Ornithine Cyclodeaminase:  Structure, Mechanism of Action, and Implications for the μ-Crystallin Family^,

Abstract

Ornithine cyclodeaminase catalyzes the conversion of L-ornithine to L-proline by an NAD(+)-dependent hydride transfer reaction that culminates in ammonia elimination. Phylogenetic comparisons of amino acid sequences revealed that the enzyme belongs to the mu-crystallin protein family whose three-dimensional fold has not been reported. Here we describe the crystal structure of ornithine cyclodeaminase in complex with NADH, refined to 1.80 A resolution. The enzyme consists of a homodimeric fold whose subunits comprise two functional regions: (i) a novel substrate-binding domain whose antiparallel beta-strands form a 14-stranded barrel at the oligomeric interface and (ii) a canonical Rossmann fold that interacts with a single dinucleotide positioned for re hydride transfer. The adenosyl moiety of the cofactor resides in a solvent-exposed crevice on the protein surface and makes contact with a "domain-swapped"-like coil-helix module originating from the dyad-related molecule. Diffraction data were also collected to 1.60 A resolution on crystals grown in the presence of l-ornithine. The structure revealed that the substrate carboxyl group interacts with the side chains of Arg45, Lys69, and Arg112. In addition, the ammonia leaving group hydrogen bonds to the side chain of Asp228 and the site of hydride transfer is 3.8 A from C4 of the nicotinamide. The absence of an appropriately positioned water suggested that a previously proposed mechanism that calls for hydrolytic elimination of the imino intermediate must be reconsidered. A more parsimonious description of the chemical mechanism is proposed and discussed in relation to the structure and function of mu-crystallins.

Crystal structure of human μ‐crystallin complexed with NADPH

“…This sequence alignment is produced by ClustalW (Thompson et al 1994) and ESPript (Gouet et al 1999). (Goodman et al 2004), yet they are replaced by Ser and Gly in human CRYM.…”

“…In comparison of the human CRYM active site with those of AfAlaDH and PpOCD (Gallagher et al 2004;Goodman et al 2004), the thyroid hormone T 3 -binding site is proposed. The fact that the T 3 -binding capacity of human CRYM is regulated by the cofactor NADPH (Vie et al 1997) suggests that the T 3 -binding site should be close to the NADPH molecule or structural changes should be triggered upon NADPH binding.…”

“…Although the crystal structures of Archaeoglobus fulgidus AlaDH (AfAlaDH) and Pseudomonas putida OCD (PpOCD) have been solved (Gallagher et al 2004;Goodman et al 2004), no CRYM structure is currently available. In order to understand the molecular mechanism of CRYM function, we overexpressed CRYM in Escherichia coli, and purified and crystallized this protein.…”

The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host

“…FTL578 is a putative ornithine cyclodeaminase (Ocd), which catalyzes the conversion of L-ornithine to L-proline in several plant and soil bacteria (62,63). An FTL578 transposon mutant of F. novicida U112 exhibited reduced growth in macrophages (64), but its activity as an ornithine cyclodeaminase remains to be defined in F. tularensis.…”

Estudos de genes envolvidos na via biossintética do antibiótico antitumoral Cosmomicina

“…Ornitina ciclodesaminase (Ocd) é um membro da família da proteína μ-cristalino de mamíferos, na qual a atividade biológica consiste na conversão de Lornitina a L-prolina e amônia (Goodman et al, 2004). Para tentar entender os grupos funcionais desta enzima que estão envolvidas na catálise foi feita a cristalização da proteína Ocd de Pseudomona putida .…”