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PDBsum entry 2qii
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References listed in PDB file
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Key reference
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Title
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Glutamate versus glutamine exchange swaps substrate selectivity in tRNA-Guanine transglycosylase: insight into the regulation of substrate selectivity by kinetic and crystallographic studies.
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Authors
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N.Tidten,
B.Stengl,
A.Heine,
G.A.Garcia,
G.Klebe,
K.Reuter.
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Ref.
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J Mol Biol, 2007,
374,
764-776.
[DOI no: ]
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PubMed id
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Abstract
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Bacterial tRNA-guanine transglycosylase (Tgt) catalyses the exchange of guanine
in the wobble position of particular tRNAs by the modified base preQ(1). In
vitro, however, the enzyme is also able to insert the immediate biosynthetic
precursor, preQ(0), into those tRNAs. This substrate promiscuity is based on a
peptide switch in the active site, gated by the general acid/base Glu235. The
switch alters the properties of the binding pocket to allow either the
accommodation of guanine or preQ(1). The peptide conformer recognising guanine,
however, is also able to bind preQ(0). To investigate selectivity regulation,
kinetic data for Zymomonas mobilis Tgt were recorded. They show that selectivity
in favour of the actual substrate preQ(1) over preQ(0) is not achieved by a
difference in affinity but via a higher turnover rate. Moreover, a
Tgt(Glu235Gln) variant was constructed. The mutation was intended to stabilise
the peptide switch in the conformation favouring guanine and preQ(0) binding.
Kinetic characterisation of the mutated enzyme revealed that the Glu235Gln
exchange has, with respect to all substrate bases, no significant influence on
k(cat). In contrast, K(M)(preQ(1)) is drastically increased, while K(M)(preQ(0))
seems to be decreased. Hence, regarding k(cat)/K(M) as an indicator for
catalytic efficiency, selectivity of Tgt in favour of preQ(1) is abolished or
even inverted in favour of preQ(0) for Tgt(Glu235Gln). Crystal structures of the
mutated enzyme confirm that the mutation strongly favours the binding pocket
conformation required for the accommodation of guanine and preQ(0). The way this
is achieved, however, significantly differs from that predicted based on crystal
structures of wild-type Tgt.
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Figure 1.
Fig. 1. Queuine modification pathway. GTP, guanosine
triphosphate AdoMet, S-adenosylmethionine; B[12], coenzyme
B[12]; oQ, epoxyqueuine.
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Figure 2.
Fig. 2. Base exchange mechanism in bacterial Tgt.
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The above figures are
reprinted
from an Open Access publication published by Elsevier:
J Mol Biol
(2007,
374,
764-776)
copyright 2007.
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