This study demonstrates how a multi-theoretical, multilevel process evaluation was used to assess implementation of Families Improving Together (FIT) for weight loss intervention. FIT is a randomized controlled trial evaluating a culturally tailored, motivational plus family-based program on weight loss in African American adolescents and their parents. Social Cognitive, Self Determination, Family Systems theories and cultural tailoring principles guided the conceptualization of essential elements across individual/family, facilitator, and group levels. Data collection included an observational rating tool, attendance records, and a validated psychosocial measure. Results. Attendance records (0=absent, 1=present, criteria=≥70%) indicated that 71.5% of families attended each session. The survey (1=false, 6=true, criteria=≥4.5) indicated that participants perceived a positive group climate (M=5.16, SD=.69). A trained evaluator reported that facilitator dose delivered (0=no, 1=yes, criteria=≥75%) was high (99.6%), and fidelity (1=none to 4=all, criteria=≥3) was adequate at facilitator (M=3.63, SD=.41) and group levels (M=3.35, SD=.49). Five cultural topics were raised by participants related to eating (n=3) and physical activity (n=2) behaviors and were integrated as part of the final curriculum. Discussion. Results identify areas for program improvement related to delivery of multi-theoretical and cultural tailoring elements. Findings may inform future strategies for implementing effective weight loss programs for ethnic minority families.

Iron-sulfur (Fe-S) clusters are necessary for the proper functioning of numerous metalloproteins. Fe-S cluster (Isc) and sulfur utilization factor (Suf) pathways are the key biosynthetic routes responsible for generating these Fe-S cluster prosthetic groups in Escherichia coli. Although Isc dominates under normal conditions, Suf takes over during periods of iron depletion and oxidative stress. Sulfur acquisition via these systems relies on the ability to remove sulfur from free cysteine using a cysteine desulfurase mechanism. In the Suf pathway, the dimeric SufS protein uses the cofactor pyridoxal 5-phosphate (PLP) to abstract sulfur from free cysteine, resulting in the production of alanine and persulfide. Despite much progress, the stepwise mechanism by which this PLP-dependent enzyme operates remains unclear. Here, using rapid-mixing kinetics in conjunction with X-ray crystallography, we analyzed the pre-steadystate kinetics of this process while assigning early intermediates of the mechanism. We employed H123A and C364A SufS variants to trap Cys-aldimine and Cys-ketimine intermediates of the cysteine desulfurase reaction, enabling direct observations of these intermediates and associated conformational changes of the SufS active site. Of note, we propose that Cys-364 is essential for positioning the Cys-aldimine for C␣ deprotonation, His-123 acts to protonate the Ala-enamine intermediate, and Arg-56 facilitates catalysis by hydrogen bonding with the sulfhydryl of Cys-aldimine. Our results, along with previous SufS structural findings, suggest a detailed model of the SufS-catalyzed reaction from Cys binding to C-S bond cleavage and indicate that Arg-56, His-123, and Cys-364 are critical SufS residues in this C-S bond cleavage pathway.

OleT, a recently discovered member of the CYP152 family of cytochrome P450s, catalyzes a unique decarboxylation reaction, converting free fatty acids into 1-olefins and carbon dioxide using H2O2 as an oxidant. The C–C cleavage reaction proceeds through hydrogen atom abstraction by an iron­(IV)-oxo intermediate known as Compound I. The capacity of the enzyme for generating important commodity chemicals and liquid biofuels has inspired a flurry of investigations seeking to maximize its biosynthetic potential. One common approach has sought to address the limitations imposed by the H2O2 cosubstrate, particularly for in vivo applications. Numerous reports have shown relatively efficient decarboxylation activity with various combinations of the enzyme with pyridine nucleotides, biological redox donors, and dioxygen, implicating a mechanism whereby OleT can generate Compound I via a canonical P450 O2 dependent reaction scheme. Here, we have applied transient kinetics, cryoradiolysis, and steady state turnover studies to probe the precise origins of OleT turnover from surrogate redox systems. Electron transfer from several redox donors is prohibitively sluggish, and the enzyme is unable to form the hydroperoxo-ferric adduct that serves as a critical precursor to Compound I. Despite the ability for OleT to readily bind O2 once it is reduced, autoxidation of the enzyme and redox partners leads to the generation of H2O2, which is ultimately responsible for the vast majority of turnover. These results illuminate several strategies for improving OleT for downstream biocatalytic applications.

Increasing levels of energy consumption, dwindling resources, and environmental considerations have served as compelling motivations to explore renewable alternatives to petroleum-based fuels, including enzymatic routes for hydrocarbon synthesis. Phylogenetically diverse species have long been recognized to produce hydrocarbons, but many of the enzymes responsible have been identified within the past decade. The enzymatic conversion of C chain length fatty aldehydes (or acids) to C hydrocarbons, alkanes or alkenes, involves a C-C scission reaction. Surprisingly, the enzymes involved in hydrocarbon synthesis utilize non-heme mononuclear iron, dinuclear iron, and thiolate-ligated heme cofactors that are most often associated with monooxygenation reactions. In this review, we examine the mechanisms of several enzymes involved in hydrocarbon biosynthesis, with specific emphasis on the structural and electronic changes that enable this functional switch.

The β-hydroxylation of l-histidine is the first step in the biosynthesis of the imidazolone base of the antifungal drug nikkomycin. The cytochrome P450 (NikQ) hydroxylates the amino acid while it is appended via a phosphopantetheine linker to the non-ribosomal peptide synthetase (NRPS) NikP1. The latter enzyme is comprised of an MbtH and single adenylation and thiolation domains, a minimal composition that allows for detailed binding and kinetics studies using an intact and homogeneous NRPS substrate. Electron paramagnetic resonance studies confirm that a stable complex is formed with NikQ and NikP1 when the amino acid is tethered. Size exclusion chromatography is used to further refine the principal components that are required for this interaction. NikQ binds NikP1 in the fully charged state, but binding also occurs when NikP1 is lacking both the phosphopantetheine arm and appended amino acid. This demonstrates that the interaction is mainly guided by presentation of the thiolation domain interface, rather than the attached amino acid. Electrochemistry and transient kinetics have been used to probe the influence of l-His-NikP1 binding on catalysis by NikQ. Unlike many P450s, the binding of substrate fails to induce significant changes on the redox potential and autoxidation properties of NikQ and slows down the binding of dioxygen to the ferrous enzyme to initiate catalysis. Collectively, these studies demonstrate a complex interplay between the NRPS maturation process and the recruitment and regulation of an auxiliary tailoring enzyme required for natural product biosynthesis.