Phthalate Dioxygenase Reductase: a Modular Structure for Electron Transfer from Pyridine Nucleotides to [2Fe-2S]

Correll, Carl C.; Batie, Christopher J.; Ballou, David P.; Ludwig, Martha

doi:10.1126/science.1280857

Cited by 308 publications

(328 citation statements)

References 61 publications

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“…The other oxidoreductases shown in Figure 1 catalyze electron transfer reactions between NAD(P)H and iron-containing enzymes such as the cis-diol-forming aromatic ring dioxygenases (43)(44)(45), the bacterial diiron center hydroxylases (34,46) and plant acyl-ACP desaturases (17). Among the three-domain reductases, phthalate dioxygenase reductase [2PIA, (42) …”

Section: Bioinformatic Analysis Of Rv3230cmentioning

confidence: 99%

Identification of Rv3230c as the NADPH Oxidoreductase of a Two-Protein DesA3 Acyl-CoA Desaturase inMycobacterium tuberculosisH37Rv

Chen

Fox

2006

Biochemistry

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DesA3 is a membrane-bound stearoyl-CoA Δ9 desaturase that produces oleic acid, a precursor of mycobacterial membrane phospholipids and triglycerides. DesA3 has sequence homology with other membrane desaturases including the presence of the eight His motif proposed to bind the diiron center active site. This family of desaturases function as multi-component complexes, and thus require electron transfer proteins for efficient catalytic turnover. Here we present evidence that Rv3230c from Mycobacterium tuberculosis H37Rv is a biologically relevant electron transfer partner for DesA3 from the same pathogen. For these studies, Rv3230c was expressed as a partially soluble protein in Escherichia coli; recombinant DesA3 was expressed in Mycobacterium smegmatis as a catalytically active membrane protein. The addition of E. coli lysates containing Rv3230c to lysates of M. smegmatis expressing DesA3 gave strong conversion of [1-14 C]-18:0-CoA to [1-14 C]-cis-Δ 9 -18:1-CoA and of [1-14 C]-16:0-CoA to [1-14 C]-cis-Δ 9 -16:1-CoA. Both M. tuberculosis proteins were required to reconstitute activity, as various combinations of control lysates lacking either Rv3230c or DesA3 gave minimal or no activity. Furthermore, the specificity of interaction between Rv3230c and DesA3 was implied by the inability of other related redox systems to substitute for Rv3230c. The reconstituted activity was dependent upon the presence of NADPH, could be saturated by increasing the amount of Rv3230c added and was also sensitive to the salt concentration in the buffer. The results are consistent with the formation of a protein-protein complex, possibly with electrostatic character. This work defines a multi-protein, acyl-CoA desaturase complex from M. tuberculosis H37Rv to minimally consist of a soluble Rv3230c reductase and integral membrane DesA3 desaturase. Further implications of this finding relative to the properties of other multi-protein iron enzyme complexes are discussed.Mycobacterium tuberculosis is a human pathogen that causes one of the world's deadliest diseases, tuberculosis (TB 1 ). Current estimates are that up to one-third of the world's population may be infected with M. tuberculosis and that over two million people die from TB-related diseases each year (1-4). TB has resurged, in part due to the appearance of multipledrug-resistant strains. Consequently, the development of new drugs and the reexamination of drugs formerly deemed effective have become major focuses of world-wide TB research (3)(4)(5). This intense effort has included genome sequencing (6), proteome functional analysis by micro-array approaches (7-11), the TB Protein Structure Initiative [(12), * To whom correspondence should be addressed: Department of Biochemistry, 433 Babcock Drive, Madison, WI 53706. Telephone: (608) . E-mail: bgfox@biochem.wisc.edu. † This work was supported by the National Institutes of Health Grant R01 GM-50853 to B.G.F.1 Abbreviations: AlkB, alkane ω-hydroxylase; DesA3, product of Mycobacterium tuberculosis H37Rv gene rv3229c, a...

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Section: Bioinformatic Analysis Of Rv3230cmentioning

confidence: 99%

Identification of Rv3230c as the NADPH Oxidoreductase of a Two-Protein DesA3 Acyl-CoA Desaturase inMycobacterium tuberculosisH37Rv

Chen

Fox

2006

Biochemistry

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“…These enzymes are all members of a large family of flavoenzymes related to ferredoxin reductase (FNR) [33] which includes, e.g. phenol hydroxylase P5 protein, xylene monooxygenase, toluate 1,2-dioxygenase, benzoate 1,2-dioxygenase, CDP-6-deoxy-3,4-glucoseen reductase and methane monooxygenase component C. Most importantly, a number of crystal structures are available for interpretation of conserved regions [34,35].…”

Section: Sequence Homologies With Other Proteins and Cofactorbinding mentioning

confidence: 99%

Predicted structure and possible ionmotive mechanism of the sodium‐linked NADH‐ubiquinone oxidoreductase of Vibrio alginolyticus

1995

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Two groups have now published sequences of the six genes contained in the operon coding for the sodium-linked NADH-ubiquinone oxidoreductase of Vibrio aiginolyticus. Sequence analyses indicate that this enzyme is unrelated to other known respiratory NADH dehydrogenases. A search for cofactor motifs suggests that the enzyme contains only one FAD, a ferredoxin-type iron sulphur centre, and the NADH-binding site. These are all located on NqrF, a subunit that can be recognized as a new member of a large diverse family of NAD(P)H-oxidizing flavoenzymes. A possible model of ion-coupling is presented, based upon this new information.Key words." NADH-ubiquinone oxidoreductase; NADH dehydrogenase; Sodium ion translocation BackgroundSeveral types of enzyme (NQR) are known to catalyse the respiratory reaction of oxidation of NADH by membranebound ubiquinone. Only one, termed complex I, is found in mammalian mitochondria, where it oxidizes internally generated NADH. Bovine complex I is remarkably complicated, with seven mitochondrially encoded and at least 34 further nuclear encoded subunits [1]. Gene clusters coding for proton translocating NADH-ubiquinone oxidoreductases have also been identified in the prokaryotes Paracoccus denitrificans, Rhodobacter capsulatus and Escherichia coli [24]. These comprise homologues of only 14 of the mammalian subunits [5] and, presumably, represent a more minimal core catalytic structure. The purified enzyme from E. eoli can be subfractionated into three domains, viz. a fragment (FP) of three subunits which contains the NADH site, FMN and iron sulphur centres; a more amphipathic fragment (IP) of four subunits also containing iron sulphur centres; and a very hydrophobic fragment (HP) of seven proteins which are homologues of the mitochondrially encoded subunits of eukaryotes. In the mitochondrial enzyme, one HP subunit (ND 1) contains a ubiquinone-binding site while another (ND2) is reactive with rotenone and DCCD [1]. All homologues of complex I are presumed to be coupled to proton translocation and have been generically termed NDH-1 types [5,6]. *Corresponding author. Fax: (44) (1208) 821 575. E-mail: mbprr@seqnet.dl.ac.uk Two other types of NADH dehydrogenase can be found in mitochondria from fungal and plant sources [7]. One of these oxidizes internally generated NADH but, in contrast to complex I, it is not coupled to proton translocation. It is likely to be homologous to a non-protonmotive NQR in bacteria, termed NDH-2 [6]. The enzyme may be a single polypeptide, whose sequence has been established [8][9][10], and with FAD as the only redox cofactor [6]. A third NADH dehydrogenase in the inner membrane of plant and fungal mitochondria, which we propose might be termed NDH-3, has an externally facing NADH site and can directly oxidize cytosolic NADH [11]. This enzyme is also not coupled to proton translocation [12]. A bacterial homologue has not been identified and its detailed structure and composition remain unknown.A further type of NQR was first found in the marine bacter...

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“…27 , 28 Distinct flavin mononucleotide (FMN), NADH binding, and [2Fe-2S] domains are revealed in the crystal structure of Burkholderia cepacia phthalate dioxygenase reductase (PDR), demonstrating the structural and functional modularity of FNR proteins. 28 To simplify and extend the characterization of MMOR, the ferredoxin (MMOR-Fd, referred to hereafter as MMOR-Fd (1-98)) and FAD/NADH (MMOR-FAD) domains were expressed as separate proteins. 29 Except for interdomain electron transfer, the reductase domain proteins exhibit redox, spectroscopic, and kinetic properties nearly identical to those of full-length MMOR.…”

Section: Introductionmentioning

confidence: 99%

Intermolecular Electron-Transfer Reactions in Soluble Methane Monooxygenase: A Role for Hysteresis in Protein Function

2005

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Electron transfer from reduced nicotinamide adenine dinucleotide (NADH) to the hydroxylase component (MMOH) of soluble methane monooxygenase (sMMO) primes its non-heme diiron centers for reaction with dioxygen to generate high-valent iron intermediates that convert methane to methanol. This intermolecular electron-transfer step is facilitated by a reductase (MMOR), which contains [2Fe-2S] and flavin adenine dinucleotide (FAD) prosthetic groups. To investigate interprotein electron transfer, chemically reduced MMOR was mixed rapidly with oxidized MMOH in a stopped-flow apparatus, and optical changes associated with reductase oxidation were recorded. The reaction proceeds via four discrete kinetic phases corresponding to the transfer of four electrons into the two dinuclear iron sites of MMOH. Pre-equilibrating the hydroxylase with sMMO auxiliary proteins MMOB or MMOD severely diminishes electron-transfer throughput from MMOR, primarily by shifting the bulk of electron transfer to the slowest pathway. The biphasic reactions for electron transfer to MMOH from several MMOR ferredoxin analogues are also inhibited by MMOB and MMOD. These results, in conjunction with the previous finding that MMOB enhances electrontransfer rates from MMOR to MMOH when preformed MMOR-MMOH-MMOB complexes are allowed to react with NADH [Gassner, G. T.; Lippard, S. J. Biochemistry 1999, 38, 12768-12785], suggest that isomerization of the initial ternary complex is required for maximal electron-transfer rates. To account for the slow electron transfer observed for the ternary precomplex in this work, a model is proposed in which conformational changes imparted to the hydroxylase by MMOR are retained throughout the catalytic cycle. Several electron-transfer schemes are discussed with emphasis on those that invoke multiple interconverting MMOH populations.

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Phthalate Dioxygenase Reductase: a Modular Structure for Electron Transfer from Pyridine Nucleotides to [2Fe-2S]

Cited by 308 publications

References 61 publications

Identification of Rv3230c as the NADPH Oxidoreductase of a Two-Protein DesA3 Acyl-CoA Desaturase inMycobacterium tuberculosisH37Rv

Identification of Rv3230c as the NADPH Oxidoreductase of a Two-Protein DesA3 Acyl-CoA Desaturase inMycobacterium tuberculosisH37Rv

Predicted structure and possible ionmotive mechanism of the sodium‐linked NADH‐ubiquinone oxidoreductase of Vibrio alginolyticus

Intermolecular Electron-Transfer Reactions in Soluble Methane Monooxygenase: A Role for Hysteresis in Protein Function

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