Biomedical Engineering Reference
In-Depth Information
the evolutionary history of Y synthases. The
truD
and
truA
superfamilies are highly
divergent from all other Y synthases, whereas
rsuA
and
rluA
share the highest degree
of homology (Koonin
1996
). Although the superfamilies display sequence diversity,
structural comparisons of proteins from each superfamily reveal that all Y synthases
share a core consisting of b-sheets and several a-helices, which flank a cleft harbor-
ing a catalytically essential aspartate residue (Hoang and Ferre-D'Amare
2001
) .
Most Y synthases, with the exception of the archael and eukaryotic
truB
homo-
logues, are capable of selecting target uridine residues without auxiliary guide
RNAs, thus acting as stand-alone enzymes (Ofengand
2002
) . The Y synthases of the
archael and eukaryotic
truB
family, including Cbf5, have evolved a specific RNA-
binding domain, known as a
p
seudo
u
ridine synthase and
a
rcheosine-transglycosy-
lases (PUA) domain (Aravind and Koonin
1999
), that has paramount importance for
the function of these enzymes in the isomerization of substrate RNA.
13.2.2.3
Mechanism of
Y
Synthesis
All Y synthases convert uridine to Y through a “base-flipping” mechanism that
promotes detachment and rotation of the uridine base, a process coordinated by a
critical aspartate residue in the active site of the Y synthase (Fig.
13.1
) (Hoang and
Ferre-D'Amare
2001
). Mutagenesis studies combined with structural analyses indi-
cate that the aspartate residue is dispensable for the structural integrity of the enzyme
active site but is absolutely essential for enzymatic activity (Del Campo et al.
2001
;
Hoang et al.
2005
; Huang et al.
1998
; Ramamurthy et al.
1999
; Raychaudhuri et al.
1999
). This catalytic aspartate uridine faces the uridine at the site of modification
and the Y synthase accesses the target uridine by “flipping” the uridine residue
away from the RNA helical stack. This “base-flipping” mechanism was first demon-
strated in
truB
-mediated isomerization of uridine at position 55 within the tRNA
T-loop. More precisely, the uridine at the site of pseudouridylation together with the
two adjacent residues are “flipped out” from the interior to the exterior of the helical
stack, thereby becoming available to the active site of the Y synthase (Hoang and
Ferre-D'Amare
2001
). Importantly, a conserved histidine residue located near the
catalytic aspartate mediates the stabilization of the Y synthase-RNA complex and
facilitates the spatial rearrangement of the substrate uridine (Gu et al.
1998,
1999
;
Hoang and Ferre-D'Amare
2001
) . The “base- fl ipping” mechanism is also performed
by DNA methyltransferases, thus suggesting a conserved role for this process in
modifying nucleic acids (Klimasauskas et al.
1994
) .
Although the detailed kinetic and catalytic mechanism(s) responsible for Y con-
version remain only partially understood, it is known that the reaction must proceed
through a series of events that include cleavage of the
N
-glycosyl link, rotation of the
detached uracil base, and formation of a
C
-glycosyl bond to reconnect the uracil base
to the ribose (Spedaliere et al.
2004
) (Fig.
13.1
). Two models describing the molecu-
lar events underlying the pseudouridylation reaction have been proposed. The first
involves a Michael-type attack by the catalytic aspartate on the carbon at position six
(C6) of the uracil base. According to the “Michael mechanism,” once the glycosidic
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