Deubiquitination complex platform: A plausible mechanism for regulating the substrate specificity of deubiquitinating enzymes

Deubiquitinating enzymes (DUBs) or deubiquitinases facilitate the escape of multiple proteins from ubiquitin‒proteasome degradation and are critical for regulating protein expression levels in vivo. Therefore, dissecting the underlying mechanism of DUB recognition is needed to advance the development of drugs related to DUB signaling pathways. To data, extensive studies on the ubiquitin chain specificity of DUBs have been reported, but substrate protein recognition is still not clearly understood. As a breakthrough, the scaffolding role may be significant to substrate protein selectivity. From this perspective, we systematically characterized the scaffolding proteins and complexes contributing to DUB substrate selectivity. Furthermore, we proposed a deubiquitination complex platform (DCP) as a potentially generic mechanism for DUB substrate recognition based on known examples, which might fill the gaps in the understanding of DUB substrate specificity.

Biochemical characterization with kinetic studies of melanogenic enzyme tyrosinase from white button mushroom, Agaricus bisporus

In Agaricus bisporus, color is a key determinant for marketability and consumer acceptability. However, postharvest browning has become a major concern, affecting the overall economics of the mushroom industry. In button mushrooms, the tyrosinase enzyme (E.C.1.14.18.1) is responsible for the browning reactions by catalyzing the conversion of monophenols and diphenols into quinones which polymerize to form melanin. Thus, the present study focused on the purification and characterization of tyrosinase from A. bisporus. This enzyme was purified with a final yield of 19.71% and 32.05 purification fold. The study of enzymatic activity over a temperature (5-45°C) and pH range (3-10) showed that the optimum temperature was 35°C with pH 7. The kinetic studies revealed that Km values were different for catechol (0.71 mM) and L-dopa (0.87 mM), which indicated a higher affinity of the enzyme for catechol. Inhibition studies showed that cinnamic acid is a non-competitive inhibitor while salicylic acid is a competitive inhibitor of tyrosinase. The molecular weight of the enzyme was found to be 43 kDa and different amide regions were reflected by the FTIR spectra of the enzyme. This study may provide valuable insights into the structure, biochemical properties, and inhibition of tyrosinase enzyme for controlling mushroom browning.

Research progress in structure and function of pectin methylesterase / 生物工程学报

Pectin methylesterase (PME) is an important pectinase that hydrolyzes methyl esters in pectin to release methanol and reduce the degree of methylation of pectin. At present, it has broad application prospects in food processing, tea beverage, paper making and other production processes. With the in-depth study of PME, the crystal structures with different sources have been reported. Analysis of these resolved crystal structures reveals that PME belongs to the right-hand parallel β-helix structure, and its catalytic residues are two aspartic acids and a glutamine, which play the role of general acid-base, nucleophile and stable intermediate, in the catalytic process. At the same time, the substrate specificity is analyzed to understand the recognition mechanism of the substrate and active sites. This paper systematically reviews these related aspects.

Advances in molecular modification of ω-transaminase / 生物工程学报

ω-Transaminase catalyzes the asymmetric reductive amination of carbonyl compounds, and has great application prospect in the preparation of chiral amines. The application in synthesis of bulky chiral amines is limited by the special structure of substrate binding region in the wild-type enzyme. Moreover, there are also some drawbacks in the stereoselectivity and stability of ω-transaminase. So far, -tωransaminase satisfying the industrial requirements is still rare. In this review, we first introduce the structure and catalytic mechanism of ω-transaminase, and then discuss the structural differences between S-selective and R-selective enzymes. Molecular modification of ω-transaminase was introduced in detail, by focusing on the structure and mechanism-based molecular modification, including substrate specificity, stereoselectivity, and stability.

Phospholipase C from Pseudomonas aeruginosa and Bacillus cereus; characterization of catalytic activity

OBJECTIVE@#To study characteristics of phospholipases C (PLCs), their importance for producing microorganisms as well as the potential of their use for industrial purposes.@*METHOD@#PLC from Bacillus cereus (B. cereus) D101 was selected as an example of Gram-positive PLCs and PLC from Pseudomonas aeruginosa (P. aeruginosa) D183 of Gram-negative ones. Enzymes were partially purified by ammonium sulfate precipitation followed by membrane dialysis. Partially purified preparations were used to study effect of different factors on activities as well as in substrate specificity tests which were conducted using a turbidimetric assay method.@*RESULTS@#Maximum activity was at pH 7 and 8 and 40 °C for P. aeruginosa PLC, and pH 8-10 and 37 °C for B. cereus PLC. Both PLCs were inhibited by Pi at 5 mM or higher, whereas, PLC from B. cereus only was inhibited by EDTA. Activity of P. aeruginosa PLC was not affected by removing Zn(2+) ions from reaction mixture or their replacement with Ca(2+), Ba(2+), Mg(2+) or Mn(2+) ions. Vis-à-vis, activity of B. cereus PLC was found to be metal ion dependent. PLCs from both isolates were relatively thermostable and showed maximum affinity toward phosphatidylcholine. Sphingomyelin and phosphatidylethanolamine were not good substrates and phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and cardiolipin could be considered non-substrates.@*CONCLUSION@#Human body physiological conditions could favor activity of P. aeruginosa and B. cereus PLCs. These enzymes may participate in phosphate scavenging and virulence of producing isolates but not in autolysis. PLCs from both isolates are potential candidates for industrial use.

Phospholipase C from Pseudomonas aeruginosa and Bacillus cereus; characterization of catalytic activity

Objective: To study characteristics of phospholipases C (PLCs), their importance for producing microorganisms as well as the potential of their use for industrial purposes. Method: PLC from Bacillus cereus (B. cereus) D101 was selected as an example of Gram-positive PLCs and PLC from Pseudomonas aeruginosa (P. aeruginosa) D183 of Gram-negative ones. Enzymes were partially purified by ammonium sulfate precipitation followed by membrane dialysis. Partially purified preparations were used to study effect of different factors on activities as well as in substrate specificity tests which were conducted using a turbidimetric assay method. Results: Maximum activity was at pH 7 and 8 and 40 °C for P. aeruginosa PLC, and pH 8-10 and 37 °C for B. cereus PLC. Both PLCs were inhibited by Pi at 5 mM or higher, whereas, PLC from B. cereus only was inhibited by EDTA. Activity of P. aeruginosa PLC was not affected by removing Zn

Characterization of the interdependency between residues that bind the substrate in a â-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the â-glycosidase from Spodoptera frugiperda (Sfâgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfâgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl â-galactoside and p-nitrophenyl â-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfâgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on â-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

Purification and characterization of beta-glucosidase from Melanocarpus sp. MTCC 3922

This study reports the purification and characterization of beta-glucosidase from a newly isolated thermophilic fungus, Melanocarpus sp. Microbial Type Culture Collection (MTCC) 3922. The molecular weight of beta-glucosidase was determined to be ~ 92 and 102 kDa with SDS PAGE and gel filtration, respectively, and pI of ~ 4.1. It was optimally active at 60 C and pH 6.0, though was stable at 50 C and pH 5.0 - 6.0. The presence of DTT, mercaptoethanol and metal ions such as Na+, K+, Ca2+, Mg2+and Zn2+ positively influenced the activity of beta-glucosidase but the activity was inhibited in the presence of CuSO4. beta-Glucosidase recognized pNP- beta-glucopyranoside (pNPG) as the preferred substrate, and showed very low affinity for pNP- beta-D-cellobioside. Km and Vmax for the hydrolysis of pNPG by beta-glucosidase was calculated as 3.3 mM and 43.68 ‘molmin-1mg protein-1, respectively and k cat was quantified as 4 x 10³ min-1. beta-Glucosidase activity was enhanced appreciably in the presence of alcohols (methanol and ethanol) moreover, purified beta-glucosidase showed putative transglycosylation activity that was positively catalyzed in presence of methanol as an acceptor molecule

Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a consensus Chk phosphorylation motif

Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1alpha were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1alpha were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1alpha were specific targets for Chk1 and/or Chk2 at least in vitro.

The Substrate Specificity of Cyclic Imide Hydrolase Mutants / 中国生物工程杂志

The effect of C-terminal region residues on the substrate specificity of a novel cyclic imide hydrolase (CIH), a recombinant cyclic imide hydrolase (CIH293), and its mutants deleted or substituted at C-terminus (CIH291, CIH290, KK292-293EE) was reported. The substrate specificity and kinetic parameters of the mutants were analyzed by both the spectrophotometric assay and high-performance liquid chromatography. Results show that the substrate specificity of mutants was not obviously changed, but slightly low for the affinity between the substrate and enzyme, compared with the wild-type enzyme, CIH293. In conclusion, the last three residues of CIH293 play an important role for the enzyme activity.

Alteration of Substrate Specificity by Common Variants, E158K/E308G and V257M, in Human Hepatic Drug-metabolizing Enzyme, Flavin-containing Monooxygenase 3

Our earlier studies found a significant correlation between the activities of ranitidine N-oxidation catalyzed by hepatic flavin-containing monooxygenase (FMO) and the presence of mutations in exon 4 (E158K) and exon 7 (E308G) of the FMO3 gene in Korean volunteers. However, caffeine N-1 demethylation (which is also partially catalyzed by FMO) was not significantly correlated with these FMO3 mutations. In this study, we examined another common mutation (V257M) in exon 6 of FMO3 gene. The V257M variant, which is caused by a point mutation (G769A), was commonly observed (13.21% allele frequency) in our subjects (n=159). This point mutation causes a substitution of Val257 to Met257, with transformation of the secondary structure. The presence of this mutant allele correlated significantly with a reduction in caffeine N-1-demethylating activity, but was not correlated with the activity of N-oxidation of ranitidine. In a family study, the low FMO activity observed in a person heterozygous for a nonsense mutation in exon 4 (G148X) and heterozygous for missense mutation in exon 6 (V257M) of FMO3 was attributed to the mutations. Our results suggest that various point mutations in the coding regions of FMO3 may influence FMO3 activity according to the probe substrates of varying chemical structure that correlate with each mutation on the FMO3 gene.

The research actualities of chitinase and prospects / 中国海洋药物

Chitinase (EC3.2.1.14) catalyses the reaction of hydrolyzing the chitin into N acetylglucosamine (GlcNac) and (GlcNac) n.With the in deep study of chitinase,more and more biological function of chitinase appeared obviously.Now,we introduce the actualities of chitinase research,including substrate specificity,physiological function,antifungi function and transgenics, et al .