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1.
Crystal structure of human AKT1 with an allosteric inhibitor reveals a new mode of kinase inhibition
AKT1 () is a member of the serine/threonine AGC protein kinase family involved in cellular metabolism, growth, proliferation and survival. The three human AKT isozymes are highly homologous multi-domain proteins with both overlapping and distinct cellular functions. Dysregulation of the AKT pathway has been identified in multiple human cancers. Several clinical trials are in progress to test the efficacy of AKT pathway inhibitors in treating cancer. Recently, a series of AKT isozyme-selective allosteric inhibitors have been reported. They require the presence of both the pleckstrin-homology (PH) and kinase domains of AKT, but their binding mode has not yet been elucidated. We present here a 2.7 Å resolution co-crystal structure of human AKT1 containing both the PH and kinase domains with a selective allosteric inhibitor bound in the interface. The structure reveals the interactions between the PH and kinase domains, as well as the critical amino residues that mediate binding of the inhibitor to AKT1. Our work also reveals an intricate balance in the enzymatic regulation of AKT, where the PH domain appears to lock the kinase in an inactive conformation and the kinase domain disrupts the phospholipid binding site of the PH domain. This information advances our knowledge in AKT1 structure and regulation, thereby providing a structural foundation for interpreting the effects of different classes of AKT inhibitors and designing selective ones. NP_005154.2相似文献
2.
Kausiki Datta Neil P. Johnson Vince J. LiCata Peter H. von Hippel 《The Journal of biological chemistry》2009,284(25):17180-17193
In addition to their capacity for template-directed 5′ → 3′ DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3′ → 5′ exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3′ terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis.Escherichia coli DNA polymerase (DNAP)2 I is a single subunit polymerase that is organized into three functional domains: an N-terminal domain that is associated with 5′ → 3′ exonuclease activity, an intermediate domain that carries the 3′ → 5′ proofreading activity, and a C-terminal domain that is associated with the 5′ → 3′ template-directed polymerization activity. An important role of DNAP I is to remove the RNA primers of the Okazaki fragments formed during lagging strand DNA synthesis in E. coli replication and to fill in the resulting gaps by template-directed DNA synthesis (1). An N-terminal deletion mutant of DNAP I, known as the “large fragment” or Klenow form of the enzyme, contains only the polymerase (pol) and the 3′ → 5′ exonuclease (exo) domains. The Klenow polymerase has served and continues to serve as an excellent model system for isolating and defining general structure-function relationships in polymerases and in the supporting machinery of DNA replication.The main function of the 3′ → 5′ exonuclease activity of DNAP I is to remove misincorporated nucleotide residues from the 3′-end of the primer (2), thus contributing significantly to the overall fidelity of DNA replication (3). Contrary to initial expectations, crystallographic studies showed that the pol and exo active sites are quite far apart in replication polymerases, about 30 Å in Klenow (4). As a consequence, the ability of polymerases to “shuttle” the 3′-end of the primer strand efficiently between the pol and the exo sites in order to rectify misincorporation events during polymerization is critical to maintaining the overall accuracy of template-directed replication. Elucidation of the mechanisms of this shuttling and determination of the factors that control the rates (and equilibria) of the active site switching reaction will certainly increase our understanding of fidelity control by DNA polymerases.An early crystallographic study of the Klenow polymerase complexed with fully paired primer-template (P/T) DNA revealed that 3–4 nt of the 3′-primer terminus had been unwound from the template stand and partitioned into the exo site and that an extended single-stranded DNA (ssDNA) binding pocket of the exo site appeared to make position-specific hydrophobic contacts with the unstacked bases at the 3′-end of the primer (4). A separate crystallographic study of an editing complex confirmed that an ssDNA fragment 4 nt in length was bound at the exo site in the same conformation as seen for the single-stranded 3′-primer sequence unwound from P/T DNA (5). A structure of Klenow polymerase with the DNA bound at the pol site has not yet been reported, although such structures have been obtained for other homologous polymerases, including Klentaq (the “large fragment” of Thermus aquaticus (Taq) DNAP), Bacillus stearothermophilus (Bst) “large fragment” polymerase, and the T7 DNAP (6–8), all of which are members of the polymerase family that includes Klenow.The amino acid residues involved in the binding of DNA at the pol site in these polymerases (determined from co-crystal structures) and those of Klenow (determined by site-directed mutagenesis studies (9, 10)) are highly conserved, suggesting that a similar DNA binding mode at the pol site may apply to all of the DNAP I polymerases. The crystal structure of Klenow revealed that the polymerization domain has a shape reminiscent of a right hand in which the palm, fingers, and thumb domains form the DNA-binding crevice. Structural studies with various DNAP I polymerases in the presence of P/T DNA constructs yielded an “open” binary complex, whereas the addition of the next correct dNTP (as a chain-terminating dideoxy-NTP) resulted in the formation of a catalytically competent “closed” ternary complex (6–8). In the latter complex, the 3′-primer terminus was base-paired with the template DNA, and the templating base was poised for incorporation of the next correct nucleotide. These structures showed that the conformation of the DNA primer terminus bound at the pol site is markedly different from that of the “frayed open” primer observed at the exo site in Klenow (4, 5).Although crystallographic studies have provided a wealth of information about the conformations of the DNA substrates bound at the active sites of DNAP, replication itself is a dynamic process (reviewed in Ref. 11), and it is critical to be able to distinguish between various forms of DNA-polymerase complexes in solution in order to fully understand the mechanistic details of the replication process. A solution approach used by Millar and co-workers (reviewed in Ref. 12) for studying the conformation of DNA in these complexes involved measuring the time-resolved fluorescence anisotropy properties of a dansyl fluorophore attached to a DNA base located 8 bp upstream of the P/T DNA junction. The changes in the lifetime of the fluorophore, which appeared to depend mostly on the local environment occupied by the probe within the protein (i.e. buried versus partially exposed), were correlated with specific binding conformations of the primer to provide an estimate of the fractional occupancy of the pol and the exo sites. Reha-Krantz and co-workers (13) more recently used a related approach, here involving the monitoring of changes in the fluorescent lifetimes of a single 2-aminopurine (2-AP) base (a fluorescent analogue of adenine) site-specifically substituted in the template strand at the P/T junction, to make similar fractional occupancy measurements. However, we note that structural interpretations of these fluorescence experiments relied heavily on the available crystal structures, and it remained to be shown directly that the 3′-end of the primer in P/T DNA constructs assumes the same distribution of conformations when bound to the protein in solution.To get around this problem, as well as to directly investigate the conformations of the primer DNA in both active sites of the Klenow and Klentaq polymerases, we have used a novel CD spectroscopic approach to characterize the solution conformations of primer DNA bound to Klenow and Klentaq DNAPs. Previously, we had shown that CD spectroscopy, in conjunction with fluorescence measurements, can be used to examine changes in local DNA and RNA conformations at 2-AP dimer probes inserted at specified positions within the nucleic acid frameworks of a variety of macromolecular machines functioning in solution (14–16). 2-AP is a structural isomer of adenine that forms base pairs with thymine in DNA (and uridine in RNA), and the substitution of 2-AP for adenine in such bp does not significantly perturb the structure or stability of the resultant double helix. Furthermore, when these probes are used as dimer pairs, the CD spectrum primarily reflects the interaction of the transition dipoles of the two probes themselves and thus the local conformation of the DNA at those positions within the P/T DNA. The characteristic CD and fluorescence signals for 2-AP probes in nucleic acids occur at wavelengths of >300 nm, a spectral region in which the protein and the canonical nucleic acid components of the “macromolecular machines of gene expression” are otherwise transparent. In this study, we have examined the binding of Klenow and Klentaq polymerases to P/T DNA constructs that were designed to be comparable with the nucleic acid components of functioning replication complexes. By examining the low energy CD spectra of site-specifically placed 2-AP probes, we have been able to characterize base conformations at defined positions within the DNA to reveal conformational features of specific DNA bases bound at and near both the pol and the exo active sites of these polymerases. These measurements, in that they directly reflect the actual conformations of the DNA chains bound within the active sites of the functioning polymerase, have also provided a direct means to estimate the equilibrium distributions of primer ends between the two active sites for various P/T DNA constructs. 相似文献
3.
Ravi Datta Sharma Andrew M Lynn Pradeep Kumar Sharma Rajnee Safdar Jawaid 《Bioinformation》2009,3(10):430-434
The stability of amidase-03 structure (a cell wall hydrolase protein) from Bacillus anthracis was studied using classical
molecular dynamics (MD) simulation. This protein (GenBank accession number: ) contains an amidase-03
domain which is known to exhibit the catalytic activity of N-acetylmuramoyl-L-alanine amidase (digesting MurNAc-Lalanine
linkage of bacterial cell wall). The amidase-03 enzyme has stability at high temperature due to the core formed by
the combination of several secondary structure elements made of β-sheets. We used root-mean-square-displacement (RMSD)
of the simulated structure from its initial state to demonstrate the unfolding of the enzyme using its secondary structural
elements. Results show that amidase-03 unfolds in transition state ensemble (TSE). The data suggests that α-helices unfold
before β-sheets from the core during simulation. NP_844822相似文献
4.
Javier?A. Couto Matthew?P. Vivero Harry?P.W. Kozakewich Amir?H. Taghinia John?B. Mulliken Matthew?L. Warman Arin?K. Greene 《American journal of human genetics》2015,96(3):480-486
Verrucous venous malformation (VVM), also called “verrucous hemangioma,” is a non-hereditary, congenital, vascular anomaly comprised of aberrant clusters of malformed dermal venule-like channels underlying hyperkeratotic skin. We tested the hypothesis that VVM lesions arise as a consequence of a somatic mutation. We performed whole-exome sequencing (WES) on VVM tissue from six unrelated individuals and looked for somatic mutations affecting the same gene in specimens from multiple persons. We observed mosaicism for a missense mutation (, c.1323C>G; NM_002401.3, p.Iso441Met) in mitogen-activated protein kinase kinase kinase 3 (MAP3K3) in three of six individuals. We confirmed the presence of this mutation via droplet digital PCR (ddPCR) in the three subjects and found the mutation in three additional specimens from another four participants. Mutant allele frequencies ranged from 6% to 19% in affected tissue. We did not observe this mutant allele in unaffected tissue or in affected tissue from individuals with other types of vascular anomalies. Studies using global and conditional Map3k3 knockout mice have previously implicated MAP3K3 in vascular development. MAP3K3 dysfunction probably causes VVM in humans. NP_002392相似文献
5.
The uroporphyrinogen III synthase (UROS) enzyme (also known as hydroxymethylbilane hydrolyase) catalyzes the cyclization of
hydroxymethylbilane to uroporphyrinogen III during heme biosynthesis. A deficiency of this enzyme is associated with the very
rare Gunther''s disease or congenital erythropoietic porphyria, an autosomal recessive inborn error of metabolism. The current
study investigated the possible role of UROS (Homo sapiens [EC: 4.2.1.75; 265 aa; 1371 bp mRNA; Entrez Pubmed ref ,
NP_000366.1]) in evolution by studying the phylogenetic relationship and divergence of this gene using computational methods.
The UROS protein sequences from various taxa were retrieved from GenBank database and were compared using Clustal-W
(multiple sequence alignment) with defaults and a first-pass phylogenetic tree was built using neighbor-joining method as in
DELTA BLAST 2.2.27+ version. A total of 163 BLAST hits were found for the uroporphyrinogen III synthase query sequence and
these hits showed putative conserved domain, HemD superfamily (as on 14th Nov 2012). We then narrowed down the search by
manually deleting the proteins which were not UROS sequences and sequences belonging to phyla other than Chordata were
deleted. A repeat phylogenetic analysis of 39 taxa was performed using PhyML and TreeDyn software to confirm that UROS is a
highly conserved protein with approximately 85% conserved sequences in almost all chordate taxons emphasizing its importance
in heme synthesis. NM_000375.2相似文献
6.
Chao Wang Sihong Chen Xi Wang Lei Wang A. Katrine Wallis Robert B. Freedman Chih-chen Wang 《The Journal of biological chemistry》2010,285(35):26788-26797
Protein disulfide isomerase (PDI), which consists of multiple domains arranged as abb′xa′c, is a key enzyme responsible for oxidative folding in the endoplasmic reticulum. In this work we focus on the conformational plasticity of this enzyme. Proteolysis of native human PDI (hPDI) by several proteases consistently targets sites in the C-terminal half of the molecule (x-linker and a′ domain) leaving large fragments in which the N terminus is intact. Fluorescence studies on the W111F/W390F mutant of full-length PDI show that its fluorescence is dominated by Trp-347 in the x-linker which acts as an intrinsic reporter and indicates that this linker can move between “capped” and “uncapped” conformations in which it either occupies or exposes the major ligand binding site on the b′ domain of hPDI. Studies with a range of constructs and mutants using intrinsic fluorescence, collision quenching, and extrinsic probe fluorescence (1-anilino-8-naphthalene sulfonate) show that the presence of the a′ domain in full-length hPDI moderates the ability of the x-linker to generate the capped conformation (compared with shorter fragments) but does not abolish it. Hence, unlike yeast PDI, the major conformational plasticity of full-length hPDI concerns the mobility of the a′ domain “arm” relative to the bb′ “trunk” mediated by the x-linker. The chaperone and enzymatic activities of these constructs and mutants are consistent with the interpretation that the reversible interaction of the x-linker with the ligand binding site mediates access of protein substrates to this site. 相似文献
7.
The Family X DNA Polymerase from Deinococcus radiodurans Adopts
a Non-standard Extended Conformation
Nicolas Leulliot Lionel Cladière Fran?ois Lecointe Dominique Durand Ulrich Hübscher Herman van Tilbeurgh 《The Journal of biological chemistry》2009,284(18):11992-11999
Deinococcus radiodurans is an extraordinarily radioresistant
bacterium that is able to repair hundreds of radiation-induced double-stranded
DNA breaks. One of the players in this pathway is an X family DNA polymerase
(PolXDr). Deletion of PolXDr has been shown to decrease
the rate of repair of double-stranded DNA breaks and increase cell sensitivity
to gamma-rays. A 3′→5′ exonuclease activity that stops
cutting close to DNA loops has also been demonstrated. The present crystal
structure of PolXDr solved at 2.46-Å resolution reveals that
PolXDr has a novel extended conformation in stark contrast to the
closed “right hand” conformation commonly observed for DNA
polymerases. This extended conformation is stabilized by the C-terminal PHP
domain, whose putative nuclease active site is obstructed by its interaction
with the polymerase domain. The overall conformation and the presence of non
standard residues in the active site of the polymerase X domain makes
PolXDr the founding member of a novel class of polymerases involved
in DNA repair but whose detailed mode of action still remains enigmatic.DNA replication and repair are functions that are of vital importance for
the maintenance of cellular life. These functions are carried out by various
DNA replicating engines, most of them acting as multiprotein complexes.
Deinococcus radiodurans, a Gram-positive bacterium, is characterized
by an extraordinary resistance to ionizing radiation and desiccation. After
radiation induced cutting of its 3.28-megabase genome into hundreds of small
fragments, it is capable of reassembling it completely
(1). Different hypotheses have
been suggested to explain this radioresistance. A recently proposed mechanism
involves the creation of long linear DNA intermediates by an extended
synthesis-dependent strand annealing process, where overlapping chromosomal
fragments are used both as primers and as templates for synthesis of
complementary single strands
(2). Recircularization of
chromosomes would be assured by homologous recombination. Although DNA
polymerase I is one of the main enzymes involved in this process, it was shown
that other proteins affect double strand break repair efficiency in D.
radiodurans. One of these is an X family DNA polymerase
(PolXDr)5
(3). Cells devoid of
PolXDr protein show increased sensitivity to γ-irradiation
and a longer delay in the restoration of an intact genome after irradiation.
It was therefore proposed that PolXDr has an important role in
double strand break repair in D. radiodurans. The contribution of
PolXDr may become essential for instance when damage gets too
important or, alternatively, it may act in different repair pathways from
polymerase I. Indeed, some of the X DNA polymerases, such as Saccharomyces
cerevisiae Pol4 and human polymerase λ
(4) have been proposed to play
important roles in different DNA repair processes, including non-homologous
end-joining (5). It was shown
that PolXDr also has strong 3′→5′ exonuclease
activity that is stimulated by Mn2+
(6). This activity is
associated with proofreading mechanisms in other polymerase families and
encoded by protein domains or subunits distinct from the polymerase catalytic
domain (7). Curiously the
exonuclease activity of PolXDr is modulated upon encounter of a
stem-loop structure. The combination of both activities leads to the
hypothesis that PolXDr might be involved in DNA repair, potentially
non-homologous end-joining, by processing damaged DNA or repair intermediates,
thus generating substrates for other repair proteins
(6). Very recently an
orthologue of PolX from Bacillus subtilis was characterized. It was
shown that PolXBs is a template-directed DNA polymerase acting on
DNA gaps with a downstream 5′ phosphate group, suggesting it may play a
role in base excision repair
(8).DNA polymerases all combine a catalytic palm domain, a thumb domain,
binding double-stranded DNA, and a finger domain that fixes the incoming
nucleotide. The polymerase domain of the X family belongs to the
Polβ-like nucleotidyltransferase superfamily, sharing ∼25% amino acid
identity with the DNA polymerase domains of Polλ, Pol4, and Polβ.
PolXDr has a second domain at the C terminus called PHP, with
strong sequence identity with the histidinol phosphatase involved in histidine
transport in bacteria. Due to its similarity to histidinol phosphatase and the
presence of a trinuclear zinc site, the PolXDr PHP domain is
thought to function as phosphoesterase
(9). In the context of DNA
polymerases, this activity might be responsible for the degradation of
pyrophosphate, thus driving the polymerization reaction, or contributes to a
nuclease reaction that would be involved in proofreading the newly synthesized
strand. The deletion of the PHP domain also had a negative effect on survival
of γ-irradiated cells suggesting that this domain possesses a function
in DNA repair. Unexpectedly, deletion of the PHP domain destroys structure
modulated but not the general 3′→5′ exonuclease activity
(6). No activity could be
demonstrated for the PHP domain alone.In this report we present the crystal structure of PolXDr at
2.46-Å resolution. Surprisingly, PolXDr adopts a stretched
out conformation instead of the commonly observed closed right hand
conformation. In the active site of the polymerase catalytic domain, the two
universally conserved aspartates are replaced by two glutamates, whereas the
active site of the PHP domain is obstructed by its interaction with the
polymerase domain. 相似文献
8.
Yohtaro Saito Hiroki Ashida Tomoko Sakiyama Nicole Tandeau de Marsac Antoine Danchin Agnieszka Sekowska Akiho Yokota 《The Journal of biological chemistry》2009,284(19):13256-13264
The sequences classified as genes for various ribulose-1,5-bisphosphate
(RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely
distributed among bacteria, archaea, and eukaryota. In the phylogenic tree
constructed with these sequences, RuBisCOs and RLPs are grouped into four
separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III
sequences, 19 conserved amino acid residues are essential for CO2
fixation; however, 1-11 of these 19 residues are substituted with other amino
acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity
detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P)
enolase reaction catalyzed by Bacillus subtilis, Microcystis
aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from
Rhodospirillum rubrum, Rhodopseudomonas palustris,
Chlorobium tepidum, and Bordetella bronchiseptica were
inactive in the enolase reaction. DK-MTP-1-P enolase activity of B.
subtilis RLP required Mg2+ for catalysis and, like RuBisCO,
was stimulated by CO2. Four residues that are essential for the
enolization reaction of RuBisCO, Lys175, Lys201,
Asp203, and Glu204, were conserved in RLPs and were
essential for DK-MTP-1-P enolase catalysis. Lys123, the residue
conserved in DK-MTP-1-P enolases, was also essential for B. subtilis
RLP enolase activity. Similarities between the active site structures of
RuBisCO and B. subtilis RLP were examined by analyzing the effects of
structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state
analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in
the DK-MTP-1-P enolase reaction with a Ki value of 103
μm. RuBP and d-phosphoglyceric acid, the substrate
and product, respectively, of RuBisCO, were weaker competitive inhibitors.
These results suggest that the amino acid residues utilized in the B.
subtilis RLP enolase reaction are the same as those utilized in the
RuBisCO RuBP enolization reaction.Ribulose-1,5-bisphosphate carboxylase/oxygenase
(RuBisCO)4 catalyzes
the carboxylation and oxygenation reactions of ribulose 1,5-bisphosphate
(RuBP) in photosynthesis
(1-4).
This enzyme is the sole CO2-fixing enzyme in plants; however, it
has certain inefficiencies. It has a very low turnover rate, a low affinity
for the substrate, CO2, and low specificity between the
carboxylation and oxygenation reactions
(5-7).
Thus, the intrinsic enzymatic properties of RuBisCO are inadequate for
efficient incorporation of CO2 into organic matter in
photosynthesis (7). However,
plants have overcome these disadvantages by investing a huge amount of leaf
nitrogen in RuBisCO synthesis
(8).In nature, there are wide variations in the properties and primary
sequences of RuBisCO among different photosynthetic organisms
(9-12).
The primary sequences vary as much as 73% without loss of activity. The
relative specificity ranges from ∼0.5 in a small subunitless RuBisCO to
238 in a red algal, hexadecameric RuBisCO
(13,
14). The affinity for
CO2 varies some 100-fold
(15). Comparisons between
these kinetic parameters and the primary sequences are expected to reveal
promising strategies for improving the enzyme, and many studies have been
conducted on this topic (7,
16-18).A RuBisCO-like protein (RLP) with no CO2-fixing activity was
first demonstrated in Chlorobium tepidum
(19), and a similar protein in
Bacillus subtilis was found to be involved in the methionine salvage
pathway (20). These findings
have pointed to a new direction in RuBisCO research
(17,
21). The phylogenetic tree of
the catalytic subunits of RuBisCOs and their homologs shows four major
clusters, forms I-III, and form IV (Fig.
1A). Form I and II RuBisCOs are involved in
photosynthetic or chemosynthetic CO2 fixation, whereas the
metabolic function of form III RuBisCOs remains unclear, although they can fix
CO2 on RuBP (9,
22). Forms I-III conserve
almost all 19 amino acid residues that are essential for CO2
fixation in RuBisCO (Fig.
1B). The form IV cluster in the phylogenetic tree
consists of RLPs that show ∼20% homology to plant form I or bacterial form
II RuBisCOs (12,
20,
21,
23-25).
There are 8-18 RuBisCO-essential residues that are conserved in RLPs
(Fig. 1B). Form IV
RLPs are further subdivided into four groups; α1, α2, β, and
γ (21). The RLP of
B. subtilis is classified in α1 and catalyzes the enolization
reaction of 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) but not the
carboxylation of RuBP (Fig.
2A) (20,
21,
23). The absence of
CO2-fixing activity in the B. subtilis RLP may be ascribed
to changes in 8 of the 19 amino acid residues essential for CO2
fixation in RuBisCO (Fig.
1B). Several of these residues are located at the
C-terminal domains of B. subtilis RLP and RuBisCO. The dimeric
RuBisCO from Rhodospirillum rubrum catalyzes the DK-MTP-1-P enolase
reaction with very low activity
(20). These findings, together
with the similarity in the chemical structures of substrates for B.
subtilis RLP and RuBisCO (Fig.
2A), suggest that they may have a close evolutionary
relationship (12,
21,
23-25).Open in a separate windowFIGURE 1.Homology between RLPs and RuBisCOs. A, phylogenetic tree of
RLPs and RuBisCOs. Deduced amino acid sequence of B. subtilis subsp.
subtilis str. 168 RLP () was compared with sequences of RLPs
of Thermotoga lettingae TMO ( NP_389242), Beggiatoa sp. SS
( YP_001471302), Ostreococcus tauri (Ostreococcus tauri IV,
ZP_01997270), Alkalilimnicola ehrlichei MLHE-1 ( CAL54998), R.
rubrum ATCC 11170 (R. rubrum IV, YP_742007), R.
palustris CGA009 (R. palustris IV-1, YP_427085),
Archaeoglobus fulgidus DSM 4304 (A. fulgidus IV, NP_947514),
M. aeruginosa PCC 7806 (M. aeruginosa IV, NP_070416), G.
kaustophilus HTA426 ( CAJ43366), Bacillus cereus ATCC 14579
( YP_146806), B. bronchiseptica RB50 ( NP_833754), Polaromonas
sp. JS666 ( NP_887583), C. tepidum TLS ( YP_546958), and R.
palustris CGA009 (R. palustris IV-2, NP_662651) and of RuBisCOs
of R. palustris CGA009 (R. palustris II, NP_945615), R.
rubrum ATCC 11170 (R. rubrum II, NP_949975), M.
jannaschii DSM 2661 ( YP_427487), A. fulgidus DSM 4304 (A.
fulgidus III, NP_248230), Thermococcus kodakaraensis KOD1
( NP_070466), Galdieria partita ( YP_184703), R. palustris
CGA009 (R. palustris I, BAA75796), M. aeruginosa PCC 7806
(M. aeruginosa I, NP_946905), O. tauri (O. tauri IV,
CAJ43363), and S. oleracea ( YP_717262). When an organism has more
than one RuBisCO and/or RLP sequence, the form number of each sequence in the
RuBisCO family follows the name of the organism. ClustalW and TreeView
programs (available on the World Wide Web) were used to construct the
phylogenetic tree. B, multiple alignments of sequences underlined in
A. Identical amino acid residues are indicated by black
shading, and similar amino acid residues are indicated by gray
shading. Sequences are numbered according to the S.
oleracea sequence. Catalytic and RuBP-binding residues deduced for
RuBisCO are indicated by open triangles and filled
triangles, respectively. Alignment was visualized with the BOXSHADE
program (available on the World Wide Web). NP_054944Open in a separate windowFIGURE 2.Catalytic and structural similarity of RLPs and RuBisCOs.
A, catalytic reactions of RuBisCO and RLP. B, comparison of
active sites between S. oleracea RuBisCO binding CABP (8RUC) and
G. kaustophilus RLP (2OEM) modeled to bind DK-MTP-1-P. DK-MTP-1-P in
G. kaustophilus RLP was depicted by substituting the methyl group of
DK-H-1-P in 2OEM with the thiomethyl group of MTRu-1-P bound to MtnA
(28). Side chains of active
site residues and ligands are shown as sticks. These five residues of
B. subtilis were substituted with other amino acids in this study.
CABP and DK-MTP-1-P are shown in white, and their phosphate groups
are shown in red and orange, respectively. Mg2+
atoms are shown in yellow. Protein structures were drawn with PyMOL
(available on the World Wide Web).The RuBisCO reaction starts with the abstraction of the C3 proton from RuBP
to form the cis-enediol(ate) of RuBP
(Fig. 2A)
(26). Using the spinach
numbering format to identify RuBisCO and RLP residues, the carbamate formed on
the ε-amino group of Lys201 may be the general base to
abstract the proton, and the cis-enediol(ate) form of RuBP is
stabilized in the combination of side chains from Lys175 and
His294 (27).
Asp203, Glu204, and the carbamate Lys201 of
the enzyme active site stabilize the cis-enediol(ate) and
CO2 through the Mg2+ ion
(26). The B. subtilis
RLP abstracts the C1 proton of its substrate DK-MTP-1-P to start the
DK-MTP-1-P enolization reaction
(12,
21,
23). The ε-amino group of
Lys123 is thought to be required for the abstraction of the
1-proS proton in the Geobacillus kaustophilus RLP, which
belongs to group α1, together with the B. subtilis RLP
(Fig. 2B)
(25). Lys123 is
conserved among DK-MTP-1-P enolases and resides very near the C1 of
2,3-diketohexane 1-phosphate (DK-H-1-P), a structural analogue of DK-MTP-1-P.
As is the case in RuBisCO, the enolate intermediate is stabilized by
Mg2+ and several amino acid residues: Lys175,
Asp203, Glu204, His294, and the carbamylated
Lys201.The results of these studies suggest that the DK-MTP-1-P enolase is
structurally and functionally related to photosynthetic RuBisCO. However,
research on the G. kaustophilus RLP revealed that the
proton-abstracting, reaction-starting residues differed between the DK-MTP-1-P
enolase and RuBisCO (25). It
has been reported that when lysine at 201 is substituted with an alanine in
the G. kaustophilus RLP, the enzyme is still capable of catalyzing
enolization of DK-MTP-1-P
(25). This result raises a
question about the above hypothesis on the close evolutionary relationship
between the RLP and RuBisCO, because a carbamylated lysine residue would be
required at this position to form the Mg2+-chelating triad linkage
together with Asp203 and Glu204 and to stabilize the
reaction intermediate in the RuBP enolization reaction of RuBisCO.Evolutionary relationships of genes with similar sequences are deduced by
comparing gene sequence homology of the genes and amino acid sequence homology
of the predicted proteins and by analyzing conservation of functional motifs
of the predicted proteins in silico. Comparison of protein structures
at the active sites also provides important information. However, it may
difficult to predict their mutual evolutionary relationship more precisely
when they catalyze different reactions in individual metabolic pathways. The
present research adopted a new method to resolve such an issue.We studied the structural and functional interrelationships of RLP and
RuBisCO after enzymological characterization of B. subtilis RLP as
the DK-MTP-1-P enolase enzyme. The results showed that DK-MTP-1-P enolase
activity was limited to some RLPs in the cluster, including B.
subtilis in form IV RLPs. All of the catalytic residues for the RuBisCO
reaction were also indispensable for DK-MTP-1-P enolase activity. The
architecture of the B. subtilis RLP substrate-binding residues
stereospecifically stabilized the transition state analog in CO2
fixation of RuBisCO. The fact that the transition state analog of RuBisCO
interacts with the active site of Bacillus RLP strongly supports
their evolutionary proximity. 相似文献
9.
10.
11.
Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (kcat 415 s−1), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3′ → 5′ translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the “leading” AdnB and “lagging” AdnA motor domains track in tandem, 3′ to 5′, along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5′ and 3′ terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5′ strand, while the AdnB nuclease cleaves the displaced 3′ strand. We present evidence that the distinctive “molecular ruler” function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5′-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme. 相似文献
12.
Background
Fluctuating asymmetry is a contentious indicator of stress in populations of animals and plants. Nevertheless, it is a measure of developmental noise, typically obtained by measuring asymmetry across an individual organism''s left-right axis of symmetry. These individual, signed asymmetries are symmetrically distributed around a mean of zero. Fluctuating asymmetry, however, has rarely been studied in microorganisms, and never in fungi.Objective and Methods
We examined colony growth and random phenotypic variation of five soil microfungal species isolated from the opposing slopes of “Evolution Canyon,” Mount Carmel, Israel. This canyon provides an opportunity to study diverse taxa inhabiting a single microsite, under different kinds and intensities of abiotic and biotic stress. The south-facing “African” slope of “Evolution Canyon” is xeric, warm, and tropical. It is only 200 m, on average, from the north-facing “European” slope, which is mesic, cool, and temperate. Five fungal species inhabiting both the south-facing “African” slope, and the north-facing “European” slope of the canyon were grown under controlled laboratory conditions, where we measured the fluctuating radial asymmetry and sizes of their colonies.Results
Different species displayed different amounts of radial asymmetry (and colony size). Moreover, there were highly significant slope by species interactions for size, and marginally significant ones for fluctuating asymmetry. There were no universal differences (i.e., across all species) in radial asymmetry and colony size between strains from “African” and “European” slopes, but colonies of Clonostachys rosea from the “African” slope were more asymmetric than those from the “European” slope.Conclusions and Significance
Our study suggests that fluctuating radial asymmetry has potential as an indicator of random phenotypic variation and stress in soil microfungi. Interaction of slope and species for both growth rate and asymmetry of microfungi in a common environment is evidence of genetic differences between the “African” and “European” slopes of “Evolution Canyon.” 相似文献13.
Jaeyong Lee Joanne Johnson Ziwei Ding Mark Paetzel Rosemary B. Cornell 《The Journal of biological chemistry》2009,284(48):33535-33548
CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in the synthesis of phosphatidylcholine, the most abundant phospholipid in eukaryotic cell membranes. The CCT-catalyzed transfer of a cytidylyl group from CTP to phosphocholine to form CDP-choline is regulated by a membrane lipid-dependent mechanism imparted by its C-terminal membrane binding domain. We present the first analysis of a crystal structure of a eukaryotic CCT. A deletion construct of rat CCTα spanning residues 1–236 (CCT236) lacks the regulatory domain and as a result displays constitutive activity. The 2.2-Å structure reveals a CCT236 homodimer in complex with the reaction product, CDP-choline. Each chain is composed of a complete catalytic domain with an intimately associated N-terminal extension, which together with the catalytic domain contributes to the dimer interface. Although the CCT236 structure reveals elements involved in binding cytidine that are conserved with other members of the cytidylyltransferase superfamily, it also features nonconserved active site residues, His-168 and Tyr-173, that make key interactions with the β-phosphate of CDP-choline. Mutagenesis and kinetic analyses confirmed their role in phosphocholine binding and catalysis. These results demonstrate structural and mechanistic differences in a broadly conserved protein fold across the cytidylyltransferase family. Comparison of the CCT236 structure with those of other nucleotidyltransferases provides evidence for substrate-induced active site loop movements and a disorder-to-order transition of a loop element in the catalytic mechanism. 相似文献
14.
Restriction endonuclease BpuJI specific for the 5'-CCCGT sequence is related to the archaeal Holliday junction resolvase family
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Sukackaite R Lagunavicius A Stankevicius K Urbanke C Venclovas C Siksnys V 《Nucleic acids research》2007,35(7):2377-2389
Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5′-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3nt from the 3′-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins. 相似文献
15.
She M Decker CJ Chen N Tumati S Parker R Song H 《Nature structural & molecular biology》2006,13(1):63-70
Decapping is a key step in both general and nonsense-mediated 5' --> 3' mRNA-decay pathways. Removal of the cap structure is catalyzed by the Dcp1-Dcp2 complex. The crystal structure of a C-terminally truncated Schizosaccharomyces pombe Dcp2p reveals two distinct domains: an all-helical N-terminal domain and a C-terminal domain that is a classic Nudix fold. The C-terminal domain of both Saccharomyces cerevisiae and S. pombe Dcp2p proteins is sufficient for decapping activity, although the N-terminal domain can affect the efficiency of Dcp2p function. The binding of Dcp2p to Dcp1p is mediated by a conserved surface on its N-terminal domain, and the N-terminal domain is required for Dcp1p to stimulate Dcp2p activity. The flexible nature of the N-terminal domain relative to the C-terminal domain suggests that Dcp1p binding to Dcp2p may regulate Dcp2p activity through conformational changes of the two domains. 相似文献
16.
The crystal structure and mutational analysis of human NUDT9 总被引:2,自引:0,他引:2
Human ADP-ribose pyrophosphatase NUDT9 belongs to a superfamily of Nudix hydrolases that catabolize potentially toxic compounds in the cell. The enzyme hydrolyzes ADP-ribose (ADPR) to AMP and ribose 5'-phosphate. NUDT9 shares 39% sequence identity with the C-terminal cytoplasmic domain of the ADPR-gated calcium channel TRPM2, which exhibits low but specific enzyme activity. We determined crystal structures of NUDT9 in the presence and in the absence of the reaction product ribose 5'-phosphate. On the basis of these structures and comparison with a bacterial homologue, a model of the substrate complex was built. The structure and activity of a double point mutant (R(229)E(230)F(231) to R(229)I(230)L(231)), which mimics the Nudix signature of the ion channel domain, was determined. Finally, the activities of a pair of additional mutated constructs were compared to the wild-type enzyme. The first corresponds to a minimal Nudix domain missing an N-terminal domain and C-terminal tail; the second disrupts two potential general bases in the active site. NUDT9 contains an N-terminal domain with a novel fold and a catalytic C-terminal Nudix domain. Unlike its closest functional homologue (homodimeric Escherichia coli ADPRase), it is active as a monomer, and the substrate is bound in a cleft between the domains. The structure of the RIL mutant provides structural basis for the reduced activity of the TRPM2 ion channel. The conformation and binding interactions of ADPR substrate are predicted to differ from those observed for E.coli ADPRase; mutation of structurally aligned acidic residues in their active sites produce significantly different effects on catalytic efficiency, indicating that their reaction pathways and mechanisms may have diverged. 相似文献
17.
Acceptability of Carraguard vaginal microbicide gel among HIV-infected women in Chiang Rai, Thailand
Whitehead SJ McLean C Chaikummao S Braunstein S Utaivoravit W van de Wijgert JH Mock PA Siraprapasiri T Friedland BA Kilmarx PH Markowitz LE 《PloS one》2011,6(9):e14831
Background
Few studies of microbicide acceptability among HIV-infected women have been done. We assessed Carraguard® vaginal gel acceptability among participants in a randomized, controlled, crossover safety trial in HIV-infected women in Thailand.Methodology/Principal Findings
Participants used each of 3 treatments (Carraguard gel, methylcellulose placebo gel, and no product) for 7 days, were randomized to one of six treatment sequences, and were blinded to the type of gel they received in the two gel-use periods. After both gel-use periods, acceptability was assessed by face-to-face interview. Responses were compared to those of women participating in two previous Carraguard safety studies at the same study site. Sixty women enrolled with a median age of 34 years; 25% were sexually active. Self-reported adherence (98%) and overall satisfaction rating of the gels (87% liked “somewhat” or “very much”) were high, and most (77%) considered the volume of gel “just right.” For most characteristics, crossover trial participants evaluated the gels more favorably than women in the other two trials, but there were few differences in the desired characteristics of a hypothetical microbicide. Almost half (48%) of crossover trial participants noticed a difference between Carraguard and placebo gels; 33% preferred Carraguard while 12% preferred placebo (p = 0.01).Conclusions/Significance
Daily Carraguard vaginal gel use was highly acceptable in this population of HIV-infected women, who assessed the gels more positively than women in two other trials at the site. This may be attributable to higher perceived need for protection among HIV-infected women, as well as to study design differences. This trial was registered in the U.S. National Institutes of Health clinical trials registry under registration number . NCT00213044相似文献18.
19.
20.
Alena Siarheyeva Ronghua Liu Frances J. Sharom 《The Journal of biological chemistry》2010,285(10):7575-7586
P-glycoprotein (ABCB1), a member of the ABC superfamily, functions as an ATP-driven multidrug efflux pump. The catalytic cycle of ABC proteins is believed to involve formation of a sandwich dimer in which two ATP molecules are bound at the interface of the nucleotide binding domains (NBDs). However, such dimers have only been observed in isolated NBD subunits and catalytically arrested mutants, and it is still not understood how ATP hydrolysis is coordinated between the two NBDs. We report for the first time the characterization of an asymmetric state of catalytically active native P-glycoprotein with two bound molecules of adenosine 5′-(γ-thio)triphosphate (ATPγS), one of low affinity (Kd 0.74 mm), and one “occluded” nucleotide of 120-fold higher affinity (Kd 6 μm). ATPγS also interacts with P-glycoprotein with high affinity as assessed by inhibition of ATP hydrolysis and protection from covalent labeling of a Walker A Cys residue, whereas other non-hydrolyzable ATP analogues do not. Binding of ATPγS (but not ATP) causes Trp residue heterogeneity, as indicated by collisional quenching, suggesting that it may induce conformational asymmetry. Asymmetric ATPγS-bound P-glycoprotein does not display reduced binding affinity for drugs, implying that transport is not driven by ATP binding and likely takes place at a later stage of the catalytic cycle. We propose that this asymmetric state with two bound nucleotides represents the next intermediate on the path toward ATP hydrolysis after nucleotide binding, and an alternating sites mode of action is achieved by simultaneous switching of the two active sites between high and low affinity states. 相似文献