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Functional mechanism of C-terminal tail in the enzymatic role of porcine testicular carbonyl reductase: a combined experiment and molecular dynamics simulation study of the C-terminal tail in the enzymatic role of PTCR.

ABSTRACT: Porcine testicular carbonyl reductase, PTCR which is one of the short chain dehydrogenases/reductases (SDR) superfamily catalyzes the NADPH-dependent reduction of carbonyl compounds including steroids and prostaglandins. Previously we reported C-terminal tail of PTCR was deleted due to a nonsynonymous single nucleotide variation (nsSNV). Here we identified from kinetic studies that the enzymatic properties for 5α-dihydrotestosterone (5α-DHT) were different between wild-type and C-terminal-deleted PTCRs. Compared to wild-type PTCR, C-terminal-deleted PTCR has much higher reduction rate. To investigate structural difference between wild-type and C-terminal-deleted PTCRs upon 5α-DHT binding, we performed molecular dynamics simulations for two complexes. Using trajectories, molecular interactions including hydrogen bonding patterns, distance between 5α-DHT and catalytic Tyr193, and interaction energies are analyzed and compared. During the MD simulation time, the dynamic behavior of C-terminal tail in wild-type PTCR is also examined using essential dynamics analysis. The results of our simulations reveal that the binding conformation of 5α-DHT in C-terminal-deleted PTCR is more favorable for reduction reaction in PTCR, which shows strong agreement with kinetic data. These structural findings provide valuable information to understand substrate specificity of PTCR and further kinetic properties of enzymes belonging to the SDR superfamily.

SUBMITTER: Son M

PROVIDER: S-EPMC3960098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Enzymatic characterization of a recombinant carbonyl reductase from <i>Acetobacter</i> sp. CCTCC M209061.

Project description:BackgroundAcetobacter sp. CCTCC M209061 could catalyze carbonyl compounds to chiral alcohols following anti-Prelog rule with excellent enantioselectivity. Therefore, the enzymatic characterization of carbonyl reductase (CR) from Acetobacter sp. CCTCC M209061 needs to be investigated.ResultsA CR from Acetobacter sp. CCTCC M209061 (AcCR) was cloned and expressed in E. coli. AcCR was purified and characterized, finding that AcCR as a dual coenzyme-dependent short-chain dehydrogenase/reductase (SDR) was more preferred to NADH for biocatalytic reactions. The AcCR was activated and stable when the temperature was under 35 °C and the pH range was from 6.0 to 8.0 for the reduction of 4'-chloroacetophenone with NADH as coenzyme, and the optimal temperature and pH were 45 °C and 8.5, respectively, for the oxidation reaction of isopropanol with NAD+. The enzyme showed moderate thermostability with half-lives of 25.75 h at 35 °C and 13.93 h at 45 °C, respectively. Moreover, the AcCR has broad substrate specificity to a range of ketones and ketoesters, and could catalyze to produce chiral alcohol with e.e. >99% for the majority of tested substrates following the anti-Prelog rule.ConclusionsThe recombinant AcCR exhibited excellent enantioselectivity, broad substrate spectrum, and highly stereoselective anti-Prelog reduction of prochiral ketones. These results suggest that AcCR is a powerful catalyst for the production of anti-Prelog alcohols.Graphical abstractThe biocatalytic reactions conducted with the recombinant AcCR.

| S-EPMC5573764 | biostudies-literature

Functional characterization of the promoter of carbonyl reductase 1 gene in porcine endometrial cells.

Project description:Prostaglandins (PGs) play a critical role in porcine reproduction, of which prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) exert antiluteolytic and luteolysis actions, respectively. As a rate-limiting enzyme, carbonyl reductase 1 (CBR1) catalyzes the conversion of PGE2 to PGF2α. A high ratio of PGE2:PGF2α is beneficial to the establishment and maintenance of porcine pregnancy. PG is essential for the establishment of pregnancy which resembles the proinflammatory response and nuclear factor κB (NF-κB) is involved in the process. Bioinformatic analysis has shown that NF-κB is a possible factor bound to two cis-regulatory elements in CBR1 promoter. In this study, we cloned the 2997 bp (-2875/+122) of the promoter, and constructed six 5'-deleted dual-luciferase reporter recombinant vectors. In endometrial cells, the region of P2 (-1640/+7) exhibited the greatest transcriptional activity at driving luciferase expression, but not significantly different from that of P1 (-2089/+7). The activity of P1, P2, and P3 (-1019/+7) was highly significantly higher than that of others (P<0.01), suggesting that two positive regulatory elements were likely present in the regions of -1640/-1019 and -1019/-647. The results also showed that the -1640/-647 region was indispensable for the promoter. The results of chromatin immunoprecipitation (ChIP) demonstrated that the NF-κB subunit p65 binds to one site around -1545/-1531. Using four reference genes, we found that the over-expression of p65 enhanced the expression of CBR1 (P<0.05) in porcine endometrial epithelial cells, while knockdown of the p65 did not down-regulate the CBR1 expression. These results indicated that NF-κB (p65) could bind to the special element of CBR1 gene promoter in porcine endometrial epithelial cells in vitro. The binding site of NF-κB was a positive regulator for the CBR1 gene promoter, but was not necessary for the basic expression.

| S-EPMC5498843 | biostudies-literature

Human carbonyl reductase 1 is an S-nitrosoglutathione reductase.

Project description:Human carbonyl reductase 1 (hCBR1) is an NADPH-dependent short chain dehydrogenase/reductase with broad substrate specificity and is thought to be responsible for the in vivo reduction of quinones, prostaglandins, and other carbonyl-containing compounds including xenobiotics. In addition, hCBR1 possesses a glutathione binding site that allows for increased affinity toward GSH-conjugated molecules. It has been suggested that the GSH-binding site is near the active site; however, no structures with GSH or GSH conjugates have been reported. We have solved the x-ray crystal structures of hCBR1 and a substrate mimic in complex with GSH and the catalytically inert GSH conjugate hydroxymethylglutathione (HMGSH). The structures reveal the GSH-binding site and provide insight into the affinity determinants for GSH-conjugated substrates. We further demonstrate that the structural isostere of HMGSH, S-nitrosoglutathione, is an ideal hCBR1 substrate (Km = 30 microm, kcat = 450 min(-1)) with kinetic constants comparable with the best known hCBR1 substrates. Furthermore, we demonstrate that hCBR1 dependent GSNO reduction occurs in A549 lung adenocarcinoma cell lysates and suggest that hCBR1 may be involved in regulation of tissue levels of GSNO.

| S-EPMC2602912 | biostudies-literature

Spike-in Experiment for ChIP-chip Simulation

Project description:The most widely-used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms and analysis algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. All commercial tiling array platforms performed well, although each platform and analysis algorithm had distinct performance and cost characteristics. Simple sequence repeats and genome redundancy tend to result in false positives on oligonucleotide platforms. The spike-in DNA samples and the resulting array data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. Keywords: chip-ChIP simulation For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

2007-12-01 | GSE9732 | GEO

Functional role of C-terminal cytoplasmic tail of rat vanilloid receptor 1.

Project description:The vanilloid receptor [transient receptor potential (TRP)V1, also known as VR1] is a member of the TRP channel family. These receptors share a significant sequence homology, a similar predicted structure with six transmembrane-spanning domains (S1-S6), a pore-forming region between S5 and S6, and the cytoplasmically oriented C- and N-terminal regions. Although structural/functional studies have identified some of the key amino acids influencing the gating of the TRPV1 ion channel, the possible contributions of terminal regions to vanilloid receptor function remain elusive. In the present study, C-terminal truncations of rat TRPV1 have been constructed to characterize the contribution of the cytoplasmic C-terminal region to TRPV1 function and to delineate the minimum amount of C tail necessary to form a functional channel. The truncation of 31 residues was sufficient to induce changes in functional properties of TRPV1 channel. More pronounced effects of C-terminal truncation were seen in mutants lacking the final 72 aa. These changes were characterized by a decline of capsaicin-, pH-, and heat-sensitivity; progressive reduction of the activation thermal threshold (from 41.5 to 28.6 degrees C); and slowing of the activation rate of heat-evoked membrane currents (Q10 from 25.6 to 4.7). The voltage-induced currents of the truncated mutants exhibited a slower onset, markedly reduced outward rectification, and significantly smaller peak tail current amplitudes. Truncation of the entire TRPV1 C-terminal domain (155 residues) resulted in a nonfunctional channel. These results indicate that the cytoplasmic COOH-terminal domain strongly influences the TRPV1 channel activity, and that the distal half of this structural domain confers specific thermal sensitivity.

| S-EPMC6742269 | biostudies-literature

The role of the C-terminal tail of flavocytochrome b2.

Project description:A flavocytochrome b2 (L-lactate dehydrogenase) mutant was constructed in which the C-terminal tail (23 amino acid residues) had been deleted (Gly-489----Stop). This tail appears to form many intersubunit contacts in the tetrameric wild-type protein, and it was expected that its removal might lead to the formation of monomeric flavocytochrome b2. The isolated tail-deleted mutant enzyme (TD-b2), however, was found to be tetrameric (Mr 220,000). TD-b2 shows Km and kcat. values (at 25 degrees C and pH 7.5) of 0.96 +/- 0.06 mM and 165 +/- 6 s-1 respectively compared with 0.49 +/- 0.04 mM and 200 +/- 10 s-1 for the wild-type enzyme. The kinetic isotope effect with [2-2H]lactate as substrate seen for TD-b2, with ferricyanide as electron acceptor, was essentially the same as that observed for the wild-type enzyme. TD-b2 exhibited loss of activity during turnover in a biphasic process. The rate of the faster of the two phases was dependent on L-lactate concentration and at saturating concentrations showed a first-order deactivation rate constant, kf(deact.), of 0.029 s-1 (at 25 degrees C and pH 7.5). The slower phase, however, was independent of L-lactate concentration and gave a first-order deactivation rate constant, ks(deact.), of 0.01 s-1 (at 25 degrees C and pH 7.5). This slower phase was found to correlate with dissociation of FMN, which is one of the prosthetic groups of the enzyme. Thus fully deactivated TD-b2, which was also tetrameric, was found to be completely devoid of FMN. Much of the original activity of TD-b2 could be recovered by re-incorporation of FMN. Thus the C-terminal tail of flavocytochrome b2 appears to be required for the structural integrity of the enzyme around the flavin active site even though the two are well separated in space.

| S-EPMC1133508 | biostudies-other

Molecular dynamics simulation of Escherichia coli dihydrofolate reductase and its protein fragments: relative stabilities in experiment and simulations.

Project description:We have carried out molecular dynamics simulations of the native dihydrofolate reductase from Escherichia coli and several of its folded protein fragments at standard temperature. The simulations have shown fragments 1--36, 37--88, and 89--159 to be unstable, with a C(alpha)RMSD (C(alpha) root mean squared deviation) >5 A after 3.0 nsec of simulation. The unfolding of fragment 1--36 was immediate, whereas fragments 37--88 and 89--159 gradually unfolded because of the presence of the beta-sheet core structure. In the absence of residues 1--36, the two distinct domains comprising fragment 39--159 associated with each other, resulting in a stable conformation. This conformation retained most of its native structural elements. We have further simulated fragments derived from computational protein cutting. These were also found to be unstable, with the exception of fragment 104--159. In the absence of alpha(4), the loose loop region of residues 120--127 exhibited a beta-strand-like behavior, associating itself with the beta-sheet core of the protein fragment. The current study suggests that the folding of dihydrofolate reductase involves cooperative folding of distinct domains which otherwise would have been unstable as independent folded units in solution. Finally, the critical role of residues 1--36 in allowing the two distinct domains of fragment 104--159 to fold into the final native conformation is discussed.

| S-EPMC2249846 | biostudies-literature

A potentially crucial role of the PKD1 C-terminal tail in renal prognosis.

Project description:BackgroundAutosomal dominant polycystic disease (ADPKD) often results in renal failure. Recently, allelic influences of PKD1 mutation types on renal survival were extensively investigated. Here, we analyzed integrated influences of PKD1 mutation types and positions on renal survival.MethodsWe included 338 (82 pedigrees) and 72 (12 pedigrees) patients with PKD1 and PKD2 mutations, respectively, identified through comprehensive gene analysis of 101 probands with ADPKD. Genetic testing was performed using next-generation sequencing, long-range PCR, and multiplex ligation-dependent probe amplification. Pathogenic mutations were identified by a software package-integrated seven databases and provided access to five cloud-based computing systems.ResultsMean renal survivals of carriers with PKD1 non-truncating-type mutations at positions upstream of G-protein-coupled receptor proteolytic site (GPS-upstream domain), transmembrane domain, or cytoplasmic C-terminal tail (CTT) domain were 70.2, 67.0, and 50.1 years, respectively (P < 0.0001); renal survival was shorter for mutation positions closer to CTT domain, suggesting its crucial role in renal prognosis. Furthermore, in truncating-type mutations, strong inactivation is anticipated on nucleotides downstream from the mutation site, implying CTT domain inactivation irrespective of mutation site. Shorter mean renal survival was found for PKD1 truncating-type than non-truncating-type mutation carriers (P = 0.0348); mean renal survival was not different between PKD1 3'- and 5'-region truncating-type mutation carriers (P = 0.4375), but was shorter in PKD1 3'-region than in 5'-region non-truncating-type mutation carriers (P = 0.0014). Variable strength of CTT domain inactivation might account for these results.ConclusionsAforementioned findings indicate that CTT domain's crucial role in renal prognosis needs further investigation by larger studies (ClinicalTrials.gov; NCT02322385).

| S-EPMC5838153 | biostudies-literature

Histone H4 N-terminal tail mutants

Project description:A series of yeast carrying K->R mutations in the histone H4 N-terminal tail were profiled by gene expression array in mid-log phase.

2010-06-10 | E-GEOD-2324 | biostudies-arrayexpress

GRK2 activation by receptors: role of the kinase large lobe and carboxyl-terminal tail.

Project description:G protein-coupled receptor (GPCR) kinases (GRKs) were discovered by virtue of their ability to phosphorylate activated GPCRs. They constitute a branch of the AGC kinase superfamily, but their mechanism of activation is largely unknown. To initiate a study of GRK2 activation, we sought to identify sites on GRK2 remote from the active site that are involved in interactions with their substrate receptors. Using the atomic structure of GRK2 in complex with Gbetagamma as a guide, we predicted that residues on the surface of the kinase domain that face the cell membrane would interact with the intracellular loops and carboxyl-terminal tail of the GPCR. Our study focused on two regions: the kinase large lobe and an extension of the kinase domain known as the C-tail. Residues in the GRK2 large lobe whose side chains are solvent exposed and facing the membrane were targeted for mutagenesis. Residues in the C-tail of GRK2, although not ordered in the crystal structure, were also targeted because this region has been implicated in receptor binding and in the regulation of AGC kinase activity. Four substitutions out of 20, all within or adjacent to the C-tail, resulted in significant deficiencies in the ability of the enzyme to phosphorylate two different GPCRS: rhodopsin, and the beta(2)-adrenergic receptor. The mutant exhibiting the most dramatic impairment, V477D, also showed significant defects in phosphorylation of nonreceptor substrates. Interestingly, Michaelis-Menten kinetics suggested that V477D had a 12-fold lower k(cat), but no changes in K(M), suggesting a defect in acquisition or stabilization of the closed state of the kinase domain. V477D was also resistant to activation by agonist-treated beta(2)AR. Therefore, Val477 and other residues in the C-tail are expected to play a role in the activation of GRK2 by GPCRs.

| S-EPMC2744320 | biostudies-literature

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