Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi

Gene sequences annotated as proteins of unknown or non-specific function and hypothetical proteins account for a large fraction of most genomes. In the strictly anaerobic and organohalide respiring Dehalococcoides mccartyi, this lack of annotation plagues almost half the genome. Using a combination of bioinformatics analyses and genome-wide metabolic modelling, new or more specific annotations were proposed for about 80 of these poorly annotated genes in previous investigations of D. mccartyi metabolism. Herein, we report the experimental validation of the proposed reannotations for two such genes (KB1_0495 and KB1_0553) from D. mccartyi strains in the KB-1 community. KB1_0495 or DmIDH was originally annotated as an NAD+-dependent isocitrate dehydrogenase, but biochemical assays revealed its activity primarily with NADP+ as a cofactor. KB1_0553, also denoted as DmPMI, was originally annotated as a hypothetical protein/sugar isomerase domain protein. We previously proposed that it was a bifunctional phosphoglucose isomerase/phosphomannose isomerase, but only phosphomannose isomerase activity was identified and confirmed experimentally. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families

Application of Time-of-Flight-Secondary Ion Mass Spectrometry for the Detection of Enzyme Activity on Solid Wood Substrates

Time-of-flight-secondary ion mass spectrometry (TOF-SIMS) is a surface analysis technique that is herein demonstrated to be a viable tool for the detection of enzyme activity on solid substrates. Proof-of-principle experiments are presented that utilize commercial cellulase and laccase enzymes, which are known to modify major polymeric components of wood (i.e., cellulose and lignin, respectively). Enzyme activity is assessed through principle component analysis (PCA) as well as through peak ratios intended to measure selective enzymatic wood degradation. Spectral reproducibility of the complex wood substrates is found to be within 5% relative standard deviation (RSD), allowing for relative quantification of changes in wood composition. Procedures are also presented to identify and avoid the influence of mass interferences from protein adsorption by the enzyme solutions. The activity of a cellulase cocktail is clearly evident through the TOF-SIMS spectra and is supported by high-pressure liquid chromatography (HPLC) measurements of sugar release and by complementary X-ray photoelectron spectroscopy (XPS) measurements of the wood surfaces. Laccase activity, which is mediated through small organic molecules, can be detected in the TOF-SIMS spectra through a decrease in G and S lignin peaks. This work has positive implications for the development of qualitative, high-throughput screening assays for enzyme activity on industrially relevant, lignocellulosic substrates