Arg279 is the key regulator of coenzyme selectivity in the flavin-dependent ornithine monooxygenase SidA

Robinson, Reeder M.; Franceschini, S.; Fedkenheuer, M.; Rodrı́guez, P.; Ellerbrock, Jacob; Romero, Elvira; Echandi, Maria Paulina; Campo, Julia S. Martín del; Sobrado, Pablo

doi:10.1016/j.bbapap.2014.02.005

Cited by 16 publications

(33 citation statements)

References 26 publications

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“…63–65 The NMOs EtcB, PvdA, SidA, and CchB are specific for NADPH over NADH in this reduction reaction. 45,48,54,66,67 Next, oxygen is proposed to diffuse from the bulk solvent into the enzyme active site housing the flavin cofactor through enzyme multichannels. 68 A one-electron transfer reaction from 14 to molecular oxygen results in a radical pair consisting of flavin semiquinone ( 15 ) and superoxide anion.…”

Section: N–o Bond Forming Enzymesmentioning

confidence: 99%

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

et al. 2017

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Natural products that contain functional groups with heteroatom-heteroatom linkages (X–X, where X = N, O, S, and P) are a small yet intriguing group of metabolites. The reactivity and diversity of these structural motifs has captured the interest of synthetic and biological chemists alike. Functional groups containing X–X bonds are found in all major classes of natural products and often impart significant biological activity. This review presents our current understanding of the biosynthetic logic and enzymatic chemistry involved in the construction of X–X bond containing functional groups within natural products. Elucidating and characterizing biosynthetic pathways that generate X–X bonds could both provide tools for biocatalysis and synthetic biology, as well as guide efforts to uncover new natural products containing these structural features.

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Section: N–o Bond Forming Enzymesmentioning

confidence: 99%

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

et al. 2017

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“…In the presence of D-Lys the k cat /K NADPH value was ϳ4-fold higher. Thus, NbtG does not have significant coen- zyme selectivity, in contrast to other Class B flavin monooxygenases, which are all highly selective for NADPH (1,(37)(38)(39). Lysine Hydroxylation-NbtG displayed significant activity as measured by the oxygen consumption assay.…”

Section: Purification Of Wild-type Nbtg and Variants-wild-typementioning

confidence: 99%

“…In SidA the position of the NADP ϩ adenine-ribose moiety is stabilized by a network of interactions that also involve the phosphate. In particular, the ribose 3Ј-OH is H-bonded to both the side chain and the peptidic nitrogen of Ser-254, whereas the phosphate interacts with Ser-325 and with Arg-279 (37). In NbtG, Arg-279 is substituted by Pro-238, and Ser-325 is substituted by Asp-277, whose longer side chain is likely to interact with the ribose rather than with the phosphate.…”

Section: Purification Of Wild-type Nbtg and Variants-wild-typementioning

confidence: 99%

An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

Binda

Robinson

Campo

et al. 2015

Journal of Biological Chemistry

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Background: Flavin-dependent lysine monooxygenases are involved in siderophore biosynthesis and are promising bacterial drug targets. Results: Biochemical and structural characterization of lysine monooxygenase from Nocardia farcinica (NbtG) is presented. Conclusion: An unprecedented domain conformation blocks the proper binding of NAD(P)H in the active site, which explains the high level of uncoupling observed in NbtG. Significance: The structural and biochemical data should aid in drug design.

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“…SidA is specific for Orn and is almost 100% coupled, meaning that for every one molecule of oxygen consumed, a nearly stoichiometric amount of HO-N 5 -Orn is formed [10, 11, 18]. The strict substrate specificity of SidA differs from related members of Class B monooxygenases, which can hydroxylate a large number of substrates [19, 20].…”

Section: Introductionmentioning

confidence: 99%

Contribution to catalysis of ornithine binding residues in ornithine N5-monooxygenase

Robinson

Qureshi

Klancher

et al. 2015

Archives of Biochemistry and Biophysics

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The SidA ornithine N5-monooxygenase from Aspergillus fumigatus is a flavin monooxygenase that catalyzes the NADPH-dependent hydroxylation of ornithine. Herein we report a mutagenesis study targeting four residues that contact ornithine in crystal structures of SidA: Lys107, Asn293, Asn323, and Ser469. Mutation of Lys107 to Ala abolishes activity as measured in steady-state oxygen consumption and ornithine hydroxylation assays, indicating that the ionic interaction of Lys107 with the carboxylate of ornithine is essential for catalysis. Mutation of Asn293, Asn323, or Ser469 individually to Ala results in >14-fold increases in Km values for ornithine. Asn323 to Ala also increases the rate constant for flavin reduction by NADPH by 18-fold. Asn323 is unique among the four ornithine binding residues in that it also interacts with NADPH by forming a hydrogen bond with the nicotinamide ribose. The crystal structure of N323A complexed with NADP(+) and ornithine shows that the nicontinamide riboside group of NADP is disordered. This result suggests that the increase in flavin reduction rate results from an increase in conformational space available to the enzyme-bound NADP(H). Asn323 thus facilitates ornithine binding at the expense of hindering flavin reduction, which demonstrates the delicate balance that exists within protein-ligand interaction networks in enzyme active sites.

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Arg279 is the key regulator of coenzyme selectivity in the flavin-dependent ornithine monooxygenase SidA

Cited by 16 publications

References 26 publications

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

Contribution to catalysis of ornithine binding residues in ornithine N5-monooxygenase

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