31phospho-NMR demonstration of phosphocysteine as a catalytic intermediate on the Escherichia coli phosphotransferase system EIIMtl.

The mannitol-specific phosphotransferase system transport protein, Enzyme IIMtl, contains two catalytically important phosphorylated amino acid residues, both present on the cytoplasmic part of the enzyme. Recently, this portion has been subcloned, purified, and shown to be an enzymatically active domain. The N-terminal half has also been subcloned and shown to be the mannitol-binding domain. When combined the two domains catalyze mannitol phosphorylation at the expense of phospho-HPr (van Weeghel, R. P., Meyer, G. H., Pas, H. H., Keck, W. H., and Robillard, G. T., Biochemistry in press). The phospho-NMR spectrum of the purified phosphorylated cytoplasmic domain, taken at pH 8.0, shows two signals, one at -6.9 ppm compared with inorganic phosphate resulting from phosphohistidine and one at +11.9 ppm originating from phosphocysteine. Addition of mannitol plus membranes containing the N-terminal mannitol-binding domain results in the formation of mannitol 1-phosphate and the disappearance of the two signals at -6.9 and +11.9 ppm.

Phosphoenolpyruvate-dependent mannitol phosphotransferase system of Escherichia coli: overexpression, purification, and characterization of the enzymatically active C-terminal domain of enzyme IImtl equivalent to enzyme IIImtl.

The extreme C-terminus (Ser-490 to Lys-637) of the Escherichia coli EIImtl was subcloned to test structural and mechanistic proposals about the existence of an EIII-like domain in this enzyme. Oligonucleotide-directed mutagenesis was used to produce a unique NcoI restriction site and, at the same time, to change Ser-490 into methionine in a flexible region in front of the proposed EIII-like domain. The 16-kDa C-terminal domain (CI) was overexpressed in Escherichia coli, purified, and analyzed in vitro for catalytic activity in the presence of an EIImtl mutated at its first phosphorylation site, His-554 (EII-H554A). The results presented show that this domain can be expressed as a structurally stable, enzymatically active entity which is able to restore the PEP-dependent phosphorylation activity of the mutant EIImtl-H554A to 25% of wild-type levels. To demonstrate the EIII activity of the CI domain in a more direct way, we also substituted it for EIIImtl in the Staphylococcus carnosus system. The CI domain was active in transferring the phosphoryl group to Staph. carnosus EII; however, it was 6.5 times less active compared to Staph. carnosus EIIImtl itself. EIIImtl from Staph. carnosus, on the other hand, was able to substitute for the isolated C-terminal domain in the E. coli mannitol phosphorylation assay; however, it appeared to be 2 or 3 times less effective.

Airborne fungi--a resurvey.

A 15-month survey of airborne fungi at 14 geographical stations was conducted to determine the incidence of different fungal genera. Five of these stations were surveyed 25 years earlier. A comparison between previous studies and present surveys revealed similar organisms at each station with slight shifts in frequency of dominant genera.

An up-to-date look at mold allergy.

The general distribution of the more frequently encountered airborne molds is reviewed. Improved diagnostic and therapeutic mold allergens, a knowledge of botanical relationships among molds and new injection techniques facilitate handling of clinical problems caused by molds.