Whole Cell Active Inhibitors of Mycobacterial Lipoamide Dehydrogenase Afford Selectivity over the Human Enzyme through Tight Binding Interactions.

Tuberculosis remains a leading cause of death from a single bacterial infection worldwide. Efforts to develop new treatment options call for expansion into an unexplored target space to expand the drug pipeline and bypass resistance to current antibiotics. Lipoamide dehydrogenase is a metabolic and antioxidant enzyme critical for mycobacterial growth and survival in mice. Sulfonamide analogs were previously identified as potent and selective inhibitors of mycobacterial lipoamide dehydrogenase in vitro but lacked activity against whole mycobacteria. Here we present the development of analogs with improved permeability, potency, and selectivity, which inhibit the growth of Mycobacterium tuberculosis in axenic culture on carbohydrates and within mouse primary macrophages. They increase intrabacterial pyruvate levels, supporting their on-target activity within mycobacteria. Distinct modalities of binding between the mycobacterial and human enzymes contribute to improved potency and hence selectivity through induced-fit tight binding interactions within the mycobacterial but not human enzyme, as indicated by kinetic analysis and crystallography.

HorTILLUS-A Rich and Renewable Source of Induced Mutations for Forward/Reverse Genetics and Pre-breeding Programs in Barley (Hordeum vulgare L.).

TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS ( Hordeum-TILLING-University of Silesia) population created for spring barley cultivar "Sebastian" after double-treatment of seeds with two chemical mutagens: sodium azide (NaN3) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M2 plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M3 progeny of 3,481 M2 individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072-6,912 M2 plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.

Co-suppression of synthesis of major α-kafirin sub-class together with γ-kafirin-1 and γ-kafirin-2 required for substantially improved protein digestibility in transgenic sorghum.

KEY MESSAGE: Co-suppressing major kafirin sub-classes is fundamental to improved protein digestibility and nutritional value of sorghum. The improvement is linked to an irregularly invaginated phenotype of protein bodies. ABSTRACT: The combined suppression of only two genes, γ kafirin-1 (25 kDa) and γ-kafirin-2 (50 kDa), significantly increases sorghum kafirin in vitro digestibility. Co-suppression of a third gene, α-kafirin A1 (25 kDa), in addition to the two genes increases the digestibility further. The high-digestibility trait has previously only been obtained either through the co-suppression of six kafirin genes (α-A1, 25 kDa; α-B1, 19 kDa; α-B2, 22 kDa; γ-kaf1, 27 kDa; γ-kaf 2, 50 kDa; and δ-kaf 2, 18 kDa) or through random chemical-induced mutations (for example, the high protein digestibility mutant). We present further evidence that suppressing just three of these genes alters kafirin protein cross-linking and protein body microstructure to an irregularly invaginated phenotype. The irregular invaginations are consistent with high pepsin enzyme accessibility and hence high digestibility. The approach we adopted towards increasing sorghum protein digestibility appears to be an effective tool in improving the status of sorghum as a principal supplier of energy and protein in poor communities residing in marginal agro-ecological zones of Africa.

Induced protein polymorphisms and nutritional quality of gamma irradiation mutants of sorghum.

Physical and biochemical analysis of protein polymorphisms in seed storage proteins of a mutant population of sorghum revealed a mutant with redirected accumulation of kafirin proteins in the germ. The change in storage proteins was accompanied by an unusually high level accumulation of free lysine and other essential amino acids in the endosperm. This mutant further displayed a significant suppression in the synthesis and accumulation of the 27kDa γ-, 24kDa α-A1 and the 22kDa α-A2 kafirins in the endosperm. The suppression of kafirins was counteracted by an upsurge in the synthesis and accumulation of albumins, globulins and other proteins. The data collectively suggest that sorghum has huge genetic potential for nutritional biofortification and that induced mutations can be used as an effective tool in achieving premium nutrition in staple cereals.