Literature summary for 5.4.3.5 extracted from

Makins, C.; Whitelaw, D.A.; McGregor, M.; Petit, A.; Mothersole, R.G.; Prosser, K.E.; Wolthers, K.R.
Optimal electrostatic interactions between substrate and protein are essential for radical chemistry in ornithine 4,5-aminomutase (2017), Biochim. Biophys. Acta, 1865, 1077-1084 .

Search for more literature for 5.4.3.5

Cloned(Commentary)

Cloned (Comment)	Organism
genes oraS and oraE encode the alpha- and beta-subunit, respectively	Acetoanaerobium sticklandii

Protein Variants

Protein Variants	Comment	Organism
E81A	site-directed mutagenesis, inactive beta-subunit mutant	Acetoanaerobium sticklandii
E81D	site-directed mutagenesis, almost inactive beta-subunit mutant	Acetoanaerobium sticklandii
E81Q	site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type	Acetoanaerobium sticklandii
N226D	site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type	Acetoanaerobium sticklandii
R297K	site-directed mutagenesis, almost inactive beta-subunit mutant	Acetoanaerobium sticklandii
S162A	site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type	Acetoanaerobium sticklandii
Y160F	site-directed mutagenesis, almost inactive beta-subunit mutant	Acetoanaerobium sticklandii
Y187F	site-directed mutagenesis, the beta-subunit mutant shows highly reduced activity compared to wild-type	Acetoanaerobium sticklandii

Inhibitors

Inhibitors	Comment	Organism	Structure
2,4-diaminobutyrate	-	Acetoanaerobium sticklandii

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.029	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant S162A	Acetoanaerobium sticklandii
0.031	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant Y187F	Acetoanaerobium sticklandii
0.043	-	D-ornithine	pH 8.5, 25°C, recombinant wild-type enzyme	Acetoanaerobium sticklandii
0.12	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant N226D	Acetoanaerobium sticklandii
0.14	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant Y160F	Acetoanaerobium sticklandii
0.78	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant E81A	Acetoanaerobium sticklandii
0.87	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant E81Q	Acetoanaerobium sticklandii
4.9	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant R297K	Acetoanaerobium sticklandii

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
191400	-	-	Acetoanaerobium sticklandii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
D-ornithine	Acetoanaerobium sticklandii	-	(2R,4S)-2,4-diaminopentanoate	-	r
D-ornithine	Acetoanaerobium sticklandii DSM 519	-	(2R,4S)-2,4-diaminopentanoate	-	r

Organism

Organism	UniProt	Comment	Textmining
Acetoanaerobium sticklandii	E3PY95 AND E3PY96	subunits beta and alpha	-
Acetoanaerobium sticklandii DSM 519	E3PY95 AND E3PY96	subunits beta and alpha	-

Reaction

Reaction	Comment	Organism	Reaction ID
D-ornithine = (2R,4S)-2,4-diaminopentanoate	catalytic mechanism of ornithine 4,5-aminomutase. Substrate binding results in formation of a Schiff base between the terminal amino group of the substrate and the imine nitrogen of the PLP cofactor. Subsequent homolysis of the Co-C bond generates cob(II)alamin and the highly reactive 5'-deoxyadenosyl radical, which abstracts a hydrogen atom from the C4 of the substrate. The substrate radical intermediate then rearranges to the product-like radical intermediate via a proposed cyclic intermediate. Re-abstraction of a hydrogen atom from 5'-deoxyadenosine regenerates the 5'-deoxyadenosyl radical. Product release and recombination between cob(II)alamin and the 5'-deoxyadenosyl radical completes the catalytic cycle	Acetoanaerobium sticklandii	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
D-ornithine	-	Acetoanaerobium sticklandii	(2R,4S)-2,4-diaminopentanoate	-	r
D-ornithine	-	Acetoanaerobium sticklandii DSM 519	(2R,4S)-2,4-diaminopentanoate	-	r
additional information	enzyme OAM is highly specific for D-ornithine, which forms non-covalent interactions with R297 and E81 through its alpha-amine and alpha-carboxylate groups. Neither wild type enzyme OAM nor any of the R297 or E81 variants are active towards the larger substrate	Acetoanaerobium sticklandii	?	-	?
additional information	enzyme OAM is highly specific for D-ornithine, which forms non-covalent interactions with R297 and E81 through its alpha-amine and alpha-carboxylate groups. Neither wild type enzyme OAM nor any of the R297 or E81 variants are active towards the larger substrate	Acetoanaerobium sticklandii DSM 519	?	-	?

Subunits

Subunits	Comment	Organism
heterotetramer	alpha2beta2	Acetoanaerobium sticklandii

Synonyms

Synonyms	Comment	Organism
OAM	-	Acetoanaerobium sticklandii
ornithine 4,5-aminomutase	-	Acetoanaerobium sticklandii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Acetoanaerobium sticklandii

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.002	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant E81D	Acetoanaerobium sticklandii
0.013	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant E81A	Acetoanaerobium sticklandii
0.027	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant Y160F	Acetoanaerobium sticklandii
0.029	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant E81Q	Acetoanaerobium sticklandii
0.068	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant R297K	Acetoanaerobium sticklandii
0.12	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant Y187F	Acetoanaerobium sticklandii
0.37	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant S162A	Acetoanaerobium sticklandii
0.38	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant N226D	Acetoanaerobium sticklandii
8.2	-	D-ornithine	pH 8.5, 25°C, recombinant wild-type enzyme	Acetoanaerobium sticklandii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8.5	-	assay at	Acetoanaerobium sticklandii

Cofactor

Cofactor	Comment	Organism	Structure
5'-deoxyadenosylcobalamin	-	Acetoanaerobium sticklandii
additional information	ornithine 4,5-aminomutase is a 5'-deoxyadenosylcobalamin and pyridoxal 5'-phosphate co-dependent radical enzyme. The conserved residues include S162 and N226, which form hydrogen bonds to the pyridine nitrogen and the phenolic group of pyridoxal 5'-phosphate, respectively. Other residues include Y187 and Y160, which flank the pyridine ring. The former aromatic side chain also forms a hydrogen bond to the pyridoxal 5'-phosphate phosphate, while the latter side chain forms a hydrogen bond to AdoCbl in a modelled closed conformation of the enzyme	Acetoanaerobium sticklandii
pyridoxal 5'-phosphate	the protonation state of the pyridoxal 5'-phosphate cofactor has less of a role in radical-mediated chemistry compared to electrostatic interactions between the substrate and protein. Binding structure analysis and comparison with lysien 5,6-aminomutase, EC 5.4.3.3	Acetoanaerobium sticklandii

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
0.0046	-	2,4-diaminobutyrate	pH 8.5, 25°C, recombinant wild-type enzyme	Acetoanaerobium sticklandii
0.032	-	2,4-diaminobutyrate	pH 8.5, 25°C, recombinant mutant Y187F	Acetoanaerobium sticklandii
0.1	-	2,4-diaminobutyrate	pH 8.5, 25°C, recombinant mutant N226D	Acetoanaerobium sticklandii
0.27	-	2,4-diaminobutyrate	pH 8.5, 25°C, recombinant mutant S162A	Acetoanaerobium sticklandii
2	-	2,4-diaminobutyrate	pH 8.5, 25°C, recombinant mutant Y160F	Acetoanaerobium sticklandii

General Information

General Information	Comment	Organism
malfunction	conservative substitutions of the residues that form salt bridges to the alpha-carboxylate (R297) or the alpha-amine (E81) of D-ornithine results in a 300-600fold reduction in catalytic turnover and a more pronounced 1000 to 14000fold decrease in catalytic efficiency compared to wild-type. Mutating residues that solely interact with the pyridoxal 5'-phosphate cofactor leads to more modest decreases (10-60fold) in kcat and kcat/Km. All but one variant (S162A) elicite an increase in the kinetic isotope effect on kcat and kcat/Km with D,L-ornithine-3,3,4,4,5,5-d6 as the substrate, which indicates that hydrogen atom abstraction is more rate determining. The substitutions decrease the extent of CoeC bond homolysis, they do not affect the structural integrity of the active site	Acetoanaerobium sticklandii
additional information	ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii is an adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes a 1,2-amino shift of the delta-amino group of Dornithine to form 2S,4R-diaminopentanoate, interconverting D-ornithine and 2S,4R-diaminopentanoate. The reaction occurs via a radical-based mechanism whereby a PLP-bound substrate radical undergoes intramolecular isomerization via an azacyclopropylcarbinyl radical intermediate. Analysis of the catalytic role of active site residues that form non-covalent interactions with PLP and/or substrate, D-ornithine, and kinetic analysis, overview. Residues that form salt bridges to the alpha-carboxylate (R297) or the alpha-amine (E81) of D-ornithine are most critical for the enzyme activity. The protonation state of the pyridoxal 5'-phosphate cofactor has less of a role in radical-mediated chemistry compared to electrostatic interactions between the substrate and protein. Active site of OAM (PDB ID 3KOZ) with bound substrate D-ornithine in the modelled closed conformation, overview	Acetoanaerobium sticklandii

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
0.014	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant R297K	Acetoanaerobium sticklandii
0.016	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant E81A	Acetoanaerobium sticklandii
0.19	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant Y160F	Acetoanaerobium sticklandii
0.33	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant E81Q	Acetoanaerobium sticklandii
3.2	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant N226D	Acetoanaerobium sticklandii
3.9	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant Y187F	Acetoanaerobium sticklandii
13	-	D-ornithine	pH 8.5, 25°C, recombinant beta-subunit mutant S162A	Acetoanaerobium sticklandii
190	-	D-ornithine	pH 8.5, 25°C, recombinant wild-type enzyme	Acetoanaerobium sticklandii

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Release 2023.1 (January 2023)