The ornithine hydroxylase known as SidA is a class B flavin monooxygenase that catalyzes the first step in the biosynthesis of hydroxamate-containing siderophores in Aspergillus f umigatus. Crystallographic studies of SidA revealed that the FAD undergoes dramatic conformational changes between out and in states during the catalytic cycle. We sought insight into the origins and purpose of flavin motion in class B monooxygenases by probing the function of Met101, a residue that contacts the pyrimidine ring of the in FAD. Steady-state kinetic measurements showed that the mutant variant M101A has a 25-fold lower turnover number. Pre-steady-state kinetic measurements, pH profiles, and solvent kinetic isotope effect measurements were used to isolate the microscopic step that is responsible for the reduced steady-state activity. The data are consistent with a bottleneck in the final step of the mechanism, which involves flavin dehydration and the release of hydroxy-Lornithine and NADP + . Crystal structures were determined for M101A in the resting state and complexed with NADP + . The resting enzyme structure is similar to that of wild-type SidA, consistent with M101A exhibiting normal kinetics for flavin reduction by NADPH and wild-type affinity for NADPH. In contrast, the structure of the M101A−NADP + complex unexpectedly shows the FAD adopting the out conformation and may represent a stalled conformation that is responsible for the slow kinetics. Altogether, our data support a previous proposal that one purpose of the FAD conformational change from in to out in class B flavin monooxygenases is to eject spent NADP + in preparation for a new catalytic cycle.

<p class="p1">A pesar de la relevancia médica y el impacto económico en los sistemas de salud, las enfermedades cardiovasculares, tales como infartos cardíacos y accidentes cerebrovasculares, siguen siendo la principal causa de morbilidad y mortalidad en los países en vías de desarrollo. Esto se debe a que las bases estructurales y funcionales de los procesos de formación de los coágulos sanguíneos o trombos solo se conocen de forma incompleta. La trombina juega un papel esencial en estos procesos y los fundamentos atómico-moleculares de su interacción con otros factores que participan en el proceso de coagulación son poco conocidos, en particular el reconocimiento de importantes sustratos como los factores V y VIII, así como el receptor de plaquetas PAR1. Dada la importancia de estos factores, en esta investigación se produjeron fragmentos del factor VIII humano (FVIII) y se caracterizaron bioquímicamente para realizar ensayos de cristalización de complejos FVIII·Trombina. Para ello, (1) se sobreexpresaron heterólogamente los conectores ácidos entre los dominios del factor VIII (denominados FVIIIa1, FVIIIa2 y FVIIIa3), (2) se purificaron y caracterizaron estos fragmentos recombinantes, (3) se formaron sus complejos con la trombina y (4) se inició la búsqueda de las condiciones de cristalización de estos complejos proteicos. La producción del FVIII, y en particular la determinación de las condiciones en las que crecen cristales del tamaño y calidad apropiados, son un auténtico cuello de botella en los estudios de estructura-función, por ello se considera que la optimización de estos procesos permitirá obtener un mayor número de cristales de proteína de calidad adecuada para futuros estudios de difracción de rayos X.</p>

Uncertainties remain regarding factors influencing the Legionnaires' disease (LD) outbreaks in Flint, MI, after the city switched source waters and interrupted corrosion control. Notably, there was lower incidence of LD associated with residential versus hospital exposure in summer 2015 versus 2014. To test possible causes for this phenomenon, we designed a bench-scale study with an influent pH of 7.3, which demonstrated prolific growth of Legionella pneumophila in the presence of cross-linked polyethylene (PEX) pipe (10 3 cfu/mL), but 2-log lower L. pneumophila levels and >100 μg/L Cu 2+ with copper pipes. However, when phosphate was present with copper pipes, soluble copper and Cu 2+ levels decreased and L. pneumophila increased to a range comparable to that of PEX pipe. Additionally, we reveal that monthly LD incidence in Flint residences during the outbreak months was positively correlated with distribution system pH (R 2 = 0.67, p = 0.01). We hypothesized that copper service lines and pipes in residences provided biocidal levels of Cu 2+ at the very low pHs (7.3−6.9) in late summer 2015. Copper is rarely used in large service lines, point of entry pipes, or storage in hospitals where pH was not correlated to LD incidence. Interactions of pH, corrosion control, and pipe material should be considered in diagnosing LD outbreaks.