Improved O2-tolerance in variants of a H2-evolving [NiFe]-hydrogenase from Klebsiella oxytoca HP1.
In this study, we investigated the mechanism of O2 tolerance of Klebsiella oxytoca HP1 H2-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 O2 partial pressure also affect KHyd3 O2-tolerance to some extent. We propose that the extent of KHyd3 O2-tolerance is determined by a balance between the rate of O2 access to the active center through gas channels and the deoxidation rate of the oxidized active center. Based on our findings, two higher O2-tolerant KHyd3 mutations G300E and G300M were developed.
State Key Laboratory of Cellular Stress Biology and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361102, China.
Gang-Feng
GF
Huang
State Key Laboratory of Cellular Stress Biology and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361102, China. Electronic address: wuxiaobing@xmu.edu.cn.
Xiao-Bing
XB
Wu
State Key Laboratory of Cellular Stress Biology and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361102, China.
Li-Ping
LP
Bai
State Key Laboratory of Cellular Stress Biology and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361102, China.
Ke
K
Liu
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, China.
Li-Jing
LJ
Jiang
School of Energy Research, Xiamen University, Xiamen 361102, China.
Min-Nan
MN
Long
State Key Laboratory of Cellular Stress Biology and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361102, China. Electronic address: chenqx@xmu.edu.cn.
Qing-Xi
QX
Chen
D000693:Anaerobiosis; D055162:Biocatalysis; D004305:Dose-Response Relationship, Drug; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D041121:Klebsiella oxytoca / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D010100:Oxygen / Q000494:pharmacology*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004800:Enzymes, Immobilized; D021122:Protein Subunits; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen
Biocatalysts; Gas channel; H(2)-evolving hydrogenase; Klebsiella oxytoca HP1; Mutation; O(2)-tolerance