ADP competes with FAD binding in putrescine oxidase

Putrescine oxidase from Rhodococcus erythropolis NCIMB 11540 (PuO(Rh)) is a soluble homodimeric flavoprotein of 100 kDa, which catalyzes the oxidative deamination of putrescine and some other aliphatic amines. The initial characterization of PuO(Rh) uncovered an intriguing feature: the enzyme appeared to contain only one noncovalently bound FAD cofactor per dimer. Here we show that this low FAD/protein ratio is the result of tight binding of ADP, thereby competing with FAD binding. MS analysis revealed that the enzyme is isolated as a mixture of dimers containing two molecules of FAD, two molecules ADP, or one FAD and one ADP molecule. In addition, based on a structural model of PuO(Rh) that was built using the crystal structure of human monoamine oxidase B (MAO-B), we constructed an active mutant enzyme, PuO(Rh) A394C, that contains covalently bound FAD. These findings show that the covalent FAD-protein linkage can be formed autocatalytically and hint to a new-found rationale for covalent flavinylation: covalent flavinylation may have evolved to prevent binding of ADP or related cellular compounds, which would prohibit formation of flavinylated and functional enzyme.     

Discovery and characterization of a putrescine oxidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> NCIMB 11540

A gene encoding a putrescine oxidase (PuO_Rh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (K _M = 8.2 μM, k _cat = 26 s⁻¹). PuO_Rh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuO_Rh is a reasonably thermostable enzyme with t _1/2 at 50°C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuO_Rh, which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuO_Rh. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Abscisic acid biosynthesis and catabolism and their regulation roles in fruit ripening

Abscisic acid (ABA) plays a series of significant physiology roles in higher plants including but not limited to promote bud and seed dormancy, accelerate foliage fall, induce stomatal closure, inhibit growth and enhance resistance. Recently, it has been revealed that ABA also has an important regulator role in the growth, development and ripening of fruit. In higher plants ABA is produced from an indirect pathway from the cleavage products of carotenoids. The accumulation of endogenous ABA levels in plants is a dynamic balance controlled by the processes of biosynthesis and catabolism, through the regulation of key ABA biosynthetic gene and enzyme activities. It has been hypothesized that ABA levels could be part of the signal that trigger fruit ripening, and that ABA may play an important role in the regulation of ripening and senescence of both non-climacteric and climacteric fruit. The expensive costs of natural ABA and labile active ABA for its chemical synthesis limit its application in scientific research and agricultural production. These findings that ABA has various of important roles in the regulation of growth and development, quality formation, coloring and softening, ripening and senescence of fruit, are providing opportunities and challenges for Horticultural Science. This is to elucidate the specific mechanism of response and biosynthesis, signal transduction, and receptor recognition of ABA in fruit, employing comprehensive research methods, such as molecular biology, plant physiology and molecular genetics. Further and more in-depth research about ABA has a great, realistic significance for knowing the mechanisms behind the process of fruit ripening.     

Spatial and temporal variation in malaria transmission in a low endemicity area in northern Tanzania

Background

Current detection or screening for malaria infection necessitates drawing blood by fingerprick or venipuncture, which poses risks and limitations for repeated measurement. This study presents PCR detection of Plasmodium falciparum in human urine and saliva samples, and illustrates this potential application in genotyping malaria infections.

Methods

Urine and saliva were obtained from 47 thick film positive and 4 negative individuals one day after collection of blood slides and filter paper blood spots. P. falciparum DNA was extracted from blood, urine and saliva, in separate groups, using the Chelex method or Qiagen DNEasy^® kit (urine and saliva only). Blood, urine and saliva extracts were subjected to PCR in separate batches. Amplicons from the various sample types were examined for MSP2 polymorphisms and restriction fragment patterns on DHFR amino acid codon 59.

Results and discussion

Malaria infections exhibited primarily low-grade parasite densities, with a geometric mean of 775 asexual parasites/μl. Regularly matching polymorphic MSP2 genotypes were found between the corresponding urine, saliva and peripheral blood amplicons of each individual, with different inter-individual polymorphic genotypes. Amplicon yields were significantly dependent on DNA extraction method, parasite density and primer set (p < 0.001). A Qiagen^® kit extraction had more than 2× higher amplicon yield than the Chelex method, for both urine and saliva. Amplicon yields were 1.6 fold higher from saliva than urine. For each unit increase in log parasite density, the probability of amplicon enhanced 1.8 fold. Highest amplicon yields were obtained from the primer set with the shortest PCR product.

Conclusion

P. falciparum infection is detectable by PCR on human urine and saliva samples. Subject to further refinement of extraction technique and amplicon yields, large-scale malaria parasite screening and epidemiological surveys could be possible without the need to collect blood and use of needles or sharps.