nitrobacter winogradskyi) [12,19,20], from cyanobacteria (e.g. Cyanothece sp.…”

The homopentameric chlorite dismutase from Magnetospirillum sp.

“…Nitrobacter winogradskyi [14], Klebsiella pneumoniae [20] and Cyanothece sp. PCC7425 [21] can be found in a separated branch ( Figure S3).…”