Literature summary for 2.4.2.28 extracted from

Siu, K.K.; Lee, J.E.; Sufrin, J.R.; Moffatt, B.A.; McMillan, M.; Cornell, K.A.; Isom, C.; Howell, P.L.
Molecular determinants of substrate specificity in plant 5-methylthioadenosine nucleosidases (2008), J. Mol. Biol., 378, 112-128.

Activating Compound

Activating Compound	Comment	Organism	Structure
additional information	imidazole buffer is the best buffer, whereas phosphate and HEPES buffers result in enzyme velocities that are only 50-60% of those for imidazole	Arabidopsis thaliana

Cloned(Commentary)

Cloned (Comment)	Organism
into the pET28a+ vector at the NdeI and HindIII sites, overexpression of the enzyme with a cleavable N-terminal His6 tag in Escherichia coli BL21 Codon+ cells	Arabidopsis thaliana

Crystallization (Commentary)

Crystallization (Comment)	Organism
MTAN1 in its apo form and in complex with formycin A and 5'-methylthiotubercidin to a resolution of 2.0 A, 1.9 A and 1.8 A, respectively. By hanging-drop, vapor-diffusion technique. Belongs to space group P21. The monomer of MTAN1 has a mixed alpha/beta structure composed of a twisted, ten-stranded mixed beta sheet flanked by seven alpha helices and two short 3(10) helices. In the MTAN1 crystals, the asymmetric unit contains two monomers, which interact via an interface involving the four loops beta2-beta3, beta4-alpha2, beta6-alpha2b and beta8-alpha4, and the alpha2, alpha2b, and alpha5 helices. Both polar and apolar residues are involved in hydrogen bond and van der Waals interactions	Arabidopsis thaliana
the intersubunit interface of MTAN is highly hydrophobic and is dominated by van der Waals and hydrophobic interactions	Escherichia coli

Crystallization (Comment)

Organism

MTAN1 in its apo form and in complex with formycin A and 5'-methylthiotubercidin to a resolution of 2.0 A, 1.9 A and 1.8 A, respectively. By hanging-drop, vapor-diffusion technique. Belongs to space group P21. The monomer of MTAN1 has a mixed alpha/beta structure composed of a twisted, ten-stranded mixed beta sheet flanked by seven alpha helices and two short 3(10) helices. In the MTAN1 crystals, the asymmetric unit contains two monomers, which interact via an interface involving the four loops beta2-beta3, beta4-alpha2, beta6-alpha2b and beta8-alpha4, and the alpha2, alpha2b, and alpha5 helices. Both polar and apolar residues are involved in hydrogen bond and van der Waals interactions

Arabidopsis thaliana

the intersubunit interface of MTAN is highly hydrophobic and is dominated by van der Waals and hydrophobic interactions

Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
F135L	can reduce steric hindrance at the end of the 5'-alkylthio binding subsite, so that longer 5'-substituents may be accommodated more easily in the MTAN2 active site	Arabidopsis thaliana
F148L	can reduce steric hindrance at the end of the 5'-alkylthio binding subsite	Arabidopsis thaliana
I132V	no significant structural change	Arabidopsis thaliana
I145V	no significant structural change	Arabidopsis thaliana
L168M	has a more extended side chain, may affect the binding of ligands at the 5'-alkylthio position	Arabidopsis thaliana
L181M	has a more extended side chain, may affect the binding of ligands at the 5'-alkylthio position	Arabidopsis thaliana

Inhibitors

Inhibitors	Comment	Organism
5'-methylthiotubercidin	combination of a more occluded active site in its open state and reduced ligand-induced conformational changes in the plant in contrast to Escherichia coli MTAN	Arabidopsis thaliana
5'-methylthiotubercidin	active site of apo-MTAN is more open, and greater conformational changes upon ligand binding in contrast to Arabidopsis MTAN1	Escherichia coli
Formycin A	-	Arabidopsis thaliana
Formycin A	-	Escherichia coli
additional information	Tris buffer severely inhibits the nucleosidase reactions; Tris buffer severely inhibits the nucleosidase reactions	Arabidopsis thaliana

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.0034	-	5'-methylthioadenosine	-	Arabidopsis thaliana
0.0071	-	5'-methylthioadenosine	-	Arabidopsis thaliana

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
27460	-	sequence analysis	Arabidopsis thaliana
28450	-	sequence analysis	Arabidopsis thaliana

Organism

Organism	UniProt	Comment	Textmining
Arabidopsis thaliana	Q7XA67	-	-
Arabidopsis thaliana	Q9T0I8	-	-
Escherichia coli	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
by centrifugation and on Ni-NTA column and by gel filtration	Arabidopsis thaliana
on Ni-NTA column	Arabidopsis thaliana

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.38	-	with adenosine as substrate	Arabidopsis thaliana
0.46	-	with 5'-choroadenosine as substrate	Arabidopsis thaliana
0.83	-	with 5'-choroadenosine as substrate	Arabidopsis thaliana
0.96	-	with S-adenosylhomocysteine as substrate	Arabidopsis thaliana
1.06	-	with 5'-iodoadenosine as substrate	Arabidopsis thaliana
1.36	-	with 5'-phenylthioadenosine as substrate	Arabidopsis thaliana
1.5	-	with 5'-butylthioadenosine as substrate	Arabidopsis thaliana
1.85	-	with 5'-isobutylthioadenosine as substrate	Arabidopsis thaliana
1.98	-	with 5'-isobutylthioadenosine as substrate	Arabidopsis thaliana
2.44	-	with 5'-phenylthioadenosine as substrate	Arabidopsis thaliana
2.6	-	with 5'-ethylthioadenosine as substrate	Arabidopsis thaliana
2.72	-	with 5'-butylthioadenosine as substrate	Arabidopsis thaliana
3.14	-	with 5'-iodoadenosine as substrate	Arabidopsis thaliana
4.42	-	with 5'-ethylthioadenosine as substrate	Arabidopsis thaliana
6.25	-	with 5'-methylthioadenosine as substrate	Arabidopsis thaliana
11.5	-	with 5'-methylthioadenosine as substrate	Arabidopsis thaliana

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
5'-butylthioadenosine + phosphate	24% relative activity	Arabidopsis thaliana	adenine + 5-butylthio-D-ribose 1-phosphate	-	?
5'-butylthioadenosine + phosphate	25% relative activity	Arabidopsis thaliana	adenine + 5-butylthio-D-ribose 1-phosphate	-	?
5'-chloroadenosine + phosphate	13% relative activity	Arabidopsis thaliana	adenine + 5-deoxy-5-chloro-D-ribose 1-phosphate	-	?
5'-chloroadenosine + phosphate	4% relative activity	Arabidopsis thaliana	adenine + 5-deoxy-5-chloro-D-ribose 1-phosphate	-	?
5'-ethylthioadenosine + phosphate	40% relative activity	Arabidopsis thaliana	adenine + 5-ethylthio-D-ribose 1-phosphate	-	?
5'-ethylthioadenosine + phosphate	42% relative activity	Arabidopsis thaliana	adenine + 5-ethylthio-D-ribose 1-phosphate	-	?
5'-iodoadenosine + phosphate	17% relative activity	Arabidopsis thaliana	adenine + 5-deoxy-5-iodo-D-ribose 1-phosphate	-	?
5'-iodoadenosine + phosphate	28% relative activity	Arabidopsis thaliana	adenine + 5-deoxy-5-iodo-D-ribose 1-phosphate	-	?
5'-isobutylthioadenosine + phosphate	32% relative activity	Arabidopsis thaliana	adenine + 5-isobutylthio-D-ribose 1-phosphate	-	?
5'-isobutylthioadenosine + phosphate	17% relative activity	Arabidopsis thaliana	adenine + 5-isobutylthio-D-ribose 1-phosphate	-	?
5'-methylthioadenosine + phosphate	100% relative activity	Arabidopsis thaliana	adenine + 5-methylthio-D-ribose 1-phosphate	-	?
5'-phenylthioadenosine + phosphate	39% relative activity	Arabidopsis thaliana	adenine + 5-phenylthio-D-ribose 1-phosphate	-	?
5'-phenylthioadenosine + phosphate	12% relative activity	Arabidopsis thaliana	adenine + 5-phenylthio-D-ribose 1-phosphate	-	?
adenosine + phosphate	6% relative activity	Arabidopsis thaliana	adenine + D-ribose 1-phosphate	-	?
additional information	the plant active site is likely less flexible, which may contribute to its reduced efficiency in binding larger substrates. The 5'-alkylthio binding subsite is more constricted in MTAN1	Arabidopsis thaliana	?	-	?
S-adenosylhomocysteine + phosphate	14% relative activity	Arabidopsis thaliana	?	-	?

Subunits

Subunits	Comment	Organism
dimer	crystallography	Escherichia coli
dimer	crystallography	Arabidopsis thaliana

Synonyms

Synonyms	Comment	Organism
5'-methylthioadenosine nucleosidase	-	Escherichia coli
5'-methylthioadenosine nucleosidase	-	Arabidopsis thaliana
MTAN	-	Escherichia coli
MTAN1	-	Arabidopsis thaliana
MTAN2	-	Arabidopsis thaliana
MTN1	-	Arabidopsis thaliana
MTN2	-	Arabidopsis thaliana

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
2	-	5'-methylthioadenosine	-	Arabidopsis thaliana
18.7	-	5'-methylthioadenosine	-	Arabidopsis thaliana

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	-	-	Arabidopsis thaliana
8	-	-	Arabidopsis thaliana

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
0.0083	-	5'-methylthiotubercidin	at 0.005 mM	Arabidopsis thaliana
0.048	-	Formycin A	at 0.05 mM	Arabidopsis thaliana

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
588	-	5'-methylthioadenosine	-	Arabidopsis thaliana
2600	-	5'-methylthioadenosine	-	Arabidopsis thaliana