On the diversity of F420-dependent oxidoreductases: a sequence- and structure-based classification

The F420 deazaflavin cofactor is an intriguing molecule as it structurally resembles the canonical flavin cofactor, although biochemically behaves as a nicotinamide cofactor. Since its discovery, numerous enzymes relying on it have been described. The known deazaflavoproteins are taxonomically restricted to Archaea and Bacteria. The biochemistry of the deazaflavoenzymes is diverse and they exhibit some degree of structural variability as well. In this study a thorough sequence and structural homology evolutionary analysis was performed in order to generate an overarching classification of all known F420-dependent oxidoreductases. Five different superfamilies are described: Superfamily I, TIM-barrel F420-dependent enzymes; Superfamily II, Rossmann fold F420-dependent enzymes; Superfamily III, β-roll F420-dependent enzymes; Superfamily IV, SH3 barrel F420-dependent enzymes and Superfamily V, 3 layer ββα sandwich F420-dependent enzymes. This classification aims to be the framework for the identification, the description and the understanding the biochemistry of novel deazaflavoenzymes.

Structural, biochemical, and inhibition studies of proline biosynthetic enzymes

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of [Delta]1-pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knockout suppresses tumorigenic growth, suggesting PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 A resolution provide insight into cofactor and substrate recognition. We see NADPH bound to the Rossmann fold, over 25 A from the previously proposed site. The 1.85 A resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr238 and the substrate as well as limited cofactor discrimination. We also report kinetic and structural data for PYCR1 complexed with multiple P5C/Pro analogs to probe the potential of PYCR1 as a cancer therapy target. Crystal structures of binary complexes of PYCR1 with L-tetrahydro-2-furoic acid (THFA),N-formyl L-proline (NFLP), thiazolidine-2-carboxylate (T2C), and thiazolidine-4-carboxylate (T4C) have been determined at 1.80-2.35 A resolution. We also present inhibition data for the forward reaction of P5C reduction to proline in the presence of each analog.