Kinetic mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae
- PMID: 25712468
- DOI: 10.1111/febs.13247
Kinetic mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae
Abstract
L-α-glycerophosphate oxidase is an FAD-dependent enzyme that catalyzes the oxidation of L-α-glycerophosphate (Glp) by molecular oxygen to generate dihydroxyacetone phosphate (DHAP) and hydrogen peroxide (H2O2). The catalytic properties of recombinant His6-GlpO from Mycoplasma pneumoniae (His6-MpGlpO) were investigated through transient and steady-state kinetics and ligand binding studies. The results indicate that the reaction mechanism of His6-MpGlpO follows a ping-pong model. Double-mixing mode stopped-flow experiments show that, after flavin-mediated substrate oxidation, DHAP leaves rapidly prior to the oxygen reaction. The values determined for the individual rate constants and kcat (4.2 s(-1) at 4 °C), in addition to the finding that H2 O2 binds to the oxidized enzyme, suggest that H2O2 release is the rate-limiting step for the overall reaction. The results indicate that His6 -MpGlpO contains mixed populations of fast- and slow-reacting species. It is predominantly the fast-reacting species that participates in turnover. In contrast to other GlpO enzymes previously described, His6-MpGlpO is able to catalyze the reverse reaction of reduced enzyme and DHAP. This result may be explained by the standard reduction potential value of His6-MpGlpO (-167 ± 1 mV), which is lower than those of GlpO from other species. We found that D,L-glyceraldehyde 3-phosphate (GAP) may be used as a substrate in the His6-MpGlpO reaction, although it exhibited an approximately 100-fold lower kcat value in comparison with the reaction of Glp. These results also imply involvement of GlpO in glycolysis, as well as in lipid and glycerol metabolism. The kinetic models and distinctive properties of His6-MpGlpO reported here should be useful for future drug development against Mycoplasma pneumoniae infection.
Keywords: Mycoplasma pneumoniae; flavin adenine dinucleotide (FAD); flavoprotein oxidase; l-α-glycerophosphate oxidase; transient kinetics.
© 2015 FEBS.
Similar articles
-
Structure and proposed mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae.FEBS J. 2015 Aug;282(16):3030-42. doi: 10.1111/febs.13233. Epub 2015 Mar 14. FEBS J. 2015. PMID: 25688572
-
Structure of alpha-glycerophosphate oxidase from Streptococcus sp.: a template for the mitochondrial alpha-glycerophosphate dehydrogenase.Biochemistry. 2008 Jan 22;47(3):965-77. doi: 10.1021/bi701685u. Epub 2007 Dec 23. Biochemistry. 2008. PMID: 18154320
-
The soluble alpha-glycerophosphate oxidase from Enterococcus casseliflavus. Sequence homology with the membrane-associated dehydrogenase and kinetic analysis of the recombinant enzyme.J Biol Chem. 1998 Sep 11;273(37):23812-22. doi: 10.1074/jbc.273.37.23812. J Biol Chem. 1998. PMID: 9726992
-
Glycerol metabolism and its implication in virulence in Mycoplasma.FEMS Microbiol Rev. 2017 Sep 1;41(5):640-652. doi: 10.1093/femsre/fux033. FEMS Microbiol Rev. 2017. PMID: 28961963 Review.
-
Amine oxidation by d-arginine dehydrogenase in Pseudomonas aeruginosa.Arch Biochem Biophys. 2017 Oct 15;632:192-201. doi: 10.1016/j.abb.2017.06.013. Epub 2017 Jun 15. Arch Biochem Biophys. 2017. PMID: 28625766 Review.
Cited by
-
Potential Molecular Targets for Narrow-Spectrum Agents to Combat Mycoplasma pneumoniae Infection and Disease.Front Microbiol. 2016 Feb 25;7:205. doi: 10.3389/fmicb.2016.00205. eCollection 2016. Front Microbiol. 2016. PMID: 26941728 Free PMC article. Review.
-
Mycoplasma pneumoniae from the Respiratory Tract and Beyond.Clin Microbiol Rev. 2017 Jul;30(3):747-809. doi: 10.1128/CMR.00114-16. Clin Microbiol Rev. 2017. PMID: 28539503 Free PMC article. Review.
-
Mycoplasma ovipneumoniae induces sheep airway epithelial cell apoptosis through an ERK signalling-mediated mitochondria pathway.BMC Microbiol. 2016 Sep 23;16(1):222. doi: 10.1186/s12866-016-0842-0. BMC Microbiol. 2016. PMID: 27663303 Free PMC article.
-
Proteomic and Phenotypic Studies of Mycoplasma pneumoniae Revealed Macrolide-Resistant Mutation (A2063G) Associated Changes in Protein Composition and Pathogenicity of Type I Strains.Microbiol Spectr. 2023 Aug 17;11(4):e0461322. doi: 10.1128/spectrum.04613-22. Epub 2023 Jun 28. Microbiol Spectr. 2023. PMID: 37378520 Free PMC article.
-
Kukoamine A Improves Mycoplasma pneumoniae Pneumonia by Regulating miR-222-3p/Superoxide Dismutase 2.Biomed Res Int. 2022 Jun 21;2022:2064013. doi: 10.1155/2022/2064013. eCollection 2022. Biomed Res Int. 2022. Retraction in: Biomed Res Int. 2023 Dec 6;2023:9786745. doi: 10.1155/2023/9786745. PMID: 35774277 Free PMC article. Retracted.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases
Miscellaneous
