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The interaction between CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase (E1) with its reductase (E3) was investigated to determine whether the E1*E3 complex formation is needed for the C-3 deoxygenation activity. The detection of a new species when a mixture of E1 and E3 was examined by size-exclusion chromatography was the initial indication supporting the supposed complex formation. In conjunction with early mechanistic studies, the findings indicated the importance of the E1*E3 complex formation for catalysis and substantiate the mechanism suggested for the overall deoxygenation process.

The reaction catalyzed by CDP-6-deoxy-L-threo-D-gylcero-4-hexulose 3-dehydrase (E1) is an unusual one because it depends on pyridoxamine 5'-phosphate (PMP) and involves a glucoseen radical intermediate in the active site of the E1. Although the reactions in which coenzyme B6 phosphate act as the intermediate are mainly those involving amino acids and amines, the study of the biosynthesis of CDP-3,6-dideoxy-L-arabino-hexopyranose led to the discovery of an exception in which a PMP dependent reaction is catalyzed by E1. The His-220 is suggested for the active-site base using site-directed mutagenesis because the H220N mutant had reduced catalytic activity.

The inactivation of the flavoenzyme, CDP-6-deoxy-delta(super 3,4)-glucoseen reductase (E3) possessing one ferredoxin type (2Fe-2S) cluster, with 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide is a pseudo first order reaction. Except the cysteine residues, the flavin and (Fe2-2S) cluster suffer no modification. Stabilization of the charge transfer complex between E3 and NADH is possible by Cys-296. The Cys-75 restricts the proper adjoining of the redox centers, causing reduced electron transfer and results in enzyme inactivation.

Molecular cloning and sequencing of the asc gene cluster of Yersinia Pseudotuberculosis sero group VA help develop a CDP-ascarylose biosynthetic pathway model to elucidate 3,6-dideoxyhexoses synthesis and compare the asc cluster with the rfb cluster of Salmonella spp. Sequence analysis of the asc gene cluster indicates a low level of G+C concentration. The functional cloning of the gene clusters indicates that the S. typhimurine rfb ORF10.4 codes for Salmonella E1 equivalent and Salmonella rfb ORF7.6 for the E3 equivalent, denoted as rfbH and rfbI, respectively.

Ascarylose is biosynthesized to reveal the catalyzation of cytidine diphosphate(CDP)-6-deoxy-D-glycero-L-threo-4-hexulose-3-dehydrase and CDP-6-deoxy-delta-glucoseen reductase by C-3 deoxygenation. Yersinia pseudotuberculosis and Pasteurella pseudotuberculosis yield E3 but it is not completely distilled. The completely distilled form is a small part of the mixture so obtained. Purified E3 is separated by a refurbished purification sequence. Cloning and expression of the associated gene confirms the purity of E3.

The complete sequence of the gene cluster responsible for the biosynthesis of the cylindrospermopsin, a cytotoxin, in Oscillatoria sp. strain PCC 6506 is described. All the cylindrospermopsin producers have given a PCR product by using specific primers for the amplification of the cryJ gene from genomic DNA.

The in vitro reconstitution of the first steps in the biosynthesis for Oscillatoria PCC 6506 and the identification of the gene coding in PCC 6506 for a Sfp-like phosphopantetheinyl transferase is reported. AnaB which belongs to the acyl-CoA dehydrogenase superfamily might likely be involved in the catalysis of an [alpha]-[beta]-dehydrogenation of the thioester-bound proline followed by an aza-allylic isomerization to yield (S)-pyrroline-5-carboxyl-AnaD, the proposed starter for the subsequent polyketide synthase, AnaE.

The identification of a sequence from the genome of Oscillatoria sp. strain PCC 6506 coding for a polyketide synthase is described. Axenic cyanobacteria is used to show this sequence only in the genomes of Oscillatoria strains producing anatoxin-a or homoanatoxin-a, which has indicated its likely environment in the biosynthesis of these toxins.

Anatoxin-a and homoanatoxin-a are potent neurotoxins that are produced by cyanobacteria and the GC-MS and NMR analyses using labeled precursors have confirmed that proline might be the starter of these polyketide synthases. The data have provided evidence that the studies have identified the gene cluster responsible for the production of anatoxin-a and homoanatoxin-a in Oscillatoria PCC 6506.

Mutagenetic and chemical rescue experiments that confirm an important role for bicarbonate in Escherichia coli cyclopropane fatty acid synthase (CFAS) catalysis is reported. A proposition that residue E239, one of the bicarbonate ligands, could form a catalytic dyad with his ion is presented.

We report a draft sequence of the genome of Oscillatoria sp. PCC 6506, a cyanobacterium that produces anatoxin-a and homoanatoxin-a, two neurotoxins, and cylindrospermopsin, a cytotoxin. Beside the clusters of genes responsible for the biosynthesis of these toxins, we have found other clusters of genes likely involved in the biosynthesis of not-yet-identified secondary metabolites. doi: 10.1128/JB.00704-10