Investigating the amino acid sequences of membrane bound dihydroorotate:quinone oxidoreductases (DHOQOs): Structural and functional implications.

Dihydroorotate:quinone oxidoreductases (DHOQOs) are membrane bound enzymes responsible for oxidizing dihydroorotate (DHO) to orotate with concomitant reduction of quinone to quinol. They have FMN as prosthetic group and are part of the monotopic quinone reductase superfamily. These enzymes are also members of the dihydroorotate dehydrogenases (DHODHs) family, which besides membrane bound DHOQOs, class 2, includes soluble enzymes which reduce either NAD+ or fumarate, class 1. As key enzymes in both the de novo pyrimidine biosynthetic pathway as well as in the energetic metabolism, inhibitors of DHOQOs have been investigated as leads for therapeutics in cancer, immunological disorders and bacterial/viral infections. This work is a thorough bioinformatic approach on the structural conservation and taxonomic distribution of DHOQOs. We explored previously established structural/functional hallmarks of these enzymes, while searching for uncharacterized common elements. We also discuss the cellular role of DHOQOs and organize the identified protein sequences within six sub-classes 2A to 2F, according to their taxonomic origin and sequence traits. We concluded that DHOQOs are present in Archaea, Eukarya and Bacteria, including the first recognition in Gram-positive organisms. DHOQOs can be the single dihydroorotate dehydrogenase encoded in the genome of a species, or they can coexist with other DHODHs, as the NAD+ or fumarate reducing enzymes. Furthermore, we show that the type of catalytic base present in the active site is not an absolute criterium to distinguish between class 1 and class 2 enzymes. We propose the existence of a quinone binding motif ("ExAH") adjacent to a hydrophobic cavity present in the membrane interacting N-terminal domain.

Multivariate sequence analysis reveals additional function impacting residues in the SDR superfamily.

The "extended" type of short chain dehydrogenases/reductases (SDR), share a remarkable similarity in their tertiary structures inspite of being highly divergent in their functions and sequences. We have carried out principal component analysis (PCA) on structurally equivalent residue positions of 10 SDR families using information theoretic measures like Jensen-Shannon divergence and average shannon entropy as variables. The results classify residue positions in the SDR fold into six groups, one of which is characterized by low Shannon entropies but high Jensen-Shannon divergence against the reference family SDR1E, suggesting that these positions are responsible for the specific functional identities of individual SDR families, distinguishing them from the reference family SDR1E. Site directed mutagenesis of three residues from this group in the enzyme UDP-Galactose 4-epimerase belonging to SDR1E shows that the mutants promote the formation of NADH containing abortive complexes. Finally, molecular dynamics simulations have been used to suggest a mechanism by which the mutants interfere with the re-oxidation of NADH leading to the formation of abortive complexes.

Functional divergence outlines the evolution of novel protein function in NifH/BchL protein family.

Biological nitrogen fixation is accomplished by prokaryotes through the catalytic action of complex metalloenzyme, nitrogenase. Nitrogenase is a two-protein component system comprising MoFe protein (NifD and K) and Fe protein (NifH). NifH shares structural and mechanistic similarities as well as evolutionary relationships with light-independent protochlorophyllide reductase (BchL), a photosynthesis-related metalloenzyme belonging to the same protein family. We performed a comprehensive bioinformatics analysis of the NifH/BchL family in order to elucidate the intrinsic functional diversity and the underlying evolutionary mechanism among the members. To analyse functional divergence in the NifH/ BchL family, we have conducted pair-wise estimation in altered evolutionary rates between the member proteins. We identified a number of vital amino acid sites which contribute to predicted functional diversity. We have also made use of the maximum likelihood tests for detection of positive selection at the amino acid level followed by the structure-based phylogenetic approach to draw conclusion on the ancient lineage and novel characterization of the NifH/BchL protein family. Our investigation provides ample support to the fact that NifH protein and BchL share robust structural similarities and have probably deviated from a common ancestor followed by divergence in functional properties possibly due to gene duplication.

Structure of the putative dihydroorotate dehydrogenase from Streptococcus mutans.

Streptococcus mutans is one of the pathogenic species involved in dental caries, especially in the initiation and development stages. Here, the crystal structure of SMU.595, a putative dihydroorotate dehydrogenase (DHOD) from S. mutans, is reported at 2.4 Šresolution. DHOD is a flavin mononucleotide-containing enzyme which catalyzes the oxidation of L-dihydroorotate to orotate, which is the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. The reductive lysine-methylation procedure was applied in order to improve the diffraction qualities of the crystals. Analysis of the S. mutans DHOD crystal structure shows that this enzyme is a class 1A DHOD and also suggests potential sites that could be exploited for the design of highly specific inhibitors using the structure-based chemotherapeutic design technique.

Identification and characterization of the tungsten-containing class of benzoyl-coenzyme A reductases.

Aromatic compounds are widely distributed in nature and can only be biomineralized by microorganisms. In anaerobic bacteria, benzoyl-CoA (BCoA) is a central intermediate of aromatic degradation, and serves as substrate for dearomatizing BCoA reductases (BCRs). In facultative anaerobes, the mechanistically difficult reduction of BCoA to cyclohexa-1,5-dienoyl-1-carboxyl-CoA (dienoyl-CoA) is driven by a stoichiometric ATP hydrolysis, catalyzed by a soluble, three [4Fe-4S] cluster-containing BCR. In this work, an in vitro assay for BCR from the obligately anaerobic Geobacter metallireducens was established. It followed the reverse reaction, the formation of BCoA from dienoyl-CoA in the presence of various electron acceptors. The benzoate-induced activity was highly specific for dienoyl-CoA (K(m) = 24 +/- 4 microM). The corresponding oxygen-sensitive enzyme was purified by several chromatographic steps with a 115-fold enrichment and a yield of 18%. The 185-kDa enzyme comprised 73- and 20-kDa subunits, suggesting an alpha(2)beta(2)-composition. MS analysis revealed the subunits as products of the benzoate-induced bamBC genes. The alphabeta unit contained 0.9 W, 15 Fe, and 12.5 acid-labile sulfur. Results from EPR spectroscopy suggest the presence of one [3Fe-4S](0/+1) and three [4Fe-4S](+1/+2) clusters per alphabeta unit; oxidized BamBC exhibited an EPR signal typical for a W(V) species. The FeS clusters and the W- cofactor could only be fully reduced by dienoyl-CoA. BamBC represents the prototype of a previously undescribed class of dearomatizing BCRs that differ completely from the ATP-dependent enzymes from facultative anaerobes.

The OsDHODH1 gene is involved in salt and drought tolerance in rice.

In the present paper, we identified and cloned OsDHODH1 encoding a putative cytosolic dihydroorotate dehydrogenase (DHODH) in rice. Expression analysis indicated that OsDHODH1 is upregulated by salt, drought and exogenous abscisic acid (ABA), but not by cold. By prokaryotic expression, we determined the enzymatic activity of OsDHODH1 and found that overproduction of OsDHODH1 significantly improved the tolerance of Escherichia coli cells to salt and osmotic stresses. Overexpression of the OsDHODH1 gene in rice increased the DHODH activity and enhanced plant tolerance to salt and drought stresses as compared with wild type and OsDHODH1-antisense transgenic plants. Our findings reveal, for the first time, that cytosolic dihydroorotate dehydrogenase is involved in plant stress response and that OsDHODH1 could be used in engineering crop plants with enhanced tolerance to salt and drought.

A soluble NADH-dependent fumarate reductase in the reductive tricarboxylic acid cycle of Hydrogenobacter thermophilus TK-6.

Fumarate reductase (FRD) is an enzyme that reduces fumarate to succinate. In many organisms, it is bound to the membrane and uses electron donors such as quinol. In this study, an FRD from a thermophilic chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6, was purified and characterized. FRD activity using NADH as an electron donor was not detected in the membrane fraction but was found in the soluble fraction. The purified enzyme was demonstrated to be a novel type of FRD, consisting of five subunits. One subunit showed high sequence identity to the catalytic subunits of known FRDs. Although the genes of typical FRDs are assembled in a cluster, the five genes encoding the H. thermophilus FRD were distant from each other in the genome. Furthermore, phylogenetic analysis showed that the H. thermophilus FRD was located in a distinct position from those of known soluble FRDs. This is the first report of a soluble NADH-dependent FRD in Bacteria and of the purification of a FRD that operates in the reductive tricarboxylic acid cycle.

Trypanosoma cruzi oleate desaturase: molecular characterization and comparative analysis in other trypanosomatids.

Trypanosoma cruzi lipids contain a high content of unsaturated fatty acids, primarily oleic acid (C18:1) and linoleic acid (C18:2). Previous data suggest that this parasite is able to convert oleic acid into linoleic acid; humans are not able to do this. Presently, we show that T. cruzi has a gene with high similarity to the delta12 (omega6)-oleate desaturase from plants. Northern blot analysis of the oleate desaturase gene from T. cruzi (OD(Tc)) indicated that this gene is transcribed in epimastigote, amastigote, and trypomastigote forms. Pulsed-field analysis showed that OD(Tc) is located at distinct chromosomal bands on distinct T. cruzi phylogenetic groups. In addition, the chromoblot analysis demonstrated the presence of homologous OD(Tc) genes in several trypanosomatids; namely, Crithidia fasciculata, Herpetomonas megaseliae, Leptomonas seymouri, Trypanosoma freitasi, Trypanosoma rangeli, Trypanosoma lewisi, Blastocrithidia sp., Leishmania amazonensis, Endotrypanum schaudinni, and Trypanosoma conorhini. The native OD(Tc) activity was detected by metabolic labeling and analysis of total fatty acids from epimastigotes and trypomastigotes of T. cruzi, coanomastigotes of C. fasciculata, and promastigotes of L. amazonensis, H. megaseliae, and L. seymouri. The fact that the enzyme oleate desaturase is not present in humans makes it an ideal molecular target for the development of new chemotherapeutic approaches against Chagas disease.

Structure and expression of 12-oxophytodienoate reductase (subgroup I) genes in pea, and characterization of the oxidoreductase activities of their recombinant products.

Recently, we observed that expression of a pea gene (S64) encoding an oxophytodienoic acid reductase (OPR) was induced by a suppressor of pea defense responses, secreted by the pea pathogen Mycosphaerella pinodes. Because it is known that OPRs are usually encoded by families of homologous genes, we screened for genomic and cDNA clones encoding members of this putative OPR family in pea. We isolated five members of the OPR gene family from a pea genomic DNA library, and amplified six cDNA clones, including S64, by RT-PCR (reverse transcriptase-PCR). Sequencing analysis revealed that S64 corresponds to PsOPR2, and the amino acid sequences of the predicted products of the six OPR-like genes shared more than 80% identity with each other. Based on their sequence similarity, all these OPR-like genes code for OPRs of subgroup I, i.e., enzymes which are not required for jasmonic acid biosynthesis. However, the genes varied in their exon/intron organization and in their promoter sequences. To investigate the expression of each individual OPR-like gene, RT-PCR was performed using gene-specific primers. The results indicated that the OPR-like gene most strongly induced by the inoculation of pea plants with a compatible pathogen and by treatment with the suppressor from M. pinodes was PsOPR2. Furthermore, the ability of the six recombinant OPR-like proteins to reduce a model substrate, 2-cyclohexen-1-one (2-CyHE), was investigated. The results indicated that PsOPR1, 4 and 6 display robust activity, and PsOPR2 has a most remarkable ability to reduce 2-CyHE, whereas PsOPR3 has little and PsOPR5 does not reduce this compound. Thus, the six OPR-like proteins can be classified into four types. Interestingly, the gene structures, expression profiles, and enzymatic activities used to classify each member of the pea OPR-like gene family are clearly correlated, indicating that each member of this OPR-like family has a distinct function.