Molybdopterin guanine dinucleotide: a modified form of molybdopterin identified in the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides forma specialis denitrificans.

Abstract

The nature of molybdenum cofactor in the bacterial enzyme dimethyl sulfoxide reductase has been investigated by application of alkylation conditions that convert the molybdenum cofactor in chicken liver sulfite oxidase and milk xanthine oxidase to the stable, well-characterized derivative [di(carboxamidomethyl)Jmolybdopterin. The alkylated pterin obtained from dimethyl sulfoxide reductase was shown to be a modified form of alkylated molybdopterin with increased absorption in the 250-nm region of the spectrum and altered chromatographic behavior. The complex alkylated pterin was resolved into two components by treatment with nucleotide pyrophosphatase. These were identified as di(carboxamidomethyl)molybdopterin and GMP by their absorption spectra, coelution with standard compounds, and by further degradation by alkaline phosphatase to dephospho [di(carboxamidomethyl)]molybdopterin and guanosine. The GMP moiety was sensitive to periodate, identifying it as the 5' isomer. Chemical analysis of the intact alkylated pterin showed the presence of two phosphate residues per pterin. These results established that the pterin isolated from dimethyl sulfoxide reductase contains the phosphoric anhydride of molybdopterin and 5'-GMP, which is designated molybdopterin guanine dinucleotide.The suggestion that all molybdoenzymes contain a common molybdenum cofactor consisting of a low molecular weight organic component complexed to the metal was first put forward in 1964 upon the identification of pleiotropic mutants of Aspergillus nidulans that lacked the activities of nitrate reductase and xanthine dehydrogenase (1). In 1971 it was demonstrated that acidified extracts of molybdoenzymes from diverse sources could reconstitute nitrate reductase activity in extracts of a pleiotropic mutant of Neurospora crassa deficient in functional molybdenum cofactor (2). A notable exception to the concept of a universal molybdenum cofactor was the demonstration that nitrogenase in fact contains a unique iron-molybdenum cofactor that is not interchangeable with that in other molybdoenzymes (3). The identification of a pterin as the organic component of the molybdenum cofactor, first in sulfite oxidase, xanthine oxidase, and nitrate reductase (4), and later in a host of other molybdoenzymes (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), lent further credence to the idea of a common molybdenum cofactor. However, the organic component of the molybdenum cofactor, termed molybdopterin, is extremely unstable when released from its protein environment and has never been structurally characterized in its native state. Instead its structure was deduced from analysis of a number of stable degradation products-form A, form B, and urothione (18, 19)-and was confirmed by specific derivatization of the reactive vicinal sulfhydryl groups producing the dicarboxamidomethyl derivative [di-(carboxamidomethyl)]molybdopterin (camMPT) shown in Fig. 1 (20). Generation of camMPT represented a significant advance in the understanding of molybdopterin chemistr...

Effect of molybdate and tungstate on the biosynthesis of CO dehydrogenase and the molybdopterin cytosine‐dinucleotide‐type of molybdenum cofactor in Hydrogenophaga pseudoflava

“…Extraction of pterins from CO dehydrogenase with SDS and subsequent carboxamidomethylation with iodoacetamide followed published procedures [28]. Isocratic HPLC of pterins was on a reverse-phase C 18 column (ET 250/8/4 Nucleosil 120-7 C18, Macherey & Nagel) with photodiode-array detection (model 991, Waters).…”

“…The mobile phase was 50 mM ammonium acetate, pH 6.8, at a flow rate of 1 ml min Ϫ1 . Dicarboxamidomethyl molybdopterin cytosine dinucleotide [di(cam)MCD] was quantified spectrophotometrically employing ε 367 ϭ 7.34 mM Ϫ1 cm Ϫ1 [28]. Reaction of MCD to form A with I 2 /KI or to pterin-6-carboxylic acid with alkaline permanganate followed published procedures [29].…”

Direct transfer of molybdopterin cofactor to aponitrate reductase from a carrier protein in Chlamydomonas reinhardtii

“…Except for dinitrogenase, all molybdocnzymes studied appear to contain molybdopterin [1][2][3]. Much is known about the structure of molybdopterin and one of' its precursors [4,5] and about genes involved in its biosynthesis [2,6-9] but proteins involved in the pathway from early precursors and molybdate to the final incorporation of MoCo into apoenzymes are poorly understood.…”

Sulphur shuttling across a chaperone during molybdenum cofactor maturation

“…Interestingly, the structure of an FdhD homologue in the d-proteobacteria Desulfotalea psychrophila has been solved (DpFdhD; pdb code 2PW9, unpublished) and revealed that it has structural homologies with the cytidine deaminase family of nucleotide-interacting proteins 13 . As nucleotides are chemical moieties of the Mo-bisPGD structure 14 , it has been hypothesized that EcFdhD could bind and sulphurate the cofactor before its insertion into FDHs 11 . A recent study supports this hypothesis with the copurification of Mo-bisPGD onto FdsC, an FdhD homologue from Rhodobacter capsulatus 15 .…”