ABSTRACT
The first structure of a bacterial α-phosphoglucomutase with an overall fold similar to eukaryotic phosphomannomutases is reported. Unlike most α-phosphoglucomutases within the α-D-phosphohexomutase superfamily, it belongs to subclass IIb of the haloacid dehalogenase superfamily (HADSF). It catalyzes the reversible conversion of α-glucose 1-phosphate to glucose 6-phosphate. The crystal structure of α-phosphoglucomutase from Lactococcus lactis (APGM) was determined at 1.5â Å resolution and contains a sulfate and a glycerol bound at the enzyme active site that partially mimic the substrate. A dimeric form of APGM is present in the crystal and in solution, an arrangement that may be functionally relevant. The catalytic mechanism of APGM and its strict specificity towards α-glucose 1-phosphate are discussed.
Subject(s)
Bacterial Proteins/chemistry , Lactococcus lactis/enzymology , Phosphotransferases (Phosphomutases)/chemistry , Bacterial Proteins/genetics , Catalytic Domain/genetics , Crystallography, X-Ray , Glucose-6-Phosphate/chemistry , Glucose-6-Phosphate/genetics , Glucosephosphates/chemistry , Glucosephosphates/genetics , Hydrolases/chemistry , Hydrolases/classification , Hydrolases/genetics , Lactococcus lactis/genetics , Molecular Mimicry/genetics , Multigene Family , Phosphotransferases (Phosphomutases)/classification , Phosphotransferases (Phosphomutases)/genetics , Protein Binding/genetics , Substrate Specificity/geneticsABSTRACT
Increased stress tolerance of economically important plants and microorganisms can improve yields in agriculture and industrial microbiology. The pool of resources used for the genetic modification of crops and industrial fungal strains in the past has been relatively limited, and has frequently included only stress-sensitive organisms. However, certain groups of fungi have evolved specialized mechanisms that enable them to thrive under even the most extreme of environmental conditions. These species can be considered as promising sources of biotechnologically interesting genes. Together with a powerful and convenient high-throughput functional screening method, extremotolerant fungi represent a new opportunity for the identification of stress-tolerance-conferring genes. The approaches described here should provide important contributions to the enhancing of the properties of economically important organisms in the future.
Subject(s)
Genes, Fungal , Phosphoglucomutase/genetics , Phosphotransferases (Phosphomutases)/genetics , Rhodotorula/genetics , Saccharomyces cerevisiae/genetics , Biotechnology , Droughts , Gene Library , Genetic Engineering , Phosphoglucomutase/classification , Phosphoglucomutase/metabolism , Phosphotransferases (Phosphomutases)/classification , Phosphotransferases (Phosphomutases)/metabolism , Phylogeny , Rhodotorula/metabolism , Saccharomyces cerevisiae/metabolism , Salinity , Salt Tolerance/genetics , Stress, PhysiologicalABSTRACT
BACKGROUND: Phosphomannomutase (PMM) is an essential enzyme in eukaryotes. However, little is known about PMM gene and function in crop plants. Here, we report molecular evolutionary and biochemical analysis of PMM genes in bread wheat and related Triticeae species. RESULTS: Two sets of homologous PMM genes (TaPMM-1 and 2) were found in bread wheat, and two corresponding PMM genes were identified in the diploid progenitors of bread wheat and many other diploid Triticeae species. The duplication event yielding PMM-1 and 2 occurred before the radiation of diploid Triticeae genomes. The PMM gene family in wheat and relatives may evolve largely under purifying selection. Among the six TaPMM genes, the transcript levels of PMM-1 members were comparatively high and their recombinant proteins were all enzymatically active. However, PMM-2 homologs exhibited lower transcript levels, two of which were also inactive. TaPMM-A1, B1 and D1 were probably the main active isozymes in bread wheat tissues. The three isozymes differed from their counterparts in barley and Brachypodium distachyon in being more tolerant to elevated test temperatures. CONCLUSION: Our work identified the genes encoding PMM isozymes in bread wheat and relatives, uncovered a unique PMM duplication event in diverse Triticeae species, and revealed the main active PMM isozymes in bread wheat tissues. The knowledge obtained here improves the understanding of PMM evolution in eukaryotic organisms, and may facilitate further investigations of PMM function in the temperature adaptability of bread wheat.
Subject(s)
Gene Duplication , Phosphotransferases (Phosphomutases)/genetics , Plant Proteins/genetics , Triticum/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Cloning, Molecular , Evolution, Molecular , Gene Dosage , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Genetic Complementation Test , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Mutation , Phosphotransferases (Phosphomutases)/classification , Phosphotransferases (Phosphomutases)/metabolism , Phylogeny , Plant Proteins/metabolism , Poaceae/classification , Poaceae/enzymology , Poaceae/genetics , Reverse Transcriptase Polymerase Chain Reaction , Selection, Genetic , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Species Specificity , Triticum/enzymology , Yeasts/enzymology , Yeasts/geneticsABSTRACT
The most common type of the congenital disorders of glycosylation, CDG-Ia, is caused by mutations in the human PMM2 gene, reducing phosphomannomutase (PMM) activity. The PMM2 mutations mainly lead to neurological symptoms, while other tissues are only variably affected. Another phosphomannomutase, PMM1, is present at high levels in the brain. This raises the question why PMM1 does not compensate for the reduced PMM2 activity during CDG-Ia pathogenesis. We compared the expression profile of the murine Pmm1 and Pmm2 mRNA and protein in prenatal and postnatal mouse brain at the histological level. We observed a considerable expression of both Pmms in different regions of the embryonic and adult mouse brain. Surprisingly, the expression patterns were largely overlapping. This data indicates that expression differences on the cellular and tissue level are an unlikely explanation for the absence of functional compensation. These results suggest that Pmm1 in vivo does not exert the phosphomannomutase-like activity seen in biochemical assays, but either acts on as yet unidentified specific substrates or fulfils entirely different functions.
