Biochemical and Structural Characterization of Benzenoid Carboxyl Methyltransferases Involved in Floral Scent Production in Stephanotis floribunda and Nicotiana suaveolens

Flower-specific benzenoid carboxyl methyltransferases from Stephanotis floribunda and Nicotiana suaveolens were biochemically and structurally characterized. The floral scents of both these species contain higher levels of methyl benzoate and lower levels of methyl salicylate. The S. floribunda enzyme has a 12-fold lower K(m) value for salicylic acid (SA) than for benzoic acid (BA), and results of in silico modeling of the active site of the S. floribunda enzyme, based on the crystal structure of Clarkia breweri salicylic acid methyltransferase (SAMT), are consistent with this functional observation. The enzyme was therefore designated SAMT. The internal concentration of BA in S. floribunda flowers is three orders of magnitude higher than the SA concentration, providing a rationale for the observation that these flowers synthesize and emit more methyl benzoate than methyl salicylate. The N. suaveolens enzyme has similar K(m) values for BA and SA, and the in silico modeling results are again consistent with this in vitro observation. This enzyme was therefore designated BSMT. However, the internal concentration of BA in N. suaveolens petals was also three orders of magnitude higher than the concentration of SA. Both S. floribunda SAMT and N. suaveolens BSMT are able to methylate a range of other benzenoid-related compounds and, in the case of S. floribunda SAMT, also several cinnamic acid derivatives, an observation that is consistent with the larger active site cavity of each of these two enzymes compared to the SAMT from C. breweri, as shown by the models. Broad substrate specificity may indicate recent evolution or an adaptation to changing substrate availability

In vivo osteoprogenitor potency of human stromal cells from different tissues does not correlate with expression of POU5F1 or its pseudogenes

Expression of "stemness" markers is widely used as a predictor of stem cell properties of mesenchymal stem cells (MSC). Here, we show that bone marrow-derived (BM)-MSC show stem cell-like behavior in vivo; that is, they form ossicles with formation of bone, formation of adipocytes, and establishment of the murine hematopoietic microenvironment. Multipotent umbilical vein-derived stromal cells (UVSC), on the other hand, do not form bone, nor do they give rise to adipocytes in vivo. Despite these differences in stem-cell-like behavior, BMMSC and UVSC express the two transcripts variants of POU5F1 at a similar level. Also, we found that in BMMSC and UVSC, POU5F1 is detectable. However, more than 89% of the POU5F1 transcripts correspond to the POU5F1P1, -P3, or -P4 pseudogene. Despite low-level expression of POU5F1, we were unable to precipitate POU5F1 protein in either BM-MSC or UVSC. These results demonstrate that MSC stemness does not correlate to expression of POU5F1 transcripts or its pseudogenes. ©AlphaMed Press