Laboratory Methods for the Study of Tetrapyrroles Alison G. Smith and Michael Witty Syntheses of Tetrapyrroles Kevin M. Smith General Laboratory Methods for Tetrapyrroles Jerry C. Bommer and Peter Hambright Enzymatic Preparation of Tetrapyrrole Intermediates Martin J. Warren and Peter M. Shoolingin-Jordan Analysis of Biosynthetic Intermediates, 5-Aminolevulinic Acid to Heme Chang Kee Lim Analysis of Intermediates and End Products of the Chlorophyll Biosynthetic Pathway Constantin A. Rebeiz Analysis of Heme and Hemoproteins Angela Wilks Hemoproteins Purification and Characterization by Using Aqueous Two-Phase Systems Daniel Forciniti Structural Study of Heme Proteins by Electron Microscopy of 2-Dimensional Crystals Terrence G. Frey Analysis and Reconstitution of Chlorophyll-Proteins Harald Paulsen and Volkmar H.R. Schmid Two-Dimensional Crystallization of Chlorophyll-Proteins Georgios Tsiotis Biosynthesis and Analysis of Bilins Matthew J. Terry Analysis and Reconstitution of Phytochromes Michael T. McDowell and J. Clark Lagarias Analysis and Reconstitution of Phycobiliproteins: Methods for the Characterization of Bilin Attachment Reactions Wendy M. Schluchter and Donald A. Bryant

Wolffia columbiana are duckweeds that inhabit freshwater ponds along with single celled algal species and these very small flowering plants often dominate the surface layer of undisturbed ponds. This upper layer of plant material fatally shades out lower species of plants and algae. Some epiphytic algae have adapted to benefit from the dominance of Wolffia columbiana by attaching themselves with various degrees of firmness to this duckweed. The epiphytic lifestyle has the advantage of avoiding shading out and an additional advantage which becomes apparent in Winter i.e. when Wolffia columbiana submerge to avoid frost damage. In this season their epiphytes are carried with them from the most favorable position in the water column for spring and summer, the surface, to the most favorable position in the water column for the winter, the sediment debris layer. This is an example of dynamic epiphytic symbiosis, rather than the conventional static association of epiphytes and deciduous trees which only lasts for one growing season and results in death of algal epiphytes in Fall.

Rutgers Cooperative Extension (RCE) has characterized the agricultural and recreational lands in the Wreck Pond Brook Watershed; an area that has contributed to the majority of NJ ocean beach closings in 2005. The watershed is approximately 12 sq. miles in size and is comprised of a wide variety of land uses. RCE is part of a Regional Stormwater Management Planning Committee that addresses environmental impairments and recommends Best Management Practices (BMP's) to remediate any non-point source contributions of nutrient loading and fecal coliform within the watershed. Geographic Information System (GIS) and tax assessment information were used to identify and characterize the quantity of agricultural and recreational land in the watershed. A YSI Multiparameter Probe, a Hach Colorimeter and macroinvertebrate sampling were utilized to assess nutrient levels as nitrogen and phosphorus in ponds and streams. Nutrient levels in the soil were assessed with soil probes.

Rutgers Cooperative Extension (RCE) has characterized the agricultural and recreational lands in the Wreck Pond Brook Watershed; an area that has contributed to the majority of NJ ocean beach closings in 2005. The watershed is approximately 12 sq. miles in size and is comprised of a wide variety of land uses. RCE is part of a Regional Stormwater Management Planning Committee that addresses environmental impairments and recommends Best Management Practices (BMP's) to remediate any non-point source contributions of nutrient loading and fecal coliform within the watershed. Geographic Information System (GIS) and tax assessment information were used to identify and characterize the quantity of agricultural and recreational land in the watershed. A YSI Multiparameter Probe, a Hach Colorimeter and macroinvertebrate sampling were utilized to assess nutrient levels as nitrogen and phosphorus in ponds and streams. Nutrient levels in the soil were assessed with soil probes.

Fecal pollution from human and natural sources enters soil or watercourses, mixes, then reemerges as a nuisance of unknown origin. Before remediation is attempted, the sources and identities of pollution must be identified. Previous microbial source tracking studies have relied on traditional methods of microbiology such as selective media and biochemical characteristics to quantify fecal bacteria in water samples. This is successful when single sources are responsible for pollution. However, when multiple sources are present, numbers must be subdivided into categories of pollution to define relative importance and select appropriate methods of remediation which are very different for examples such as humans and avifauna pollution. Rather than depending on a single method, we recommend a tiered approach which takes advantage of ecological parameters and conventional microbiology to provide context for more precise DNA data and related statistics.

We report the crystal structure of E. coli ketopantoate hydroxymethyltransferase (KPHMT) at 1.9 Å resolution, in complex with its product, ketopantoate. KPHMT catalyzes the first step in the biosynthesis of pantothenate (vitamin B5), the precursor of coenzyme A and the acyl carrier protein cofactor. The structure of the decameric enzyme was solved by multiwavelength anomalous dispersion to locate 160 selenomethionine sites and phase 560 kDa of protein, making it the largest structure solved by this approach. KPHMT adopts the (βα)8 barrel fold and is a member of the phosphoenolpyruvate/pyruvate superfamily. The active site contains a ketopantoate bidentately coordinated to Mg2+. Similar binding is likely for the substrate, α-ketoisovalerate, orienting the C3 for deprotonation.

Aspartate decarboxylase, which is translated as a pro-protein, undergoes intramolecular self-cleavage at Gly24-Ser25. We have determined the crystal structures of an unprocessed native precursor, in addition to Ala24 insertion, Ala26 insertion and Gly24-->Ser, His11-->Ala, Ser25-->Ala, Ser25-->Cys and Ser25-->Thr mutants. Comparative analyses of the cleavage site reveal specific conformational constraints that govern self-processing and demonstrate that considerable rearrangement must occur. We suggest that Thr57 Ogamma and a water molecule form an 'oxyanion hole' that likely stabilizes the proposed oxyoxazolidine intermediate. Thr57 and this water molecule are probable catalytic residues able to support acid-base catalysis. The conformational freedom in the loop preceding the cleavage site appears to play a determining role in the reaction. The molecular mechanism of self-processing, presented here, emphasizes the importance of stabilization of the oxyoxazolidine intermediate. Comparison of the structural features shows significant similarity to those in other self-processing systems, and suggests that models of the cleavage site of such enzymes based on Ser-->Ala or Ser-->Thr mutants alone may lead to erroneous interpretations of the mechanism.

Mal d 2 is a thaumatin-like protein and important allergen of apple fruits that is associated with IgE-mediated symptoms in apple allergic individuals. We obtained a full-length cDNA clone of Mal d 2 from RNA isolated from ripe apple (Malus domestica cv. Golden Delicious). The cDNA's open reading frame encodes a protein of 246 amino acid residues including a signal peptide of 24 residues and two putative glycosylation sites. The deduced amino acid sequence of the mature Mal d 2 protein results in a predicted molecular mass of 23,210.9Da and a calculated pI of 4.55. Sequence comparisons and molecular modeling place Mal d 2 among those pathogenesis-related thaumatin-like proteins that contain a conserved acidic cleft. In order to ensure the correct formation of the protein's eight conserved disulfide bridges we expressed Mal d 2 in Nicotiana benthamiana plants by the use of a tobacco mosaic viral vector. Transfected N.benthamiana plants accumulated Mal d 2 to levels of at least 2% of total soluble protein. MALDI-TOF mass spectrometric analyses of the recombinant Mal d 2 and its proteolytic fragments showed that the apple-specific leader peptide was correctly cleaved off by the host plant and that the mature recombinant protein was intact and not glycosylated. Purified recombinant Mal d 2 displayed the ability to bind IgE from apple-allergic individuals equivalent to natural Mal d 2. In addition, the recombinant thaumatin-like Mal d 2 exhibited antifungal activity against Fusarium oxysporum and Penicillium expansum, implying a function in plant defense against fungal pathogens.

Aspartate decarboxylase, which is translated as a pro‐protein, undergoes intramolecular self‐cleavage at Gly24–Ser25. We have determined the crystal structures of an unprocessed native precursor, in addition to Ala24 insertion, Ala26 insertion and Gly24→Ser, His11→Ala, Ser25→Ala, Ser25→Cys and Ser25→Thr mutants. Comparative analyses of the cleavage site reveal specific conformational constraints that govern self‐processing and demonstrate that considerable rearrangement must occur. We suggest that Thr57 Oγ and a water molecule form an ‘oxyanion hole’ that likely stabilizes the proposed oxyoxazolidine intermediate. Thr57 and this water molecule are probable catalytic residues able to support acid–base catalysis. The conformational freedom in the loop preceding the cleavage site appears to play a determining role in the reaction. The molecular mechanism of self‐processing, presented here, emphasizes the importance of stabilization of the oxyoxazolidine intermediate. Comparison of the structural features shows significant similarity to those in other self‐processing systems, and suggests that models of the cleavage site of such enzymes based on Ser→Ala or Ser→Thr mutants alone may lead to erroneous interpretations of the mechanism.