Dual role of NADP(H) in the reaction of a flavin dependent N-hydroxylating monooxygenase

Romero, Elvira; Fedkenheuer, M.; Chocklett, Samuel W.; Qi, Jun; Oppenheimer, Michelle; Sobrado, Pablo

doi:10.1016/j.bbapap.2012.03.004

Cited by 34 publications

(58 citation statements)

References 18 publications

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“…The affinity for NADPH appeared not to have been significantly affected because, even at the lower concentrations of NADPH that could be accurately tested (5 M), the maximum rate of reduction was obtained. Similar to the wild-type enzyme, this indicates that the affinity for NADPH is in the low micromolar concentration (16,26). The lack of effect on binding was also evident with NADH, where the K D value could be measured in the stopped-flow instrument, and it clearly did not change in the mutant enzyme.…”

Section: Discussionmentioning

confidence: 61%

“…For SidA, it has been determined that in the absence of ornithine, a species with an absorbance maximum at 380 nm is observed (16,26,27). This species is very stable and corresponds to the C4a-hydroperoxyflavin, which slowly decays to yield oxidized flavin and hydrogen peroxide.…”

Section: Resultsmentioning

confidence: 99%

“…We determined previously that the reaction is stereoselective for the transfer of the pro-R-hydrogen at C4 of NADPH to the oxidized flavin (16 Flavin Reduction-The k red value was determined under anaerobic conditions as a function of NAD(P)H in a stoppedflow spectrophotometer (16,26). The k red value for the S257A enzyme was 4-fold higher than that for the wild-type enzyme (Table 2).…”

Section: Resultsmentioning

confidence: 99%

“…The procedures for expression and purification of the recombinant proteins were essentially as previously described (6,16).…”

Section: Materials-escherichia Coli Top10 and Bl21(de3)-t1mentioning

confidence: 99%

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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

Shirey¹,

Badieyan²,

Sobrado

2013

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 61%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…The procedures for expression and purification of the recombinant proteins were essentially as previously described (6,16).…”

Section: Materials-escherichia Coli Top10 and Bl21(de3)-t1mentioning

confidence: 99%

See 2 more Smart Citations

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

Shirey¹,

Badieyan²,

Sobrado

2013

Journal of Biological Chemistry

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“…This is similar to the other flavindependent monooxygenases such as C 2 from A. baumannii (25), luciferase from Vibrio campbellii (38), RebH from Lechevalieria aerocolonigenes ATCC 39243 (42), ActVA from Streptomyces coelicolor (44), Baeyer-Villiger monooxygenase (45), N-hydroxylating hydroxylases (46,47), p-hydroxybenzoate hydroxylase (48), and 3-hydroxybenzoate-6-hydroxylase (49). Previous investigations of TftD detected a species with a high absorption around the 390-nm region during multiple turnover …”

Section: Discussionmentioning

confidence: 91%

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Pimviriyakul

Thotsaporn

Sucharitakul

et al. 2017

Journal of Biological Chemistry

108

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Edited by F. Peter GuengerichThe accumulation of chlorophenols (CPs) in the environment, due to their wide use as agrochemicals, has become a serious environmental problem. These organic halides can be degraded by aerobic microorganisms, where the initial steps of various biodegradation pathways include an oxidative dechlorinating process in which chloride is replaced by a hydroxyl substituent. Harnessing these dechlorinating processes could provide an opportunity for environmental remediation, but detailed catalytic mechanisms for these enzymes are not yet known. To close this gap, we now report transient kinetics and product analysis of the dechlorinating flavin-dependent monooxygenase, HadA, from the aerobic organism Ralstonia pickettii DTP0602, identifying several mechanistic properties that differ from other enzymes in the same class. We first overexpressed and purified HadA to homogeneity. Analyses of the products from single and multiple turnover reactions demonstrated that HadA prefers 4-CP and 2-CP over CPs with multiple substituents. Stopped-flow and rapid-quench flow experiments of HadA with 4-CP show the involvement of specific intermediates (C4a-hydroperoxy-FAD and C4a-hydroxy-FAD) in the reaction, define rate constants and the order of substrate binding, and demonstrate that the hydroxylation step occurs prior to chloride elimination. The data also identify the non-productive and productive paths of the HadA reactions and demonstrate that product formation is the rate-limiting step. This is the first elucidation of the kinetic mechanism of a two-component flavin-dependent monooxygenase that can catalyze oxidative dechlorination of various CPs, and as such it will serve as the basis for future investigation of enzyme variants that will be useful for applications in detoxifying chemicals hazardous to human health.

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Modular Oxime Formation by a trans‐AT Polyketide Synthase

et al. 2023

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Modular trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis‐AT counterparts, the trans‐AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on‐line by an unusual oxygenase‐containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site‐directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein‐protein interactions that support this chemistry. Overall, our work adds oxime‐forming machinery to the biomolecular toolbox available for trans‐AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides.

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Dual role of NADP(H) in the reaction of a flavin dependent N-hydroxylating monooxygenase

Cited by 34 publications

References 18 publications

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

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

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Modular Oxime Formation by a trans‐AT Polyketide Synthase

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