The structure of L-amino acid oxidase (LAAO) from Calloselasma rhodostoma has been determined to 2.0 A Ê resolution in the presence of two ligands: citrate and o-aminobenzoate (AB). The protomer consists of three domains: an FAD-binding domain, a substrate-binding domain and a helical domain. The interface between the substrate-binding and helical domains forms a 25 A Ê long funnel, which provides access to the active site. Three AB molecules are visible within the funnel of the LAAO±AB complex; their orientations suggest the trajectory of the substrate to the active site. The innermost AB molecule makes hydrogen bond contacts with the active site residues, Arg90 and Gly464, and the aromatic portion of the ligand is situated in a hydrophobic pocket. These contacts are proposed to mimic those of the natural substrate. Comparison of LAAO with the structure of mammalian D-amino acid oxidase reveals signi®cant differences in their modes of substrate entry. Furthermore, a mirror-symmetrical relationship between the two substrate-binding sites is observed which facilitates enantiomeric selectivity while preserving a common arrangement of the atoms involved in catalysis.

Riboflavin (vitamin B 2 ) serves as the precursor for FMN and FAD in almost all organisms that utilize the redox-active isoalloxazine ring system as a coenzyme in enzymatic reactions. The role of flavin, however, is not limited to redox processes, as 10% of flavin-dependent enzymes catalyze nonredox reactions. Moreover, the flavin cofactor is also widely used as a signaling and sensing molecule in biological processes such as phototropism and nitrogen fixation. Here, we present a study of 374 flavin-dependent proteins analyzed with regard to their function, structure and distribution among 22 archaeal, eubacterial, protozoan and eukaryotic genomes. More than 90% of flavin-dependent enzymes are oxidoreductases, and the remaining enzymes are classified as transferases (4.3%), lyases (2.9%), isomerases (1.4%) and ligases (0.4%). The majority of enzymes utilize FAD (75%) rather than FMN (25%), and bind the cofactor noncovalently (90%). High-resolution structures are available for about half of the flavoproteins. FAD-containing proteins predominantly bind the cofactor in a Rossmann fold ( 50%), whereas FMN-containing proteins preferably adopt a (ba) 8 -(TIM)-barrel-like or flavodoxin-like fold. The number of genes encoding flavin-dependent proteins varies greatly in the genomes analyzed, and covers a range from 0.1% to 3.5% of the predicted genes. It appears that some species depend heavily on flavin-dependent oxidoreductases for degradation or biosynthesis, whereas others have minimized their flavoprotein arsenal. An understanding of 'flavin-intensive' lifestyles, such as in the human pathogen Mycobacterium tuberculosis, may result in valuable new intervention strategies that target either riboflavin biosynthesis or uptake.

Summary 12-Oxophytodienoate reductases (OPRs) belong to a family of¯avin-dependent oxidoreductases. With two new tomato isoforms reported here, three OPRs have now been characterized in both tomato and Arabidopsis. Only one of these isoforms (OPR3) participates directly in the octadecanoid pathway for jasmonic acid biosynthesis, as only OPR3 reduces the 9S,13S-stereoisomer of 12-oxophytodienoic acid, the biological precursor of jasmonic acid. The subcellular localization of OPRs was analyzed in tomato and Arabidopsis. The OPR3 protein and activity were consistently found in peroxisomes where they co-localize with the enzymes of b-oxidation which catalyze the ®nal steps in the formation of jasmonic acid. The octadecanoid pathway is thus con®ned to plastids and peroxisomes and, in contrast to previous assumptions, does not involve the cytosolic compartment. The expression of tomato (Lycopersicon esculentum, Le) OPR3 was analyzed in the context of defense-related genes using a microarray comprising 233 cDNA probes. LeOPR3 was found to be up-regulated after wounding with induction kinetics resembling those of other octadecanoid pathway enzymes. In contrast to the induction of genes for wound response proteins (e.g. proteinase inhibitors), the accumulation of octadecanoid pathway transcripts was found to be more rapid and transient in wounded leaves, but hardly detectable in unwounded, systemic leaves. Consistent with the expression data, OPDA and JA were found to accumulate locally but not systemically in the leaves of wounded tomato plants. The transcriptional activation of the octadecanoid pathway and the accumulation of JA to high levels are, thus not required for the activation of defense gene expression in systemic tissues.

Here we report the crystal structure of YqjM, a homolog of Old Yellow Enzyme (OYE) that is involved in the oxidative stress response of Bacillus subtilis. In addition to the oxidized and reduced enzyme form, the structures of complexes with p-hydroxybenzaldehyde and pnitrophenol, respectively, were solved. As for other OYE family members, YqjM folds into a (␣/␤) 8 -barrel and has one molecule of flavin mononucleotide bound non-covalently at the COOH termini of the ␤-sheet. Most of the interactions that control the electronic properties of the flavin mononucleotide cofactor are conserved within the OYE family. However, in contrast to all members of the OYE family characterized to date, YqjM exhibits several unique structural features. For example, the enzyme exists as a homotetramer that is assembled as a dimer of catalytically dependent dimers. Moreover, the protein displays a shared active site architecture where an arginine finger (Arg 336 ) at the COOH terminus of one monomer extends into the active site of the adjacent monomer and is directly involved in substrate recognition. Another remarkable difference in the binding of the ligand in YqjM is represented by the contribution of the NH 2 -terminal Tyr 28 instead of a COOH-terminal tyrosine in OYE and its homologs. The structural information led to a specific data base search from which a new class of OYE oxidoreductases was identified that exhibits a strict conservation of active site residues, which are critical for this subfamily, most notably Cys 26 , Tyr 28 , Lys 109 , and Arg 336 . Therefore, YqjM is the first representative of a new bacterial subfamily of OYE homologs.