Biomedical subjects

Mahmoud W F Yaish

Publications and source records attributed to Mahmoud W F Yaish.

3 recordsLinked to original sources

PubMed2006

Cold-active winter rye glucanases with ice-binding capacity.

Extracellular pathogenesis-related proteins, including glucanases, are expressed at cold temperatures in winter rye (Secale cereale) and display antifreeze activity. We have characterized recombinant cold-induced glucanases from winter rye to further examine their roles and contributions to cold tolerance. Both basic beta-1,3-glucanases and an acidic beta-1,3;1,4-glucanase were expressed in Escherichia coli, purified, and assayed for their hydrolytic and antifreeze activities in vitro. All were found to be cold active and to retain partial hydrolytic activity at subzero temperatures (e.g. 14%-35% at -4 degrees C). The two types of glucanases had antifreeze activity as measured by their ability to modify the growth of ice crystals. Structural models for the winter rye beta-1,3-glucanases were developed on which putative ice-binding surfaces (IBSs) were identified. Residues on the putative IBSs were charge conserved for each of the expressed glucanases, with the exception of one beta-1,3-glucanase recovered from nonacclimated winter rye in which a charged amino acid was present on the putative IBS. This protein also had a reduced antifreeze activity relative to the other expressed glucanases. These results support the hypothesis that winter rye glucanases have evolved to inhibit the formation of large, potentially fatal ice crystals, in addition to having enzymatic activity with a potential role in resisting infection by psychrophilic pathogens. Glucanases of winter rye provide an interesting example of protein evolution and adaptation aimed to combat cold and freezing conditions.

Amino Acid Sequence↗

PubMed2004

Antifreeze proteins in overwintering plants: a tale of two activities.

Antifreeze proteins are found in a wide range of overwintering plants where they inhibit the growth and recrystallization of ice that forms in intercellular spaces. Unlike antifreeze proteins found in fish and insects, plant antifreeze proteins have multiple, hydrophilic ice-binding domains. Surprisingly, antifreeze proteins from plants are homologous to pathogenesis-related proteins and also provide protection against psychrophilic pathogens. In winter rye (Secale cereale), antifreeze proteins accumulate in response to cold, short daylength, dehydration and ethylene, but not pathogens. Transferring single genes encoding antifreeze proteins to freezing-sensitive plants lowered their freezing temperatures by approximately 1 degrees C. Genes encoding dual-function plant antifreeze proteins are excellent models for use in evolutionary studies to determine how genes acquire new expression patterns and how proteins acquire new activities.

Acclimatization↗

PubMed2004

Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2.

The Arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 secretes an antifreeze protein (AFP) that promotes survival at subzero temperatures. The AFP is unusual in that it also exhibits a low level of ice nucleation activity. A DNA fragment with an open reading frame encoding 473 amino acids was cloned by PCR and inverse PCR using primers designed from partial amino acid sequences of the isolated AFP. The predicted gene product, AfpA, had a molecular mass of 47.3 kDa, a pI of 3.51, and no previously known function. Although AfpA is a secreted protein, it lacked an N-terminal signal peptide and was shown by sequence analysis to have two possible secretion systems: a hemolysin-like, calcium-binding secretion domain and a type V autotransporter domain found in gram-negative bacteria. Expression of afpA in Escherichia coli yielded an intracellular 72-kDa protein modified with both sugars and lipids that exhibited lower levels of antifreeze and ice nucleation activities than the native protein. The 164-kDa AFP previously purified from P. putida GR12-2 was a lipoglycoprotein, and the carbohydrate was required for ice nucleation activity. Therefore, the recombinant protein may not have been properly posttranslationally modified. The AfpA sequence was most similar to cell wall-associated proteins and less similar to ice nucleation proteins (INPs). Hydropathy plots revealed that the amino acid sequence of AfpA was more hydrophobic than those of the INPs in the domain that forms the ice template, thus suggesting that AFPs and INPs interact differently with ice. To our knowledge, this is the first gene encoding a protein with both antifreeze and ice nucleation activities to be isolated and characterized.

Amino Acid Motifs↗