Protein‐engineering of chitosanase from Bacillus sp. MN to alter its substrate specificity

Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine, and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein‐engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N‐acetyl‐D‐glucosamine units at their subsite (?2), which is impossible for the wildtype enzyme.     

Eighty Years of <Emphasis Type="Italic">Mycopathologia</Emphasis>: A Retrospective Analysis of Progress Made in Understanding Human and Animal Fungal Pathogens

Mycopathologia was founded in 1938 to ‘diffuse the understanding of fungal diseases in man and animals among mycologists.’ This was an important mission considering that pathogenic fungi for humans and animals represent a tiny minority of the estimated 1.5–5 million fungal inhabitants on Earth. These pathogens have diverged from the usual saprotrophic lifestyles of most fungi to colonize and infect humans and animals. Medical and veterinary mycology is the subdiscipline of microbiology that dwells into the mysteries of parasitic, fungal lifestyles. Among the oldest continuing scientific publications on the subject, Mycopathologia had its share of ‘classic papers’ since the first issue was published in 1938. An analysis of the eight decades of notable contributions reveals many facets of host–pathogen interactions among 183 volumes comprising about 6885 articles. We have analyzed the impact and relevance of this body of work using a combination of citation tools (Google Scholar and Scopus) since no single citation metric gives an inclusive perspective. Among the highly cited Mycopathologia publications, those on experimental mycology accounted for the major part of the articles (36%), followed by diagnostic mycology (16%), ecology and epidemiology (15%), clinical mycology (14%), taxonomy and classification (10%), and veterinary mycology (9%). The first classic publication, collecting nearly 200 citations, appeared in 1957, while two articles published in 2010 received nearly 150 citations each, which is notable for a journal covering a highly specialized field of study. An empirical analysis of the publication trends suggests continuing interests in novel diagnostics, fungal pathogenesis, review of clinical diseases especially with relevance to the laboratory scientists, taxonomy and classification of fungal pathogens, fungal infections and carriage in pets and wildlife, and changing ecology and epidemiology of fungal diseases around the globe. We anticipate that emerging and re-emerging fungal pathogens will continue to cause significant health burden in the coming decades. It remains vital that scientists and physicians continue to collaborate by learning each other’s language for the study of fungal diseases, and Mycopathologia will strive to be their partner in this increasingly important endeavor to its 100th anniversary in 2038 and beyond.     

Genome-Wide Analysis in Wild and Cultivated <Emphasis Type="Italic">Oryza</Emphasis> Species Reveals Abundance of NBS Genes in Progenitors of Cultivated Rice

NBS-encoding genes play a critical role in the plant defense system. Wild relatives of crop plants are rich reservoirs of plant defense genes. Here, we performed a stringent genome-wide identification of NBS-encoding genes in three cultivated and eight wild Oryza species, representing three different genomes (AA, BB, and FF) from four continents. A total of 2688 NBS-encoding genes were identified from 11 Oryza genomes. All the three progenitor species of cultivated rice, namely O. barthii, O. rufipogon, and O. nivara, were the richest reservoir of NBS-encoding genes (214, 313, and 307 respectively). Interestingly, the two Asian cultivated species showed a contrasting pattern in the number of NBS-encoding genes. While indica subspecies maintained nearly equal number of NBS genes as its progenitor (309 and 313), the japonica subspecies had retained only two third in the course of evolution (213 and 307). Other major sources for NBS-encoding genes could be (i) O. longistaminata since it had the highest proportion of NBS-encoding genes and (ii) O. glumaepatula as it clustered distinctly away from the rest of the AA genome species. The present study thus revealed that NBS-encoding genes can be exploited from the primary gene pool for disease resistance breeding in rice.     

A specific amino acid residue in the catalytic site of dandelion polyphenol oxidases acts as ‘selector’ for substrate specificity

Key message

Successful site-directed mutagenesis combined with in silico modeling and docking studies for the first time offers experimental proof of the role of the ‘substrate selector’ residue in plant polyphenol oxidases.

Abstract

The plant and fungi enzymes responsible for tissue browning are called polyphenol oxidases (PPOs). In plants, PPOs often occur as families of isoenzymes which are differentially expressed, but little is known about their physiological roles or natural substrates. In a recent study that explored these structure–function relationships, the eleven known dandelion (Taraxacum officinale) PPOs were shown to separate into two different phylogenetic groups differing in catalytic cavity architecture, kinetic parameters, and substrate range. The same study proposed that the PPOs’ substrate specificity is controlled by one specific amino acid residue positioned at the entrance to the catalytic site: whereas group 1 dandelion PPOs possess a hydrophobic isoleucine (I) at position H_B2+1, group 2 PPOs exhibit a larger, positively charged arginine (R). However, this suggestion was only based on bioinformatic analyses, not experiments. To experimentally investigate this hypothesis, we converted group 1 ToPPO-2 and group 2 ToPPO-6 into PPO-2-I₂₄₄R and PPO-6-R₂₅₄I, respectively, and expressed them in E. coli. By performing detailed kinetic characterization and in silico docking studies, we found that replacing this single amino acid significantly changed the PPO’s substrate specificity. Our findings therefore proof the role of the ‘substrate selector’ in plant PPOs.

     

Identification of a Cryptococcus neoformans gene that directs expression of the cryptic Saccharomyces cerevisiae mannitol dehydrogenase gene.

The Mtl gene from Cryptococcus neoformans, which confers the ability of Saccharomyces cerevisiae Sc4l YJO to grow on mannitol with substantial NAD-dependent mannitol dehydrogenase activity, was identified. Purifications and characterizations of this enzyme show that it is found in polyploid strain BB1, and the peptide sequence of the enzyme helped identify the saccharomyces gene encoding this mannitol dehydrogenase activity. On the other hand, the Mtl gene of C. neoformans encodes a 346-amino-acid protein which is not mannitol dehydrogenase but a regulatory element which is active in a heterologous fungus.     

Expression of bacterial mtlD in Saccharomyces cerevisiae results in mannitol synthesis and protects a glycerol-defective mutant from high-salt and oxidative stress.

Polyols, or polyhydroxy alcohols, are produced by many fungi. Saccharomyces cerevisiae produces large amounts of glycerol, and several fungi that cause serious human infections produce D-arabinitol and mannitol. Glycerol functions as an intracellular osmolyte in S. cerevisiae, but the functions of D-arabinitol and mannitol in pathogenic fungi are not yet known. To investigate the functions of mannitol, we constructed a new mannitol biosynthetic pathway in S. cerevisiae. S. cerevisiae transformed with multicopy plasmids encoding the mannitol-1-phosphate dehydrogenase of Escherichia coli produced mannitol, whereas S. cerevisiae transformed with control plasmids did not. Although mannitol production had no obvious phenotypic effects in wild-type S. cerevisiae, it restored the ability of a glycerol-defective, osmosensitive osg1-1 mutant to grow in the presence of high NaCl concentrations. Moreover, osg1-1 mutants producing mannitol were more resistant to killing by oxidants produced by a cell-free H2O2-FeSO4-NaI system than were controls. These results indicate that mannitol can (i) function as an intracellular osmolyte in S. cerevisiae, (ii) substitute for glycerol as the principal intracellular osmolyte in S. cerevisiae, and (iii) protect S. cerevisiae from oxidative damage by scavenging toxic oxygen intermediates.     

Analysis and quantification of mental stress and fatigue using Maxwell relations from thermodynamics

Several experimental and theoretical techniques have been developed to analyze both physical and psychological stresses. These techniques have relied mainly on certain parameters based on physiological, behavioral, and performance related data. This study is based on a thought experiment which describes the technique to quantify mental stress based on physiological responses using the entropy concept. It relates different physiological parameters using the Maxwell relations of thermodynamics with a systems approach. Data for testing this analytical approach were obtained from an experimental study which was conducted to determine the effects of a mentally stressful situation (final examination) on the common physiological responses (blood pressure, pulse rate, and oral body temperature) of students. The results indicated that the imposed mental stress causes significant changes in physiological responses. The Maxwell relations of thermodynamics were used to quantify the level of stress under different conditions. The results obtained from these relations validated the principles of thermodynamics as applied to the human system.