Báo cáo khoa học: Lid L11 của glutamine amidotransferase domain trong CTP synthase điều hòa GTP allosteric kích hoạt hoạt tính glutaminase

Lid L11 of the glutamine amidotransferase domain

of CTP synthase mediates allosteric GTP activation

of glutaminase activity

Martin Willemoe

¨s

, Anne Mølgaard

1,2

, Eva Johansson

1,3

and Jan Martinussen

1 Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Denmark

2 Center for Biological Sequence Analysis, BioCentrum-DTU, The Technical University of Denmark, Lyngby, Denmark

3 European Synchrotron Radiation Facility, Grenoble Cedex, France

4 Microbial Physiology and Genetics, BioCentrum-DTU, The Technical University of Denmark, Lyngby, Denmark

CTP synthase (EC 6.3.4.2) catalyses the synthesis of

CTP by amination of the 4-position of the pyrimidine

moiety of UTP [1]. The enzyme is a homotetramer,

each subunit consisting of two domains; the

N-terminal synthase domain where CTP formation

takes place and the C-terminal GATase domain

Keywords

allosteric regulation; flexible loop;

Lactococcus lactis; nucleotide metabolism;

oxy-anion hole

Correspondence

M. Willemoe

¨s, Centre for Crystallographic

Studies, Department of Chemistry,

University of Copenhagen,

Universitetsparken 5, Copenhagen

DK-2100 Ø, Denmark

Fax: +45 3532 0299

Tel: +45 3532 0239

E-mail: martin@ccs.ki.ku.dk

(Received 25 November 2004, revised 8

December 2004, accepted 10 December

2004)

doi:10.1111/j.1742-4658.2004.04525.x

GTP is an allosteric activator of CTP synthase and acts to increase the k

cat

for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in

part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that

takes place in the glutamine amidotransferase class I (GATase) domain of

CTP synthase. In the GATase domain of the recently published structures

of the Escherichia coli and Thermus thermophilus CTP synthases a loop

region immediately proceeding amino acid residues forming the oxy-anion

hole and named lid L11 is shown for the latter enzyme to be ﬂexible and

change position depending on the presence or absence of glutamine in the

glutamine binding site. Displacement or rearrangement of this loop may

provide a means for the suggested role of allosteric activation by GTP to

optimize the oxy-anion hole for glutamine hydrolysis. Arg359, Gly360 and

Glu362 of the Lactococcus lactis enzyme are highly conserved residues in lid

L11 and we have analyzed their possible role in GTP activation. Characteri-

zation of the mutant enzymes R359M, R359P, G360A and G360P indicated

that both Arg359 and Gly360 are involved in the allosteric response to GTP

binding whereas the E362Q enzyme behaved like wild-type enzyme. Apart

from the G360A enzyme, the results from kinetic analysis of the enzymes

altered at position 359 and 360 showed a 10- to 50-fold decrease in GTP

activation of glutamine dependent CTP synthesis and concomitant four- to

10-fold increases in K

for GTP. The R359M, R359P and G360P also

showed no GTP activation of the uncoupled glutaminase reaction whereas

the G360A enzyme was about twofold more active than wild-type enzyme.

The elevated K

for GTP and reduced GTP activation of CTP synthesis of

the mutant enzymes are in agreement with a predicted interaction of bound

GTP with lid L11 and indicate that the GTP activation of glutamine

dependent CTP synthesis may be explained by structural rearrangements

around the oxy-anion hole of the GATase domain.

Abbreviations

ATPcS, adenosine 5¢-[c-thio]triphosphate; GATase, class I glutamine amidotransferase; PDB ID, Protein Data Bank entry.

856 FEBS Journal 272 (2005) 856–864 ª2005 FEBS

responsible for the hydrolysis of glutamine [2]. In addi-

tion the enzyme has a site for GTP that acts as an

allosteric activator. The reaction proceeds via the

intermediate 4-phosphoryl UTP generated by ATP-

dependent phosphorylation [3,4]. The amino group

transferred to this activated intermediate is either

obtained from glutamine hydrolysis or ammonia pre-

sent in the solution [1]. The rate of the glutamine

dependent CTP synthesis reaction is greatly stimulated

by the allosteric binding of GTP and this activation

has been the focus of several reports [5–9]. CTP syn-

thase is the only member of the family of GATase

domain harboring enzymes where an allosteric effector

regulates the glutaminase activity. However, only

recently has it been possible functionally to associate

individual amino acid residues, Thr-431 and Arg433 in

Lactococcus lactis CTP synthase [10] and Arg429 in

the Escherichia coli enzyme [11], with this property of

the glutamine dependent CTP formation. For the

L. lactis enzyme, allosteric binding of GTP acts in syn-

ergy with the 4-phoshorylated UTP intermediate to

activate glutamine dependent CTP synthesis [12]. In

addition, GTP appears to promote channeling of NH

derived from glutamine hydrolysis to the synthase site

[9]. The nature of the formation of this channel and

the role GTP may play in forming this, has recently

been suggested from analysis of the crystal structure of

the E. coli enzyme. Based on structural homology to

GTP binding enzymes, GTP was modeled into the

apo-structure of CTP synthase and was suggested to

bind in a cleft between the GATase domain and the

synthase domain [13] (Fig. 1A). Recently, an E. coli

mutant enzyme altered at position 109 (L109A) that is

impaired in the coupling between ammonia derived

from glutamine hydrolysis and CTP formation due to

a constricted or leaky ammonia tunnel [14], has provi-

ded evidence for this putative binding site for GTP

and the role GTP plays in coupling of glutamine

hydrolysis and CTP synthesis [13].

In a work on inhibition of the E. coli enzyme by

the analogue glutamate c-semialdehyde that mimics

intermediates of glutamine hydrolysis, Bearne and

coworkers [6] suggested that the oxy-anion hole is a

target for GTP regulation of the glutaminase activity

of CTP synthase. A comparison of part of the CTP

synthase GATase domain (Fig. 1B) with other known

structures of enzymes incorporating this catalytic

domain (Fig. 1C) showed that whereas the central

beta-sheet and the alpha helices superimpose well, the

loop regions differ, including the region that proceeds

from the oxy-anion hole into a common alpha helix.

This region with a variable loop structure has been

named lid L11 in the E. coli CTP synthase structure

[13] and we will use this nomenclature when referring

to this structural region in the following. The apo-

structure of the E. coli enzyme [13] and the structure

of the Thermus thermophilus CTP synthase [15] in a

complex with glutamine are virtually superimposable.

However, the apo-structure and the structure in com-

plex with sulfate ions of the T. thermophilus enzyme

deviates signiﬁcantly in the Ca-chain of this region

(Fig. 1B). In fact some residues of the lid L11 were

not detected in the electron density maps [15] indica-

ting that this region is highly ﬂexible and can adapt

different structures.

Even though there is currently no detailed structural

information that explains the activation of glutamine

hydrolysis in GATase domains upon binding of amino

acceptor substrates [16], it is likely to involve small

movements or re-organizations of the environment

around the oxy-anion hole [6,17–19]. The importance

of the loop corresponding to lid L11 has previously

been demonstrated for the E. coli carbamoyl phos-

phate synthase that has a cysteine residue (Cys248)

within this loop. When this residue is labeled with

N-ethylmaleimide [20] or changed to more bulky resi-

dues (Arg, Asp, Phe and Trp) [17], it results in

uncoupling of glutaminase activity from carbamoyl

phosphate synthesis. In the case of the C248D enzyme,

glutamine-dependent carbamoyl phosphate synthesis is

completely abolished and large increases in glutami-

nase activity and K

for glutamine are observed [17].

The dramatic effect on glutamine hydrolysis and coup-

ling of the reactions can be explained by local changes

in the structure of the region corresponding to lid

L11 [21].

Residues 354–357 of the L. lactis enzyme forms

part of the oxy-anion hole and homologues residues

are found with little variation in all known GATase

domains [22]. The corresponding residues from the

GATase domain of E. coli CTP synthase [23] were

previously subjected to mutational analysis. One

mutant enzyme where Gly352 was changed to a pro-

lyl (Gly355 in the L. lactis enzyme) had lost detect-

able glutamine-dependent CTP synthase activity and

could not be labeled by [

C]6-diazo-5-oxonorleucine,

but was still active with NH

Cl as a substrate. The

result from this study may be interpreted in terms of

the recent CTP synthase structure, as this residue is

positioned at a pivotal point at which the preceding

rigid part of the oxy-anion hole connects with the ﬂexi-

ble lid L11 (Fig. 1B). Flexibility or displacement of lid

L11 may be a prerequisite for the activation of the

glutaminase activity hampered by a glycine to a proline

substitution at position 352 (355 in the L. lactis

enzyme).

M. Willemoe

¨set al. GTP activation of CTP synthase

FEBS Journal 272 (2005) 856–864 ª2005 FEBS 857

Taken together, the above observations all point to

the oxy-anion hole and its immediate surroundings as

a target for regulation of GATases. This prompted us

to investigate whether highly conserved amino acid

residues surrounding the oxy-anion hole plays a role in

GTP activation of glutamine hydrolysis by the L. lactis

CTP synthase. We chose to analyze by site-directed

mutagenesis the role of the highly conserved residues

Arg359, Gly360 and Glu362 (Fig. 1D), that are part in

lid L11. Arg359 was changed to methionyl to maintain

the hydrophobic part of the arginyl side chain while

deleting the guanidinium group. The Gly360 to alanyl

and Glu362 to glutaminyl substitutions are both

allowed for at these positions (Fig. 1D), but do reduce

the backbone ﬂexibility or delete the charge of the side

chain, respectively. In addition, we replaced Arg359

and Gly360 with a prolyl to test the importance of the

ﬂexibility of lid L11 based on the assumption that a

prolyl would restrict the ﬂexibility of the Ca-chain at

these positions.

Fig. 1. The structure and role of lid L11 in CTP synthase. (A) The proposed binding of GTP modelled into the apo-structure of the E. coli CTP

synthase by Endrizzi et al. [13]. The residues are numbered according to the E. coli enzyme while numbers in subscript are according to

the L. lactis sequence. Shown are: residues 346–380 including the catalytic Cys379 of the GATase domain, residues 51–55 and residues

104–110 of the synthase domain. (B) Comparison of the structures for T. thermophilus CTP synthase (residues 358–392) in complex with

glutamine (PDB ID; 1VCO, light grey) or sulfate (PDB ID; 1VCN, dark grey). In the latter structure residues 365 and 366 (corresponding to

356 and 357 in the L. lactis enzyme) are missing from the structure as indicated by the dotted line. The residues of lid L11 are numbered

according to the T. thermophilus sequence while numbers in subscript are according to the L. lactis sequence. The side chain of the catalytic

Cys391 is shown for orientation. (C) Comparison of the oxy-anion hole and surroundings of GATase domains from CTP synthase (residues

346–380, PDP ID; 1S1M), carbamoyl phosphate synthase small domain (residues 235–270, PDB ID; 1BXR), GMP synthase (residues 53–87,

PDB ID; 1GPM) and anthranilate synthase (residues 48–85, PDB ID; 1QDL) and imidazole glycerol phosphate synthase (residues 47–87, PDB

ID; 1JVN).The oxy-anion hole and the loops corresponding to L11 in CTP synthase are encircled. (A), (B), and (C) were prepared with

MOLSCRIPT [30] and RASTER3D[31]. (D) A sequence logo [32] was generated of the sequence region of interest (L. lactis CTP synthase num-

bering), based on a BLAST [33] alignment of 43 full-length sequences annotated as CTP synthase. The height of each residue is proportional

to its frequency, and the most common residue is on top at each position. The total height of each position is adjusted to signify the extent

of conservation at that position [34].

GTP activation of CTP synthase M. Willemoe

¨set al.

858 FEBS Journal 272 (2005) 856–864 ª2005 FEBS

Báo cáo khoa học: Lid L11 of the glutamine amidotransferase domain of CTP synthase mediates allosteric GTP activation of glutaminase activity

Chủ đề:

Tài liệu liên quan

Tài liêu mới

AI tóm tắt

Giới thiệu tài liệu

Đối tượng sử dụng

Từ khoá chính

Nội dung tóm tắt

Hỗ trợ

Phương thức thanh toán

Theo dõi chúng tôi