Role of Ser-257 in the Sliding Mechanism of NADP(H) in the Reaction Catalyzed by the Aspergillus Fumigatus Flavin-dependent Ornithine N5-monooxygenase SidA

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2013 Sep 28

PMID 24072704

Citations 9

Authors

Carolyn Shirey

Somayesadat Badieyan

Pablo Sobrado

Affiliations

Soon will be listed here.

Abstract

SidA (siderophore A) is a flavin-dependent N-hydroxylating monooxygenase that is essential for virulence in Aspergillus fumigatus. SidA catalyzes the NADPH- and oxygen-dependent formation of N(5)-hydroxyornithine. In this reaction, NADPH reduces the flavin, and the resulting NADP(+) is the last product to be released. The presence of NADP(+) is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species. As part of our efforts to determine the molecular details of the role of NADP(H) in catalysis, we targeted Ser-257 for site-directed mutagenesis and performed extensive characterization of the S257A enzyme. Using a combination of steady-state and stopped-flow kinetic experiments, substrate analogs, and primary kinetic isotope effects, we show that the interaction between Ser-257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin. Molecular dynamics simulation results suggest that Ser-257 functions as a pivot point, allowing the nicotinamide of NADP(+) to slide into position for stabilization of the C4a-hydroperoxyflavin.

Citing Articles

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