Investigation of the catalytic mechanism of Mycoplasma pneumoniae L-alpha-glycerophosphate oxidase: mutation of the proposed catalytic base

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Craig Matthew Crowley II (Creator)
Western Carolina University (WCU )
Web Site:
Jamie R. Wallen

Abstract: The formation of hydrogen peroxide (H2O2) by the enzyme L-alpha-glycerophosphate oxidase(MpGlpO) is essential for the pathogenesis of Mycoplasma pneumoniae. M. pneumoniae is apathogen that targets the human respiratory tract, causing at least 40% of all pneumoniaeinfections, with the most common symptom being tracheobronchitis in children. MpGlpO relayselectrons from L-alpha-glycerophosphate to molecular oxygen to form the productsdihydroxyacetone phosphate and hydrogen peroxide. Because the formation of hydrogenperoxide is essential for M. pneumoniae pathogenesis, the MpGlpO enzyme has emerged as apotential therapeutic target for pneumoniae infections. Recently an X-ray crystal structure of theapo form MpGlpO was determined. Based on features of the proposed active site, a model ofglycerophosphate binding was proposed. This study is focused on mutating amino acids that arepredicted from the model to bind the glycerophosphate substrate to confirm their role in thecatalytic cycle. We were especially interested in investigating the hypothesis that a catalyticbase is required for deprotonation of the glycerophosphate substrate, in particular if the histidineresidue was essential for catalysis. MpGlpO wild-type and mutants H51A, H51Q, and S348A proteins were expressed and purified from E. coli resulting in 22, 2, 11 and 21 mg of proteinrespectively from 4.8 liters of Terrific Broth. The activity of the purified enzymes weredetermined by a ABTS-HRP coupled assay to be 34.7 +/- 4.6 U*mg-1, 0.45 +/- 0.18 U*mg-1,2.22 +/- 0.34 U*mg-1 and 0.62 +/- 0.22 U*mg-1 for wild-type, H51A, H51Q, and S348Arespectively. Our results, and the results of our collaborators, indicate that the reaction can stillproceed without a catalytic base, but we have identified histidine 51 and serine 348 as havingimportant roles in hydrogen bonding the glycerophosphate substrate in the MpGlpO reaction.Reactivity of the mutants indicates that neither residue acts as the essential base. Future workwill focus on understanding the contribution of other residues in the active site that are predictedto bind the substrate.

Additional Information

Language: English
Date: 2016
Microbial enzymes -- Testing
Microbial metabolism
Chemical bonds
Organic compounds -- Synthesis
Mycoplasma pneumoniae infections -- Treatment

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