Improved O₂‐tolerance in variants of a H₂‐evolving [NiFe]‐hydrogenase from Klebsiella oxytoca HP1

Abstract

a b s t r a c tIn this study, we investigated the mechanism of O 2 tolerance of Klebsiella oxytoca HP1 H 2 -evolving hydrogenase 3 (KHyd3) by mutational analysis and three-dimensional structure modeling. Results revealed that certain surface amino acid residues of KHyd3 large subunit, in particular those at the outer entrance of the gas channel, have a visible effect on its oxygen tolerance. Additionally, solution pH, immobilization and O 2 partial pressure also affect KHyd3 O 2 -tolerance to some extent. We propose that the extent of KHyd3 O 2 -tolerance is determined by a balance between the rate of O 2 access to the active center through gas channels and the deoxidation rate of the oxidized active center. Based on our findings, two higher O 2 -tolerant KHyd3 mutations G300E and G300M were developed.

Molecular Hydrogen Metabolism: a Widespread Trait of Pathogenic Bacteria and Protists

“…Typhimurium (84,237,238,294,295). However, this process has also been experimentally observed in other pathogenic Enterobacteriaceae, notably Enterobacter aerogenes (296-299), K. pneumoniae (254,258,300,301), K. oxytoca (302), and Citrobacter freundii (258,303,304).…”

An electrochemically functional layer of hydrogenase extract on an electrode of large and tunable specific surface area

“…[10][11][12] Furthermore, hydrogenases may or may not be tolerant to impurities such as CO. [13][14][15][16] Finally, their stability to aerobic oxygen represents the major challenge, 16,17 but a number of hydrogenases are quite tolerant to it. 8,16,[18][19][20][21][22][23][24][25][26] Unfortunately, an enzyme combining all of those favorable properties is yet to be discovered or engineered.…”

Dissection and engineering of theEscherichia coli formate hydrogenlyase complex

“…In that work, Huang et al [28] observed a slight increase in the air-stability of a purified G50C G120C double variant K. oxytoca Hyd-3, however a G131C variant was not tested to allow a direct comparison here. Engineering of the K. oxytoca HycE subunit, the Hyd-3 catalytic subunit, has also been reported [29]. In this case, substitution of HycE G300, predicted to be located within a gas channel leading to and from the active site, with either glutamic acid or methionine lead to increased airstability of K. oxytoca Hyd-3 [29].…”

“…Engineering of the K. oxytoca HycE subunit, the Hyd-3 catalytic subunit, has also been reported [29]. In this case, substitution of HycE G300, predicted to be located within a gas channel leading to and from the active site, with either glutamic acid or methionine lead to increased airstability of K. oxytoca Hyd-3 [29].…”