Binding of l-kynurenine to X. campestris tryptophan 2,3-dioxygenase.

The kynurenine pathway is the major route of tryptophan metabolism. The first step of this pathway is catalysed by one of two heme-dependent dioxygenase enzymes - tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) - leading initially to the formation of N-formylkynurenine (NFK). In this paper, we present a crystal structure of a bacterial TDO from X. campestris in complex with l-kynurenine, the hydrolysed product of NFK. l-kynurenine is bound at the active site in a similar location to the substrate (l-Trp). Hydrogen bonding interactions with Arg117 and the heme 7-propionate anchor the l-kynurenine molecule into the pocket. A mechanism for the hydrolysis of NFK in the active site is presented.

An Evidence-Based Walking Program in Oregon Communities: Step It Up! Survivors.

Physical activity can help mitigate the long-term symptoms and side effects of cancer and its treatment, but most cancer survivors are not active enough to achieve these benefits. An evidence-based strategy to promote physical activity among adults is a community group-based walking program. However, many evidence-based programs do not achieve intended population health outcomes because of the challenges of real-world implementation. We used the Interactive Systems Framework for Dissemination and Implementation to conceptualize implementation of a capacity-building intervention to support delivery of a community group-based walking program. We adapted an evidence-based guide for community group-based walking programs for cancer survivors and their support network. We provided a capacity-building intervention (technical assistance and small-grant funding) and evaluated this implementation intervention. We assessed effectiveness of the intervention by measuring adoption, acceptability, appropriateness, feasibility, fidelity, implementation costs, and penetration through monthly progress reports, site visit observations, interviews, and a final report. Eight organizations received a small grant and technical assistance and implemented Step It Up! Survivors (SIUS). SIUS helped cancer survivors increase their physical activity, establish social connections, and be part of a supportive environment. Despite receiving monthly technical assistance, some grantees experienced challenges in recruiting participants, developing community partnerships, and adhering to the prescribed implementation plan. Implementation facilitators included community partners and specific components (eg, incentives for participants, webinars). Organizations needed different amounts and types of assistance with adaptation and implementation. Overall fidelity to SIUS ranged from 64% to 88%. Some integrated SIUS within existing organizational programming for sustainability. The provision of funding and technical assistance was a successful implementation intervention. Our results suggest a need to better tailor technical assistance while organizations are in the process of adapting, implementing, and sustaining an evidence-based program in their local communities.

Flavin-containing heme enzymes.

There are many examples of oxidative enzymes containing both flavin and heme prosthetic groups that carry out the oxidation of their substrate. For the purpose of this article we have chosen five systems. Two of these, the L-lactate dehydrogenase flavocytochrome b(2) and cellobiose dehydrogenase, carry out the catalytic chemistry at the flavin group. In contrast, the remaining three require activation of dioxygen at the heme group in order to accomplish substrate oxidation, these being flavohemoglobin, a nitric oxide dioxygenase, and the mono-oxygenases nitric oxide synthase and flavocytochrome P450 BM3, which functions as a fatty acid hydroxylase. In the light of recent advances we will describe the structures of these enzymes, some of which share significant homology. We will also discuss their diverse and sometimes controversial catalytic mechanisms, and consider electron transfer processes between the redox cofactors in order to provide an overview of this fascinating set of enzymes.

Histidine 55 of tryptophan 2,3-dioxygenase is not an active site base but regulates catalysis by controlling substrate binding.

Tryptophan 2,3-dioxygenase (TDO) from Xanthomonas campestris is a highly specific heme-containing enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). The structure of wild type (WT TDO) in the catalytically active, ferrous (Fe (2+)) form and in complex with its substrate l-tryptophan ( l-Trp) was recently reported [Forouhar et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 473-478] and revealed that histidine 55 hydrogen bonds to l-Trp, precisely positioning it in the active site and implicating it as a possible active site base. In this study the substitution of the active site residue histidine 55 by alanine and serine (H55A and H55S) provides insight into the molecular mechanism used by the enzyme to control substrate binding. We report the crystal structure of the H55A and H55S mutant forms at 2.15 and 1.90 A resolution, respectively, in binary complexes with l-Trp. These structural data, in conjunction with potentiometric and kinetic studies on both mutants, reveal that histidine 55 is not essential for turnover but greatly disfavors the mechanistically unproductive binding of l-Trp to the oxidized enzyme allowing control of catalysis. This is demonstrated by the difference in the K d values for l-Trp binding to the two oxidation states of wild-type TDO (3.8 mM oxidized, 4.1 microM reduced), H55A TDO (11.8 microM oxidized, 3.7 microM reduced), and H55S TDO (18.4 microM oxidized, 5.3 microM reduced).