Main observation and conclusion The aminoglycoside antibiotic apramycin contains a unique bicyclic octose moiety, and biosynthesis of this moiety involves an oxidoreductase AprQ. Unlike other known Q series proteins involved in aminoglycosides biosynthesis, AprQ does not work with an aminotransferase partner, and performs a four-electron oxidation that converts a CH2OH moiety to a carboxylate group. In this study, we report mechanistic investigation of AprQ. We showed AprQ contains a flavin mononucleotide (FMN) cofactor, which is different from other known Q series enzymes that contain a flavin adenine dinucleotide (FAD) cofactor. A series of biochemical assays showed that AprQ is not a monooxygenase but a flavoprotein oxidase. Although molecular O-2 is strictly required for reaction turnover, four-electron oxidation can be achieved in the absence of O-2 in single turnover condition. These findings establish the detailed catalytic mechanism of AprQ and expand the growing family of flavoprotein oxidases, an increasingly important class of biocatalysts.

Snow cover is projected to decline during the next century in many ecosystems that currently experience a seasonal snowpack. Because snow insulates soils from frigid winter air temperatures, soils are expected to become colder and experience more winter soil freeze-thaw cycles as snow cover continues to decline. Tree roots are adversely affected by snowpack reduction, but whether loss of snow will affect root-microbe interactions remains largely unknown. The objective of this study was to distinguish and attribute direct (e.g., winter snow- and/or soil frost-mediated) vs. indirect (e.g., root-mediated) effects of winter climate change on microbial biomass, the potential activity of microbial exoenzymes, and net N mineralization and nitrification rates. Soil cores were incubated in situ in nylon mesh that either allowed roots to grow into the soil core (2mm pore size) or excluded root ingrowth (50m pore size) for up to 29months along a natural winter climate gradient at Hubbard Brook Experimental Forest, NH (USA). Microbial biomass did not differ among ingrowth or exclusion cores. Across sampling dates, the potential activities of cellobiohydrolase, phenol oxidase, and peroxidase, and net N mineralization rates were more strongly related to soil volumetric water content (P<0.05; R-2=0.25-0.46) than to root biomass, snow or soil frost, or winter soil temperature (R-2<0.10). Root ingrowth was positively related to soil frost (P<0.01; R-2=0.28), suggesting that trees compensate for overwinter root mortality caused by soil freezing by re-allocating resources towards root production. At the sites with the deepest snow cover, root ingrowth reduced nitrification rates by 30% (P<0.01), showing that tree roots exert significant influence over nitrification, which declines with reduced snow cover. If soil freezing intensifies over time, then greater compensatory root growth may reduce nitrification rates directly via plant-microbe N competition and indirectly through a negative feedback on soil moisture, resulting in lower N availability to trees in northern hardwood forests.