共查询到20条相似文献,搜索用时 31 毫秒
1.
Michal Avital-Shmilovici Jonathan Whittaker Michael A. Weiss Stephen B. H. Kent 《The Journal of biological chemistry》2014,289(34):23683-23692
Misfolding of proinsulin variants in the pancreatic β-cell, a monogenic cause of permanent neonatal-onset diabetes mellitus, provides a model for a disease of protein toxicity. A hot spot for such clinical mutations is found at position B8, conserved as glycine within the vertebrate insulin superfamily. We set out to investigate the molecular basis of the aberrant properties of a proinsulin clinical mutant in which residue GlyB8 is replaced by SerB8. Modular total chemical synthesis was used to prepare the wild-type [GlyB8]proinsulin molecule and three analogs: [d-AlaB8]proinsulin, [l-AlaB8]proinsulin, and the clinical mutant [l-SerB8]proinsulin. The protein diastereomer [d-AlaB8]proinsulin produced higher folding yields at all pH values compared with the wild-type proinsulin and the other two analogs, but showed only very weak binding to the insulin receptor. The clinical mutant [l-SerB8]proinsulin impaired folding at pH 7.5 even in the presence of protein-disulfide isomerase. Surprisingly, although [l-SerB8]proinsulin did not fold well under the physiological conditions investigated, once folded the [l-SerB8]proinsulin protein molecule bound to the insulin receptor more effectively than wild-type proinsulin. Such paradoxical gain of function (not pertinent in vivo due to impaired secretion of the mutant insulin) presumably reflects induced fit in the native mechanism of hormone-receptor engagement. This work provides insight into the molecular mechanism of a clinical mutation in the insulin gene associated with diabetes mellitus. These results dramatically illustrate the power of total protein synthesis, as enabled by modern chemical ligation methods, for the investigation of protein folding and misfolding. 相似文献
2.
Qing-xin Hua Bin Xu Kun Huang Shi-Quan Hu Satoe Nakagawa Wenhua Jia Shuhua Wang Jonathan Whittaker Panayotis G. Katsoyannis Michael A. Weiss 《The Journal of biological chemistry》2009,284(21):14586-14596
A central tenet of molecular biology holds that the function of a protein
is mediated by its structure. An inactive ground-state conformation may
nonetheless be enjoined by the interplay of competing biological constraints.
A model is provided by insulin, well characterized at atomic resolution by
x-ray crystallography. Here, we demonstrate that the activity of the hormone
is enhanced by stereospecific unfolding of a conserved structural element. A
bifunctional β-strand mediates both self-assembly (within β-cell
storage vesicles) and receptor binding (in the bloodstream). This strand is
anchored by an invariant side chain (PheB24); its substitution by
Ala leads to an unstable but native-like analog of low activity. Substitution
by d-Ala is equally destabilizing, and yet the protein diastereomer
exhibits enhanced activity with segmental unfolding of the β-strand.
Corresponding photoactivable derivatives (containing l- or
d-para-azido-Phe) cross-link to the insulin receptor with
higher d-specific efficiency. Aberrant exposure of hydrophobic
surfaces in the analogs is associated with accelerated fibrillation, a form of
aggregation-coupled misfolding associated with cellular toxicity. Conservation
of PheB24, enforced by its dual role in native self-assembly and
induced fit, thus highlights the implicit role of misfolding as an
evolutionary constraint. Whereas classical crystal structures of insulin
depict its storage form, signaling requires engagement of a detachable arm at
an extended receptor interface. Because this active conformation resembles an
amyloidogenic intermediate, we envisage that induced fit and self-assembly
represent complementary molecular adaptations to potential proteotoxicity. The
cryptic threat of misfolding poses a universal constraint in the evolution of
polypeptide sequences.How insulin binds to the insulin receptor
(IR)2 is not well
understood despite decades of investigation. The hormone is a globular protein
containing two chains, A (21 residues) and B (30 residues)
(Fig. 1A). In
pancreatic β-cells, insulin is stored as Zn2+-stabilized
hexamers (Fig. 1B),
which form microcrystal-line arrays within specialized secretory granules
(1). The hexamers dissociate
upon secretion into the portal circulation, enabling the hormone to function
as a zinc-free monomer. The monomer is proposed to undergo a change in
conformation upon receptor binding
(2). In this study, we
investigated a site of conformational change in the B-chain
(PheB24) (arrow in Fig.
1A). In classical crystal structures, this invariant
aromatic side chain (tawny in Fig.
1B) anchors an antiparallel β-sheet at the dimer
interface (blue in Fig.
1C). Total chemical synthesis is exploited to enable
comparison of corresponding d- and l-amino acid
substitutions at this site, an approach designated “chiral
mutagenesis”
(3-5).
In the accompanying article, the consequences of this conformational change
are investigated by photomapping of the receptor-binding surface
(6). Together, these studies
redefine the interrelation of structure and activity in a protein central to
the hormonal control of metabolism.Open in a separate windowFIGURE 1.Sequence and structure of insulin. A, sequences of the
B-chain (upper) and A-chain (lower) with disulfide bridges
as indicated. The arrow indicates invariant PheB24. The
B24-B28 β-strand is highlighted in blue. B, crystal structure of
the T6 zinc insulin hexamer (Protein Data Bank code 4INS): ribbon
model (left) and space-filling model (right). The B24-B28
β-strand is shown in blue, and the side chain of
PheB24 is highlighted in tawny. The B-chain is otherwise
dark gray; the A-chain, light gray; and zinc ions,
magenta. Also shown at the left are the side chains of
HisB10 at the axial zinc-binding sites. C, cylinder model
of the insulin dimer showing the B24-B26 antiparallel β-sheet
(blue) anchored by the B24 side chain (tawny circle). The A-
and B-chains are shown in light and dark gray, respectively.
The protomer at the left is shown in the R-state, in which the central
α-helix of the B-chain is elongated (B3-B19 in the frayed Rf
protomer of T3Rf3 hexamers and B1-B19 in the
R protomer of R6 hexamers). The three types of zinc insulin
hexamers share similar B24-B26 antiparallel β-sheets as conserved
dimerization elements.The structure of an insulin monomer in solution resembles a
crystallographic protomer (Fig.
2A)
(7-9).
The A-chain contains an N-terminal α-helix, non-canonical turn, and
second helix; the B-chain contains an N-terminal segment, central
α-helix, and C-terminal β-strand. The β-strand is maintained
in an isolated monomer wherein the side chain of PheB24
(tawny in Fig.
2A), packing against the central α-helix of the
B-chain, provides a “plug” to seal a crevice in the hydrophobic
core (Fig. 2B).
Anomalies encountered in previous studies of insulin analogs suggest that
PheB24 functions as a conformational switch
(4,
7,
10-14).
Whereas l-amino acid substitutions at B24 generally impair activity
(even by such similar residues as l-Tyr)
(15), a seeming paradox is
posed by the enhanced activities of nonstandard analogs containing
d-amino acids (10-12).
Open in a separate windowaAffinities are given relative to wild-type insulin (100%).bLymphocytes are human, and hepatocytes are rat; CHO designates Chinese
hamster ovary.cStandard deviations are not provided in this reference.Open in a separate windowFIGURE 2.Role of PheB24 in an insulin monomer. A, shown
is a cylinder model of insulin as a T-state protomer. The C-terminal B-chain
β-strand is shown in blue, and the PheB24 side chain
is shown in tawny. The black portion of the N-terminal
A-chain α-helix (labeled buried) indicates a hidden
receptor-binding surface (IleA2 and ValA3). B,
the schematic representation of insulin highlights the proposed role of the
PheB24 side chain as a plug that inserts into a crevice at the edge
of the hydrophobic core. C and D, whereas substitution of
PheB24 by l-Ala (C) would only partially fill
the B24-related crevice, its substitution by d-Ala (D)
would be associated with a marked packing defect. An alternative conformation,
designated the R-state, is observed in zinc insulin hexamers at high ionic
strength (74) and upon binding
of small cyclic alcohols (75)
but has not been observed in an insulin monomer.Why do d-amino acid substitutions at B24 enhance the activity of
insulin? In this study, we describe the structure and function of insulin
analogs containing l-Ala or d-Ala at B24
(Fig. 2, C and
D). Our studies were conducted within an engineered
monomer (DKP-insulin, an insulin analog containing three substitutions in the
B-chain: AspB10, LysB28, and ProB29) to
circumvent effects of self-assembly
(16). Whereas the inactive
l-analog retains a native-like structure, the active
d-analog exhibits segmental unfolding of the B-chain. Studies of
corresponding analogs containing either l- or
d-photoactivable probes
(l-para-azido-PheB24 or
d-para-azido-PheB24 (l- or
d-PapB24), obtained from photostable
para-amino-Phe (Pmp) precursors
(17)) demonstrate specific
cross-linking to the IR. Although photo-contacts map in each case to the
N-terminal domain of the receptor α-subunit (the L1 β-helix),
higher cross-linking efficiency is achieved by the d-probe.
Together, this and the following study
(6) provide evidence that
insulin deploys a detachable arm that inserts between domains of the IR.Induced fit of insulin illuminates by its scope general principles at the
intersection of protein structure and cell biology. Protein evolution is
enjoined by multiple layers of biological selection. The pathway of insulin
biosynthesis, for example, successively requires (a) specific
disulfide pairing (in the endoplasmic reticulum), (b) subcellular
targeting and prohormone processing (in the trans-Golgi network),
(c) zinc-mediated protein assembly and microcrystallization (in
secretory granules), and (d) exocytosis and rapid disassembly of
insulin hexamers (in the portal circulation), in turn enabling binding of the
monomeric hormone to target tissues
(1). Each step imposes
structural constraints, which may be at odds. This study demonstrates that
stereospecific pre-detachment of a receptor-binding arm enhances biological
activity but impairs disulfide pairing and renders the hormone susceptible to
aggregation-coupled misfolding
(18). Whereas the classical
globular structure of insulin and its self-assembly prevent proteotoxicity
(3,
19), partial unfolding enables
receptor engagement. We envisage that a choreography of conformational change
has evolved as an adaptative response to the universal threat of toxic protein
misfolding. 相似文献
TABLE 1
Previous studies of insulin analogsAnalog | Affinitya | Assayb | Ref. |
---|---|---|---|
% | |||
d-PheB24-insulin | 180 | Lymphocytes | 10 |
l-AlaB24-insulin | 1 | Hepatocytes | 68 |
l-AlaB24-insulin | 3 | Lymphocytes | 69 |
d-PheB24-insulin | 140 ± 9 | Hepatocytes | 11 |
l-AlaB24-insulin | 1.0 ± 0.1 | Hepatocytes | 11 |
d-AlaB24-insulin | 150 ± 9 | Hepatocytes | 11 |
GlyB24-insulin | 78 ± 11 | Hepatocytes | 11 |
DKP-insulin | 200c | CHO cells | 12 |
d-PheB24-DKP-insulin | 180 | CHO cells | 12 |
l-AlaB24-DKP-insulin | 7 | CHO cells | 12 |
GlyB24-DKP-insulin | 50 | CHO cells | 12 |
3.
Audrey Boniface Claudine Parquet Michel Arthur Dominique Mengin-Lecreulx Didier Blanot 《The Journal of biological chemistry》2009,284(33):21856-21862
Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of l- and d-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. The first monomer was the d-lysine-containing disaccharide-tripeptide in which the d-Glu-d-Lys bond had the unusual γ→ϵ arrangement (GlcNAc-MurNAc-l-Ala-γ-d-Glu-ϵ-d-Lys). The second monomer was the conventional disaccharide-tetrapeptide (GlcNAc-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala). The first dimer contained a disaccharide-l-Ala as the acyl donor cross-linked to the α-amine of d-Lys in a tripeptide acceptor stem with the sequence of the first monomer. In the second dimer, donor and acceptor stems with the sequences of the second and first monomers, respectively, were connected by a d-Ala4-α-d-Lys3 cross-link. The cross-linking index was 10 with an average chain length of 30 disaccharide units. The structure of the peptidoglycan of T. maritima revealed for the first time the key role of d-Lys in peptidoglycan synthesis, both as a surrogate of l-Lys or meso-diaminopimelic acid at the third position of peptide stems and in the formation of novel cross-links of the l-Ala1(α→α)d-Lys3 and d-Ala4(α→α)d-Lys3 types.Peptidoglycan (or murein) is a giant macromolecule whose main function is the protection of the cytoplasmic membrane against the internal osmotic pressure. It is composed of alternating residues of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)2 cross-linked by short peptides (1). The composition of the peptide stem in nascent peptidoglycan is l-Ala1-γ-d-Glu2-X3-d-Ala4-d-Ala5, where X is most often meso-diaminopimelic acid (meso-A2pm) or l-lysine in Gram-negative and Gram-positive species, respectively (2, 3). In the mature macromolecule, the last d-Ala residue is removed. Cross-linking of the glycan chains generally occurs between the carboxyl group of d-Ala at position 4 of a donor peptide stem and the side-chain amino group of the diamino acid at position 3 of an acceptor peptide stem (4→3 cross-links). Cross-linking is either direct or through a short peptide bridge such as pentaglycine in Staphylococcus aureus (2, 3). The enzymes for the formation of the 4→3 cross-links are active-site serine dd- transpeptidases that belong to the penicillin-binding protein (PBP) family and are the essential targets of β-lactam antibiotics in pathogenic bacteria (4). Catalysis involves the cleavage of the d-Ala4-d-Ala5 bond of a donor peptide stem and the formation of an amide bond between the carboxyl of d-Ala4 and the side chain amine at the third position of an acceptor stem. Transpeptidases of the ld specificity are active-site cysteine enzymes that were shown to act as surrogates of the PBPs in mutants of Enterococcus faecium resistant to β-lactam antibiotics (5). They cleave the X3-d-Ala4 bond of a donor stem peptide to form 3→3 cross-links. This alternate mode of cross-linking is usually marginal, although it has recently been shown to predominate in non-replicative “dormant” forms of Mycobacterium tuberculosis (6).Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors by Huber et al. (7). A morphological characteristic is the presence of an outer sheath-like envelope called “toga.” Although the organism has received considerable attention for its biotechnological potential, studies about its peptidoglycan are scarce (8–11), and in particular the fine structure of the macromolecule is still unknown. In their initial work, Huber et al. (7) showed that the composition of its peptidoglycan was unusual for a Gram-negative species, because it contained both isomers of lysine and no A2pm. Recently, we purified and studied the properties of T. maritima MurE (12); this enzyme is responsible for the addition of the amino acid residue at position 3 of the peptide stem (13, 14). We demonstrated that T. maritima MurE added in vitro l- and d-Lys to UDP-MurNAc-l-Ala-d-Glu. Although l-Lys was added in the usual way, yielding the conventional nucleotide UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys containing a d-Glu(γ→α)l-Lys amide bond, the d-isomer was added in an “upside-down” manner, yielding the novel nucleotide UDP-MurNAc-l-Ala-d-Glu(γ→ϵ)d-Lys. We also showed that the d-Lys-containing nucleotide was not a substrate for T. maritima MurF, the subsequent enzyme in the biosynthetic pathway, whereas this ligase catalyzed the addition of dipeptide d-Ala-d-Ala to the l-Lys-containing tripeptide, yielding the conventional UDP-MurNAc-pentapeptide (12).However, both the l-Lys-containing UDP-MurNAc-pentapeptide and d-Lys-containing UDP-MurNAc-tripeptide were used as substrates by T. maritima MraY with comparable efficiencies in vitro (12). This observation implies that the unusual d-Lys-containing peptide stems are likely to be translocated to the periplasmic face of the cytoplasmic membrane and to participate in peptidoglycan polymerization. Therefore, we have determined here the fine structure of T. maritima peptidoglycan and we have shown that l-Lys- and d-Lys-containing peptide stems are both present in the polymer, the latter being involved in the formation of two novel types of peptidoglycan cross-link. 相似文献
4.
Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO4. Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of l-glutamate (Glu) and its analog l-methionine-d,l-sulfoximine (l-MSO). d-Glu and the l isomers of the protein amino acids did not elicit alkalinization. l-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar l-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H+/106 cells·minute. l-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of l-[U-14C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by l-MSO. l-Glu had no influence on K+ efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific l-Glu/proton uptake process is present in Asparagus mesophyll cells. 相似文献
5.
Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [15N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[15N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[15N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants. 相似文献
6.
David B. Donner 《The Biochemical journal》1982,208(1):211-219
Native insulin inhibits the binding and degradation of 125I-labelled insulin in parallel. Half-maximal inhibition of degradation occurs with 10nm-insulin, a hormone concentration sufficient to saturate the insulin receptor. The proportion of bound hormone that is degraded increases as the insulin concentration is increased, suggesting that low-affinity uptake is functionally related to degradation. Since only a small fraction (approx. 10%) of the overall degradation occurs at the plasma membrane, or in the extracellular medium, translocation of bound hormone into the cell is the predominant mechanism mediating the degradation of insulin. In the presence of 0.6nm-insulin, a concentration at which most cell-associated hormone is receptor-bound, chloroquine increases the amount of 125I-labelled insulin retained by hepatocytes. However, chloroquine increases the retention of degradation products of insulin in incubations containing sufficient hormone (6nm) to saturate the receptor and permit occupancy of low-affinity sites. Glucagon does not compete for the interaction of 125I-labelled insulin (1nm) with the insulin receptor. In contrast, 20μm-glucagon inhibits 75% of the uptake of insulin (0.1μm) by low-affinity sites. A fraction of the cell-bound radioactivity is not intact insulin throughout a 90min association reaction at 37°C. During dissociation, fragments of 125I-labelled insulin are released to the medium more rapidly than is intact hormone. The production and transient retention of degradation products of the hormone complicates the characterization of the insulin receptor by equilibrium or kinetic methods of assay. It is proposed that insulin degradation occurs by receptor- and non-receptor-mediated pathways. The latter may be related to the action of glutathione–insulin transhydrogenase, with which both insulin and glucagon interact. 相似文献
7.
Aili Fan ) Georg Zocher Edyta Stec Thilo Stehle Shu-Ming Li ) 《The Journal of biological chemistry》2015,290(3):1364-1373
The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C4- and C7-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C3-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis. 相似文献
8.
Promotion of seed germination by cyanide 总被引:2,自引:2,他引:0
Potassium cyanide at 3 μm to 10 mm promotes germination of Amaranthus albus, Lactuca sativa, and Lepidium virginicum seeds. l-Cysteine hydrogen sulfide lyase, which catalyzes the reaction of HCN with l-cysteine to form β-l cyanoalanine, is active in the seeds. β-l-Cyanoalanine is the most effective of the 23 α-amino acids tested for promoting germination of A. albus seeds. Aspartate, which is produced by enzymatic hydrolysis of asparagine formed by hydrolysis from β-cyanoalanine, is the second most effective of the 23 amino acids. Uptake of aspartate-4-14C is much lower than of cyanide. 相似文献
9.
Tetsu Shimizu Naoki Takaya Akira Nakamura 《The Journal of biological chemistry》2012,287(48):40448-40456
An l-glucose-utilizing bacterium, Paracoccus sp. 43P, was isolated from soil by enrichment cultivation in a minimal medium containing l-glucose as the sole carbon source. In cell-free extracts from this bacterium, NAD+-dependent l-glucose dehydrogenase was detected as having sole activity toward l-glucose. This enzyme, LgdA, was purified, and the lgdA gene was found to be located in a cluster of putative inositol catabolic genes. LgdA showed similar dehydrogenase activity toward scyllo- and myo-inositols. l-Gluconate dehydrogenase activity was also detected in cell-free extracts, which represents the reaction product of LgdA activity toward l-glucose. Enzyme purification and gene cloning revealed that the corresponding gene resides in a nine-gene cluster, the lgn cluster, which may participate in aldonate incorporation and assimilation. Kinetic and reaction product analysis of each gene product in the cluster indicated that they sequentially metabolize l-gluconate to glycolytic intermediates, d-glyceraldehyde-3-phosphate, and pyruvate through reactions of C-5 epimerization by dehydrogenase/reductase, dehydration, phosphorylation, and aldolase reaction, using a pathway similar to l-galactonate catabolism in Escherichia coli. Gene disruption studies indicated that the identified genes are responsible for l-glucose catabolism. 相似文献
10.
Christos Gournas Thomas Evangelidis Alexandros Athanasopoulos Emmanuel Mikros Vicky Sophianopoulou 《The Journal of biological chemistry》2015,290(10):6141-6155
Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly56, Thr57), TMS3 (Glu138), and TMS6 (Phe248), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent Km for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle. 相似文献
11.
Yingkui Yang Vinod Mishra Chiquito J. Crasto Min Chen Reed Dimmitt Carroll M. Harmon 《The Journal of biological chemistry》2015,290(12):7685-7692
The ACTH receptor, known as the melanocortin-2 receptor (MC2R), plays an important role in regulating and maintaining adrenocortical function. MC2R is a subtype of the melanocortin receptor (MCR) family and has unique characteristics among MCRs. Endogenous ACTH is the only endogenous agonist for MC2R, whereas the melanocortin peptides α-, β-, and γ-melanocyte-stimulating hormone and ACTH are full agonists for all other MCRs. In this study, we examined the molecular basis of MC2R responsible for ligand selectivity using ACTH analogs and MC2R mutagenesis. Our results indicate that substitution of Phe7 with d-Phe or d-naphthylalanine (d-Nal(2′)) in ACTH(1–24) caused a significant decrease in ligand binding affinity and potency. Substitution of Phe7 with d-Nal(2′) in ACTH(1–24) did not switch the ligand from agonist to antagonist at MC2R, which was observed in MC3R and MC4R. Substitution of Phe7 with d-Phe7 in ACTH(1–17) resulted in the loss of ligand binding and activity. Molecular analysis of MC2R indicated that only mutation of the third transmembrane domain of MC2R resulted in a decrease in d-Phe ACTH binding affinity and potency. Our results suggest that Phe7 in ACTH plays an important role in ligand selectivity and that the third transmembrane domain of MC2R is crucial for ACTH selectivity and potency. 相似文献
12.
13.
Hao Cai Xin Wang Zhixin Zhang Juan Chen Fangbin Wang Lu Wang Jian Liu 《The Journal of biological chemistry》2022,298(4)
As a crucial metabolic intermediate, l-lactate is involved in redox balance, energy balance, and acid–base balance in organisms. Moderate exercise training transiently elevates plasma l-lactate levels and ameliorates obesity-associated type 2 diabetes. However, whether moderate l-lactate administration improves obesity-associated insulin resistance remains unclear. In this study, we defined 800 mg/kg/day as the dose of moderate l-lactate administration. In mice fed with a high-fat diet (HFD), moderate l-lactate administration for 12 weeks was shown to alleviate weight gain, fat accumulation, and insulin resistance. Along with the phenotype alterations, white adipose tissue thermogenesis was also found to be elevated in HFD-fed mice. Meanwhile, moderate l-lactate administration suppressed the infiltration and proinflammatory M1 polarization of adipose tissue macrophages (ATMs) in HFD-fed mice. Furthermore, l-lactate treatment suppressed the lipopolysaccharide-induced M1 polarization of bone marrow–derived macrophages (BMDMs). l-lactate can bind to the surface receptor GPR132, which typically drives the downstream cAMP–PKA signaling. As a nutrient sensor, AMP-activated protein kinase (AMPK) critically controls macrophage inflammatory signaling and phenotype. Thus, utilizing inhibitors of the kinases PKA and AMPK as well as siRNA against GPR132, we demonstrated that GPR132–PKA–AMPKα1 signaling mediated the suppression caused by l-lactate treatment on BMDM M1 polarization. Finally, l-lactate addition remarkably resisted the impairment of lipopolysaccharide-treated BMDM conditional media on adipocyte insulin sensitivity. In summary, moderate l-lactate administration suppresses ATM proinflammatory M1 polarization through activation of the GPR132–PKA–AMPKα1 signaling pathway to improve insulin resistance in HFD-fed mice, suggesting a new therapeutic and interventional approach to obesity-associated type 2 diabetes. 相似文献
14.
Soo-Jin Yeom Jung-Hwan Ji Nam-Hee Kim Chang-Su Park Deok-Kun Oh 《Applied and environmental microbiology》2009,75(14):4705-4710
The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co2+. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, l-ribose, as a potential starting material for many l-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter l-ribulose by mannose-6-phosphate isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter−1 h−1.l-Ribose is a potential starting material for the synthesis of many l-nucleoside-based pharmaceutical compounds, and it is not abundant in nature (5, 19). l-Ribose has been produced mainly by chemical synthesis from l-arabinose, l-xylose, d-glucose, d-galactose, d-ribose, or d-mannono-1,4-lactone (2, 17, 23). Biological l-ribose manufacture has been investigated using ribitol or l-ribulose. Recently, l-ribose was produced from ribitol by a recombinant Escherichia coli containing an NAD-dependent mannitol-1-dehydrogenase (MDH) with a 55% conversion yield when 100 g/liter ribitol was used in a 72-h fermentation (18). However, the volumetric productivity of l-ribose in the fermentation is 28-fold lower than that of the chemical method synthesized from l-arabinose (8). l-Ribulose has been biochemically converted from l-ribose using an l-ribose isomerase from an Acinetobacter sp. (9), an l-arabinose isomerase mutant from Escherichia coli (4), a d-xylose isomerase mutant from Actinoplanes missouriensis (14), and a d-lyxose isomerase from Cohnella laeviribosi (3), indicating that l-ribose can be produced from l-ribulose by these enzymes. However, the enzymatic production of l-ribulose is slow, and the enzymatic production of l-ribose from l-ribulose has been not reported.Sugar phosphate isomerases, such as ribose-5-phosphate isomerase, glucose-6-phosphate isomerase, and galactose-6-phosphate isomerase, work as general aldose-ketose isomerases and are useful tools for producing rare sugars, because they convert the substrate sugar phosphates and the substrate sugars without phosphate to have a similar configuration (11, 12, 21, 22). l-Ribose isomerase from an Acinetobacter sp. (9) and d-lyxose isomerase from C. laeviribosi (3) had activity with l-ribose, d-lyxose, and d-mannose. Thus, we can apply mannose-6-phosphate (EC 5.3.1.8) isomerase to the production of l-ribose, because there are no sugar phosphate isomerases relating to l-ribose and d-lyxose. The production of the expensive sugar l-ribose (bulk price, $1,000/kg) from the rare sugar l-ribulose by mannose-6-phosphate isomerase may prove to be a valuable industrial process, because we have produced l-ribulose from the cheap sugar l-arabinose (bulk price, $50/kg) using the l-arabinose isomerase from Geobacillus thermodenitrificans (20) (Fig. (Fig.11).Open in a separate windowFIG. 1.Schematic representation for the production of l-ribulose from l-arabinose by G. thermodenitrificans l-arabinose isomerase and the production of l-ribose from l-ribulose by B. subtilis mannose-6-phosphate isomerase.In this study, the gene encoding mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in E. coli. The substrate specificity of the recombinant enzyme for various aldoses and ketoses was investigated, and l-ribulose exhibited the highest activity among all pentoses and hexoses. Therefore, mannose-6-phosphate isomerase was applied to the production of l-ribose from l-ribulose. 相似文献
15.
Leaf discs and detached leaves exposed to l-cysteine emitted a volatile sulfur compound which was proven by gas chromatography to be H2S. This phenomenon was demonstrated in all nine species tested (Cucumis sativus, Cucurbita pepo, Nicotiana tabacum, Coleus blumei, Beta vulgaris, Phaseolus vulgaris, Medicago sativa, Hordeum vulgare, and Gossypium hirsutum). The emission of volatile sulfur by cucumber leaves occurred in the dark at a similar rate to that in the light. The emission of leaf discs reached the maximal rate, more than 40 picomoles per minute per square centimeter, 2 to 4 hours after starting exposure to l-cysteine; then it decreased. In the case of detached leaves, the maximum occurred 5 to 10 h after starting exposure. The average emission rate of H2S during the first 4 hours from leaf discs of cucurbits in response to 10 millimolar l-cysteine, was usually more than 40 picomoles per minute per square centimeter, i.e. 0.24 micromoles per hour per square decimeter. Leaf discs exposed to 1 millimolar l-cysteine emitted only 2% as much as did the discs exposed to 10 millimolar l-cysteine. The emission from leaf discs and from detached leaves lasted for at least 5 and 15 hours, respectively. However, several hours after the maximal emission, injury of the leaves, manifested as chlorosis, was evident. H2S emission was a specific consequence of exposure to l-cysteine; neither d-cysteine nor l-cystine elicited H2S emission. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate dependent enzymes, inhibited the emission. In a cell free system from cucumber leaves, H2S formation and its release occurred in response to l-cysteine. Feeding experiments with [35S]l-cysteine showed that most of the sulfur in H2S was derived from sulfur in the l-cysteine supplied and that the H2S emitted for 9 hours accounted for 7 to 10% of l-cysteine taken up. 35S-labeled SO32− and SO42− were found in the tissue extract in addition to internal soluble S2−. These findings suggest the existence of a sulfur cycle which converts l-cysteine to SO42− through cysteine desulfhydration. 相似文献
16.
Manami Oya Tetsuya Kitaguchi Ramona Pais Frank Reimann Fiona Gribble Takashi Tsuboi 《The Journal of biological chemistry》2013,288(7):4513-4521
Although amino acids are dietary nutrients that evoke the secretion of glucagon-like peptide 1 (GLP-1) from intestinal L cells, the precise molecular mechanism(s) by which amino acids regulate GLP-1 secretion from intestinal L cells remains unknown. Here, we show that the G protein-coupled receptor (GPCR), family C group 6 subtype A (GPRC6A), is involved in amino acid-induced GLP-1 secretion from the intestinal L cell line GLUTag. Application of l-ornithine caused an increase in intracellular Ca2+ concentration ([Ca2+]i) in GLUTag cells. Application of a GPRC6A receptor antagonist, a phospholipase C inhibitor, or an IP3 receptor antagonist significantly suppressed the l-ornithine-induced [Ca2+]i increase. We found that the increase in [Ca2+]i stimulated by l-ornithine correlated with GLP-1 secretion and that l-ornithine stimulation increased exocytosis in a dose-dependent manner. Furthermore, depletion of endogenous GPRC6A by a specific small interfering RNA (siRNA) inhibited the l-ornithine-induced [Ca2+]i increase and GLP-1 secretion. Taken together, these findings suggest that the GPRC6A receptor functions as an amino acid sensor in GLUTag cells that promotes GLP-1 secretion. 相似文献
17.
Krzysztof Regulski Pascal Courtin Saulius Kulakauskas Marie-Pierre Chapot-Chartier 《The Journal of biological chemistry》2013,288(28):20416-20426
Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala4→d-Asx-l-Lys3 in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala4→l-Ala-(l-Ala/l-Ser)-l-Lys3; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e.
d-Ala4→d-Asp-l-Lys3. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys3-d-Asn-l-Lys3 bridges replacing the wild-type 4→3 d-Ala4-d-Asn-l-Lys3 bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG. 相似文献
18.
The sulfur requirements of tobacco (Nicotiana tabacum L. var. Xanthi) XD cells grown in chemically defined liquid media can be satisfied by sulfate, thiosulfate, l-cyst(e)ine, l-methionine or glutathione, and somewhat less effectively by d-cyst (e) ine, d-methionine or dl-homocyst (e)ine. Sulfate uptake is inhibited after a 2 hr lag by l-cyst (e)ine, l-methionine, l-homocyst(e)ine or l-isoleucine, but not by any of the other protein amino acids, nor by d-cyst(e)ine. l-cyst(e)ine is neither a competitive nor a non-competitive inhibitor of sulfate uptake. Its action most closely resembles apparent uncompetitive inhibition. Inhibition of sulfate uptake by l-cyst(e)ine can be partially prevented by equimolar l-arginine, l-lysine, l-leucine, l-phenylalanine, l-tyrosine or l-tryptophan, but is little affected by any of the other protein amino acids. The effective amino acids are apparent competitive inhibitors of l-cyst(e)ine uptake after a 2 hr lag. Inhibition of sulfate uptake by l-methionine cannot be prevented, nor can uptake of l-methionine be inhibited by any single protein amino acid. The results suggest the occurrence of negative feedback control of sulfate assimilation by the end products, the sulfur amino acids, in cultured tobacco cells. 相似文献
19.
Li Min Zhongmin Jin Ljubica Caldovic Hiroki Morizono Norma M. Allewell Mendel Tuchman Dashuang Shi 《The Journal of biological chemistry》2009,284(8):4873-4880
N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in
l-arginine biosynthesis in plants and micro-organisms and is subject to feedback
inhibition by l-arginine. This study compares the crystal structures of NAGS from
Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with
l-arginine bound and in the active R-state complexed with CoA and
l-glutamate. Under all of the conditions examined, the enzyme consists of two stacked
trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold
but lacking kinase activity and an N-acetyltransferase (NAT) domain
homologous to other GCN5-related transferases. Binding of l-arginine to the AAK
domain induces a global conformational change that increases the diameter of the hexamer by
∼10 Å and decreases its height by
∼20Å. AAK dimers move 5Å outward along their
2-fold axes, and their tilt relative to the plane of the hexamer decreases by
∼4°. The NAT domains rotate ∼109° relative to
AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on
different subunits also change. Local motions of several loops at the
l-arginine-binding site enable the protein to close around the bound ligand, whereas
several loops at the NAT active site become disordered, markedly reducing enzymatic specific
activity.l-Arginine biosynthesis in most micro-organisms and plants involves the initial
acetylation of l-glutamate by N-acetylglutamate synthase (NAGS, EC
2.3.1.1)2 to produce N-acetylglutamate
(NAG). NAG is then converted by NAG kinase (NAGK, EC 2.7.2.8) to NAG-phosphate and subsequently
to N-acetylornithine (1, 2). Two alternative reactions are used to remove the acetyl
group from acetylornithine. The linear pathway uses N-acetylornithine
deacetylase (EC 3.5.1.16) to catalyze the metal-dependent hydrolysis of the acetyl group to form
l-ornithine and acetate, whereas the acetyl recycling pathway transfers the acetyl
group from N-acetylornithine to l-glutamate, producing
l-ornithine and NAG. This reaction is catalyzed by ornithine acetyltransferase (EC
2.3.1.35).In the linear pathway, NAGS is the only target of feedback inhibition by l-arginine.
In contrast, in the acetyl cycling pathway l-arginine may inhibit NAGS and NAGK or
ornithine acetyltransferase (3). Structure
determinations of l-arginine-insensitive (4)
and l-arginine-sensitive NAGKs (5) provided
insights into the structural basis of l-arginine inhibition of NAGK.
l-Arginine-insensitive Escherichia coli (ec) NAGK is
a homodimer (4), whereas l-arginine-sensitive
NAGKs from Thermotoga maritima (tm) and Pseudomonas
aeruginosa (pa) are hexamers formed by pair-wise interlacing of the
N-terminal helices of three ecNAGK-like dimers, to create a second type of
dimer interface. l-Arginine binding to a site close to the C terminus induces global
conformational changes that expands the ring by ∼8 Å and decreases the
tilt of the ecNAGK-like dimers relative to the plane of the ring by
∼6°. The inhibition mechanism was proposed to involve the
enlargement of an active site located close to the l-arginine-binding site.Because of the sequence similarity between NAGK and NAGS, it was speculated that they may have
similar l-arginine-binding sites and hexameric ring structures (5). However, our recent structural determination of NAGS from
Neisseria gonorrhoeae (ng) revealed the active site to be
located in the NAT domain, >25 Å away from the proposed
l-arginine-binding site (6). Therefore, the
allosteric mechanism of NAGS is likely to be different from that of
l-arginine-sensitive NAGKs. Here we compare the structures of ngNAGS
in the inactive T-state with l-arginine bound and in the R-state complexed with CoA and
l-glutamate and determine the structural basis for the allosteric inhibition of NAGS
by l-arginine. 相似文献
20.
Makoto Hibi Ryosuke Mori Ryoma Miyake Hiroshi Kawabata Shoko Kozono Satomi Takahashi Jun Ogawa 《Applied and environmental microbiology》2016,82(7):2070-2077
Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l-pipecolic acid (l-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l-Pip, and produces optically pure trans-4-hydroxy-l-pipecolic acid (trans-4-l-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had l-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-l-HyPip was successfully developed. Thus, we report a fungal family of l-Pip hydroxylases and the enzymatic preparation of trans-4-l-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities. 相似文献