Optimal electrostatic interactions between substrate and protein are essential for radical chemistry in ornithine 4,5-aminomutase

Makins, Caitlyn; Whitelaw, Douglas A.; McGregor, Michael; Petit, Alix; Mothersole, Robert G; Prosser, Kathleen E.; Wolthers, Kirsten R.

doi:10.1016/j.bbapap.2017.05.011

Cited by 4 publications

(2 citation statements)

References 22 publications

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“…The cloning, expression, and purification of 5,6-LAM (FN1863 and FN1862, KamD and KamE) and ornithine 4,5-aminomutase have been described. 27 A single transformed colony harboring either petKamBC, petKamB, petKamC, or petDAHDH was used to inoculate 100 mL of Luria Broth (LB) medium supplemented with 100 μg/mL ampicillin and 30 μg/mL chloramphenicol. The cultures were grown overnight at 37 °C at 200 rpm, and then 10 mL of the starter culture was used to inoculate 1 L of Terrific Broth also supplemented with 100 μg/ mL ampicillin and 30 μg/mL chloramphenicol.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…The cloning, expression, and purification of 5,6-LAM (FN1863 and FN1862, KamD and KamE) and ornithine 4,5-aminomutase have been described . A single transformed colony harboring either petKamBC, petKamB, petKamC, or petDAHDH was used to inoculate 100 mL of Luria Broth (LB) medium supplemented with 100 μg/mL ampicillin and 30 μg/mL chloramphenicol.…”

Section: Materials and Methodsmentioning

confidence: 99%

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Characterization of a Structurally Distinct ATP-Dependent Reactivating Factor of Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase

Darbyshire,

Wolthers

2024

Biochemistry

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Several anaerobic bacterial species, including the Gram-negative oral bacterium Fusobacterium nucleatum, ferment lysine to produce butyrate, acetate, and ammonia. The second step of the metabolic pathway�isomerization of β-L-lysine to erythro-3,5-diaminohexanoate�is catalyzed by the adenosylcobalamin (AdoCbl) and pyridoxal 5′-phosphate (PLP)-dependent enzyme, lysine 5,6-aminomutase (5,6-LAM). Similar to other AdoCbl-dependent enzymes, 5,6-LAM undergoes mechanismbased inactivation due to loss of the AdoCbl 5′-deoxyadenosyl moiety and oxidation of the cob(II)alamin intermediate to hydroxocob(III)alamin. Herein, we identified kamB and kamC, two genes responsible for ATP-dependent reactivation of 5,6-LAM. KamB and KamC, which are encoded upstream of the genes corresponding to α and β subunits of 5,6-LAM (kamD and kamE), co-purified following coexpression of the genes in Escherichia coli. KamBC exhibited a basal level of ATP-hydrolyzing activity that was increased 35% in a reaction mixture that facilitated 5,6-LAM turnover with β-L-lysine or D,L-lysine. Ultraviolet−visible (UV−vis) spectroscopic studies performed under anaerobic conditions revealed that KamBC in the presence of ATP/Mg 2+ increased the steady-state concentration of the cob(II)alamin intermediate in the presence of excess β-L-lysine. Using a coupled UV−visible spectroscopic assay, we show that KamBC is able to reactivate 5,6-LAM through exchange of the damaged hydroxocob(III)alamin for AdoCbl. KamBC is also specific for 5,6-LAM as it had no effect on the rate of substrate-induced inactivation of the homologue, ornithine 4,5-aminomutase. Based on sequence homology, KamBC is structurally distinct from previously characterized B12 chaperones and reactivases, and correspondingly adds to the list of proteins that have evolved to maintain the cellular activity of B12 enzymes.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: Materials and Methodsmentioning

confidence: 99%

Characterization of a Structurally Distinct ATP-Dependent Reactivating Factor of Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase

Darbyshire,

Wolthers

2024

Biochemistry

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Steady-state and pre-steady state kinetic analysis of ornithine 4,5-aminomutase

Darbyshire

Makins

Wolthers

2022

Methods in Enzymology

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Pyridoxal phosphate-dependent reactions in the biosynthesis of natural products

Ryan

2019

Nat. Prod. Rep.

106

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Covering: up to mid-2018Pyridoxal 5'-phosphate (PLP) is a versatile organic cofactor used to catalyze diverse reactions on amino acid, oxoacid, and amine substrates. Here we review the reactions catalyzed by PLP-dependent enzymes, highlighting enzymes reported in the natural product biosynthetic literature. We describe enzymes that catalyze transaminations, Claisen-like condensations, and β- and γ-eliminations and substitutions, along with epimerizations, decarboxylations, and transaldolations. Finally, we describe a newly reported group of O2-, PLP-dependent enzymes. Altogether, natural product biosynthesis showcases the incredible versatility of PLP-dependent transformations for building chemical complexity.

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Optimal electrostatic interactions between substrate and protein are essential for radical chemistry in ornithine 4,5-aminomutase

Cited by 4 publications

References 22 publications

Characterization of a Structurally Distinct ATP-Dependent Reactivating Factor of Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase

Characterization of a Structurally Distinct ATP-Dependent Reactivating Factor of Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase

Steady-state and pre-steady state kinetic analysis of ornithine 4,5-aminomutase

Pyridoxal phosphate-dependent reactions in the biosynthesis of natural products

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