Genetic tools for selective labeling of proteins with α-15N-amino acids

Summary A collection of genetic tools that can be used to manipulate amino acid metabolism in Escherichia coli is described. The set comprises 21 strains of bacteria, each containing a different genetic defect that is closely linked to a selectable transposon marker. These tools can be used to construct strains of E. coli with ideal genotypes for residue-specific, selective labeling of proteins with nearly any ¹⁵N-amino acid. By using strains which have been modified to contain the appropriate genetic lesions to control amino acid biosynthesis, dilution of the isotope by endogenous amino acid biosynthesis and scrambling of the label to other types of residues can be avoided.Abbreviations ¹⁵N-amino acid -¹⁵N-amino acid - Cam^R chloramphenicol-resistant - DPA diaminopimelic acid - Hfr high-frequency recombinant - LB Luria broth - Kan^R kanamycin resistant - P1 bacteriophage P1 - pfu plaque-forming units - Str^R streptomycin-resistant - Tet^R tetracycline-resistant     

Functional consequences of mutating conserved SF2 helicase motifs in the Type III restriction endonuclease EcoP15I translocase domain

For efficient DNA hydrolysis, Type III restriction endonuclease EcoP15I interacts with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of two methylation (Mod) subunits and a single restriction (Res) subunit yielding a multifunctional enzyme complex able to methylate or to hydrolyse DNA. Comprehensive sequence alignments, limited proteolysis and mass spectroscopy suggested that the Res subunit is a fusion of a motor or translocase (Tr) domain of superfamily II helicases and an endonuclease domain with a catalytic PD…EXK motif. In the Tr domain, seven predicted helicase motifs (I, Ia, II–VI), a recently discovered Q-tip motif and three additional regions (IIIa, IVa, Va) conserved among Type III restriction enzymes have been identified that are predicted to be involved in DNA binding and ATP hydrolysis. Because DNA unwinding activity for EcoP15I (as for bona fide helicases) has never been found and EcoP15I ATPase rates are only low, the functional importance of the helicase motifs and regions was questionable and has never been probed systematically. Therefore, we mutated all helicase motifs and conserved regions predicted in Type III restriction enzyme EcoP15I and examined the functional consequences on EcoP15I enzyme activity and the structural integrity of the variants by CD spectroscopy. The resulting eleven enzyme variants all, except variant IVa, are properly folded showing the same secondary structure distribution as the wild-type enzyme. Classical helicase motifs I–VI are important for ATP and DNA cleavage by EcoP15I and mutations therein led to complete loss of ATPase and cleavage activity. Among the catalytically inactive enzyme variants three preserved the ability to bind ATP. In contrast, newly assigned motifs Q-tip, Ia and Va are not essential for EcoP15I activity and the corresponding enzyme variants were still catalytically active. DNA binding was only marginally reduced (2–7 fold) in all enzyme variants tested.     

添加玉米残体对土壤-植物系统中氮素转化的影响

采用盆栽试验和^15N示踪技术对黑土添加玉米残体(秸秆和根茬)土壤-植物系统中氮素转化进行了研究,结果表明,玉米残体还田能够增加土壤氮素含量,减轻因其作为燃烧材料而造成的氮素损失和对大气的污染,玉米残体施入土壤,增加了土壤微生物氮含量,提高土壤氮活性,有利于土壤氮素养分的协调供应,玉米残体配施氮肥与氮肥单施相比,玉米植株氮素累积量相近,但氮素在玉米植株不同器官中的分配比例不同;添加玉米残体能够促进氮素从营养器官向籽粒中转移,提高氮素养分的利用效率,同时,添加玉米残体还可以降低土壤NO^-3-N的累积,减少肥料氮的损失4．7％～5．6％。     

Functional domains of bovine β-1,4 galactosyltransferase

A number of N- and C-terminal deletion and point mutants of bovine -1,4 galactosyltransferase (-1,4GT) were expressed inE. coli to determine the binding regions of the enzyme that interact withN-acetylglucosamine (NAG) and UDP-galactose. The N-terminal truncated forms of the enzyme between residues 1–129, do not show any significant difference in the apparentK _ms toward NAG or linear oligosaccharide acceptors e.g. for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15mm NAG and 50mm EDTA, like the enzymatically active protein, TL-GT129, that contains residues 130–402 of bovine -1,4GT. Also the N-terminus fragment, TL-GT129NAG, that contains residues 130–257 of the -1,4GT, binds to, and elutes with 15mm NAG and 50mm EDTA from the NAG-agarose column as efficiently as the enzymatically active TL-GT129. Unlike TL-GT129, the TL-GT129NAG binds to UDP-columns less efficiently and can be eluted from the column with only 15mm NAG. The C-terminus fragment GT-257UDP, containing residues 258–402 of -1,4GT, binds tightly to both NAG- and UDP-agarose columns. A small fraction, 5–10% of the bound protein, can be eluted from the UDP-agarose column with 50mm EDTA alone. The results show that the binding behaviour of N- and C-terminal fragments of -1,4GT towards the NAG- and UDP-agarose columns differ, the former binds preferentially to NAG-columns, while the latter binds to UDP-agarose columns via Mn²⁺.     

Substrate specificity of a recombinant chicken <Emphasis Type="Italic">β</Emphasis>-carotene 15,15′-monooxygenase that converts <Emphasis Type="Italic">β</Emphasis>-carotene into retinal

The recombinant β-carotene 15,15′-monooxygenase from chicken liver was purified as a single 60 kDa band by His-Trap HP and Resource Q chromatography. It had a molecular mass of 240 kDa by gel filtration indicating the native form to be tetramer. The enzyme converted β-carotene under maximal conditions (pH 8.0 and 37°C) with a k _cat of 1.65 min⁻¹ and a K _m of 26 μM and its conversion yield of β-carotene to retinal was 120% (mol mol⁻¹). The enzyme displayed catalytic efficiency and conversion yield for β-carotene, β-cryptoxanthin, β-apo-8′-carotenal, β-apo-4′-carotenal, α-carotene and γ-carotene in decreasing order but not for zeaxanthin, lutein, β-apo-12′-carotenal and lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₀ seems to be essential for enzyme activity.     

δ15N as an indicator of N2-fixation by cyanobacterial mats in tropical marshes

Cyanobacterial mats (CBM) are important components of wetland ecosystems in limestone-based regions of the Caribbean. During two sampling periods (July 1999 and January 2000) we measured N₂-fixation in samples from 23 different marshes simultaneously with measurements of relevant environmental factors. Samples were evaluated for abundance of five groups of cyanobacteria: (1) Leptolyngbya, (2) Oscillatoria, (3) Chroococcales, (4) Nostoc-& Stigonematales, and (5) dead sheaths. Differences in nitrogen fixation, expressed as nitrogenase activity in nmol C₂H₄ cm^–2 h^–1, were best explained by the proportion of heterocyst-forming cyanobacteria. The samples were analyzed for the natural abundance of ¹⁵N. ¹⁵N values ranged from –1.99 to 11.44 and were strongly negatively correlated with N₂-fixation. With all data included, ¹⁵N was also strongly correlated with nitrates in water. With the samples from Little Belize (high nitrate content marshes) excluded, the effect of nitrate became insignificant. N₂-fixation predicted from ¹⁵N measured on an independent data set from September 2000 was moderately accurate (r² = 0.68, 0.52 and 0.54 for predictions based on July 1999, January 2000 and combined data sets, respectively). When individual sample sets were divided into two groups with ¹⁵N < 2 and ¹⁵N > 2, the two groups were always highly significantly different in terms of their N₂-fixation. The presented evidence suggests that ¹⁵N can be used as a reliable indicator of N₂-fixation by CBM.     

Open interface and large quaternary structure movements in 3D domain swapped proteins: insights from molecular dynamics simulations of the C-terminal swapped dimer of ribonuclease A

Bovine pancreatic ribonuclease (RNase A) forms two three-dimensional (3D) domain swapped dimers. Crystallographic investigations have revealed that these dimers display completely different quaternary structures: one dimer (N-dimer), which presents the swapping of the N-terminal helix, is characterized by a compact structure, whereas the other (C-dimer), which is stabilized by the exchange of the C-terminal end, shows a rather loose assembly of the two subunits. The dynamic properties of monomeric RNase A and of the N-dimer have been extensively characterized. Here, we report a molecular dynamics investigation carried out on the C-dimer. This computational experiment indicates that the quaternary structure of the C-dimer undergoes large fluctuations. These motions do not perturb the proper folding of the two subunits, which retain the dynamic properties of RNase A and the N-dimer. Indeed, the individual subunits of the C-dimer display the breathing motion of the beta-sheet structure, which is important for the enzymatic activity of pancreatic-like ribonucleases. In contrast to what has been observed for the N-dimer, the breathing motion of the two subunits of the C-dimer is not coupled. This finding suggests that the intersubunit communications in a 3D domain swapped dimer strongly rely on the extent of the interchain interface. Furthermore, the observation that the C-dimer is endowed with a high intrinsic flexibility holds interesting implications for the specific properties of 3D domain swapped dimers. Indeed, a survey of the quaternary structures of the other 3D domain swapped dimers shows that large variations are often observed when the structural determinations are conducted in different experimental conditions. The 3D domain swapping phenomenon coupled with the high flexibility of the quaternary structure may be relevant for protein-protein recognition, and in particular for the pathological aggregations.     

Significance of rooting depth in mire plants: Evidence from natural <Superscript>15</Superscript>N abundance

Variation in stable nitrogen isotope ratios (¹⁵N) was assessed for plants comprising two wetland communities, a bog-fen system and a flood plain, in central Japan. ¹⁵N of 12 species from the bog-fen system and six species from the flood plain were remarkably variable, ranging from –5.9 to +1.1 and from +3.1 to +8.7, respectively. Phragmites australis exhibited the highest ¹⁵N value at both sites. Rooting depth also differed greatly with plant species, ranging from 5cm to over 200cm in the bog-fen system. There was a tendency for plants having deeper root systems to exhibit higher ¹⁵N values; plant ¹⁵N was positively associated with rooting depth. Moreover, an increasing gradient of peat ¹⁵N was found along with depth. This evidence, together with the fact that inorganic nitrogen was depleted under a deep-rooted Phragmites australis stand, strongly suggests that deep-rooted plants actually absorb nitrogen from the deep peat layer. Thus, we successfully demonstrated the diverse traits of nitrogen nutrition among mire plants using stable isotope analysis. The ecological significance of deep rooting in mire plants is that it enables those plants to monopolize nutrients in deep substratum layers. This advantage should compensate for any consequential structural and/or physiological costs. Good evidence of the benefits of deep rooting is provided by the fact that Phragmites australis dominates as a tall mire grass.