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The crystal structure of the tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus, a hyperthermophilic archaeon (formerly archaebacterium) that grows optimally at 100 degrees C, has been determined at 2.3 angstrom resolution by means of multiple isomorphous replacement and multiple crystal form averaging. AOR consists of two identical subunits, each containing an Fe4S4 cluster and a molybdopterin-based tungsten cofactor that is analogous to the molybdenum cofactor found in a large class of oxotransferases. Whereas the general features of the tungsten coordination in this cofactor were consistent with a previously proposed structure, each AOR subunit unexpectedly contained two molybdopterin molecules that coordinate a tungsten by a total of four sulfur ligands, and the pterin system was modified by an intramolecular cyclization that generated a three-ringed structure. In comparison to other proteins, the hyperthermophilic enzyme AOR has a relatively small solvent-exposed surface area, and a relatively large number of both ion pairs and buried atoms. These properties may contribute to the extreme thermostability of this enzyme.

An oxidoreductase with an extremely broad substrate specificity reducing reversibly 2-oxocarboxylates at the expense of reduced artificial redox mediators to (2R)-hydroxycarboxylates has been purified to a specific activity of up to 1800 mumol.min-1.mg-1 for the reduction of phenylpyruvate. The membrane-bound non-pyridine nucleotide-dependent enzyme appears in the form of various oligomers of the 80-kDa monomer. The isoelectric point is 5.1. Based on a molecular mass of 80 kDa the enzyme contains up to one molybdenum, four iron and four sulphur atoms. After growth on 99Mo-labelled molybdate, enzyme and radioactivity coincided as shown by gel electrophoresis. Permanganate oxidation delivers 0.7 mol pterin-6-carboxylic acid. The molybdenum cofactor is a mononucleotide. The enzyme is inhibited by cyanide. The first 20 amino acids have been determined. The enzyme belongs to the rare group of molybdoenzymes which possess no further prosthetic groups than the iron-sulphur clusters.

The content of specific methanogenic cofactors was assessed for a range of hydrogenotrophic and methylotrophic methanogenic bacteria grown on different substrates using high performance liquid chromatography. In general, all methanogens were found to contain coenzyme F420 analogues, methanopterin (MPT) analogues and 5-hydroxybenzimidazolylcobamide (vitamin B12-HBI). In hydrogenotrophic methanogens of the genera Methano-bacterium and Methanobrevibacter, as a rule, coenzymes F420-2 and F420-3 as well as MPT were present. Members of the closely related genera Methanospirillum, Methanogenium, Methanoculleus and Methanoplanus contained the same coenzyme F420 analogues but tatiopterin and/or thermopterin were present instead of MPT. In contrast, methylotrophic methanogens predominantly contained coenzymes F420-5 and F420-4, and sarcinapterin (SPT). In Methanolobus tindarius, both MPT and SPT were found, whereas no MPT analogue could be detected in Methanosphaera stadtmanae. In the hydrogenotroph Methanococcus voltae, SPT occurred as the sole MPT analogue. The levels of the various cofactors varied markedly among different methanogens and also for individual methanogens as a function of growth substrate or batch number. A correlation of cofactor levels and substrate utilized was not established. However, with methylotrophic methanogens it was noticed that the ratio of the contents of vitamin B12-HBI and SPT was independent of growth substrate.

All known molybdoenzymes other than nitrogenase contain the metal in association with molybdopterin or one of its dinucleotide variants. All eukaryotic molybdoproteins have been found to contain only molybdopterin, whereas the majority of bacterial enzymes contain one or another of the dinucleotides of molybdopterin. In contrast, xanthine dehydrogenase from Pseudomonas aeruginosa contains molybdopterin rather than a dinucleotide. To examine whether P. aeruginosa contains any dinucleotide of molybdopterin, cells were subjected to an analytical procedure which converts molybdopterin variants to the highly fluorescent Form A derivatives. The results showed that P. aeruginosa cells do contain molybdopterin guanine dinucleotide. The same procedure showed that rat liver does not contain any of the dinucleotides of molybdopterin.

The chlorate-resistant mutants of Escherichia coli are affected in the biosynthesis of the molybdenum cofactor and show pleiotropic loss of the activities of those enzymes which require the cofactor. The molybdenum cofactor in all molybdoenzymes other than nitrogenase is a complex of the metal with a unique pterin termed molybdopterin. The molybdenum cofactor in a number of E. coli enzymes has been shown to contain GMP in addition to the metal-molybdopterin complex, with the GMP appended in pyrophosphate linkage to the terminal phosphate ester on the molybdopterin side chain. In this paper, we have examined the biochemistry of the chlB mutant and show that the gene product of the chlB locus is essential for the addition of the GMP moiety to form molybdopterin guanine dinucleotide, a step which occurs late in the cofactor biosynthetic pathway in E. coli. Sensitive techniques were developed for the identification of fluorescent derivatives of molybdopterin and of molybdopterin guanine dinucleotide in extracts of E. coli cells. Wild type cells were shown to contain both molybdopterin and molybdopterin guanine dinucleotide, while cells of chlB mutants were found to contain elevated levels of molybdopterin but no detectable molybdopterin guanine dinucleotide.

Quantification of coenzymes and related compounds from methanogens was performed in extracts obtained from whole cells with aqueous ethanol at 80 degrees C. By means of high-performance liquid chromatography the following compounds could be detected and quantified in extracts from Methanobacterium thermoautotrophicum: coenzyme MF430, the prosthetic group of methylcoenzyme M reductase, F560, an oxidation product of this compound, coenzyme F420, F342, methanopterin, and carboxytetrahydromethanopterin, previously known as YFC. Coenzyme MF430, coenzyme F420, and methanopterin could be determined in extracts from Methanosarcina barkeri. Structural differences were noticed between the coenzymes from the methanogenic bacteria studied.

Radioactively labeled carbon monoxide (CO) dehydrogenase has been obtained in good yield and purity from Pseudomonas carboxydoflava grown in the presence of [32P]phosphate. One enzyme molecule contained an average of 8.32 molecules of phosphate. The entire phosphate content was confined to 2 molecules of FAD and 2 molecules of a pterin. These were noncovalently bound. Molybdoenzyme cofactors could be extracted into N-methyl formamide; pterins were isolated by thin-layer chromatography. CO dehydrogenase contained a novel pterin, different from molybdopterin, which was also resolved in other bacterial molybdoenzymes. Therefore, it was tentatively named bactopterin. The characteristic features of bactopterin were as follows. A relative molecular mass, Mr, of 730 which was much greater than that of molybdopterin (330) (Mr values refer to molybdenum-free forms of the cofactors; presumably, the latter were also devoid of the sulfhydryl groups contained in the native compounds). A content of 2 molecules of phosphate/molecule compared to only 1 phosphate in molybdopterin. Bactopterin was three times less susceptible to air oxidation than molybdopterin. Native bactopterin was cleaved by perchloric acid into two phosphorous-containing fragments with Mr of 330 and 420. The smaller one is believed to be very similar to molybdopterin, the larger one was not a pterin but probably contained an aromatic structure.

Bactopterin is a novel pterin occurring in bacterial molybdoenzymes as the organic portion of the molybdenum cofactor. Its structure is investigated here. The compound contains a single pterin ring and carries a side chain at carbon atom 6 of the pterin nucleus as indicated by the formation of pterin-6-carboxylic acid upon alkaline permanganate oxidation. Studies with phosphate-cleaving enzymes revealed the presence of two monophosphoric acid monoesters. The affinity of reduced bactopterin for thiol-Sepharose points to the presence of thiol(s) in active bactopterin.