Purification and Characterization of NafY (Apodinitrogenase γ Subunit) from Azotobacter vinelandii

Rubio, Luis M.; Singer, Steven W.; Ludden, Paul W.

doi:10.1074/jbc.m400965200

Cited by 34 publications

(41 citation statements)

References 36 publications

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“…Additionally, NafY could prevent insertion of clusters different from FeMo-co, such as NifB-co or other biosynthetic intermediates, into the apo-NifDK active site. Consistent with this, NafY exhibits much higher affinity for FeMo-co (K D ϭ 60 nM) than NifB-co (K D in the low micromolar range) (11).…”

Section: Discussionsupporting

confidence: 61%

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A Sterile α-Motif Domain in NafY Targets Apo-NifDK for Iron-Molybdenum Cofactor Delivery via a Tethered Domain

Hernández

Phillips

Erbil

et al. 2011

Journal of Biological Chemistry

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NafY participates in the final steps of nitrogenase maturation, having a dual role as iron-molybdenum cofactor (FeMoco) carrier and as chaperone to the FeMo-co-deficient apoNifDK (apo-dinitrogenase). NafY contains an N-terminal domain of unknown function (n-NafY) and a C-terminal domain (core-NafY) necessary for FeMo-co binding. We show here that n-NafY and core-NafY have very weak interactions in intact NafY. The NMR structure of n-NafY reveals that it belongs to the sterile ␣-motif (SAM) family of domains, which are frequently involved in protein-protein interactions. The presence of a SAM domain in NafY was unexpected and could not be inferred from its amino acid sequence. Although SAM domains are very commonly found in eukaryotic proteins, they have rarely been identified in prokaryotes. The n-NafY SAM domain binds apo-NifDK. As opposed to full-length NafY, nNafY impaired FeMo-co insertion when present in molar excess relative to FeMo-co and apo-NifDK. The implications of these observations are discussed to offer a plausible mechanism of FeMo-co insertion. NafY domain structure, molecular tumbling, and interdomain motion, as well as NafY interaction with apo-NifDK are consistent with the function of NafY in FeMo-co delivery to apo-NifDK. Complex metalloclusters are generated from simple [Fe-S] clusters by incorporating additional metals and organic/inorganic ligands. In the case of the FeMo-co of nitrogenase, simple [Fe-S] clusters are the substrates of the NifB protein, which uses them to generate NifB-co, a complex [Fe-S] cluster with a proposed [6Fe-

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Section: Discussionsupporting

confidence: 61%

“…through contacts that include a surface-exposed histidine residue (11). X-ray crystallography showed that core-NafY has a different structure than the FeMo-co-binding region of NifDK, precluding the identification of a FeMo-co-binding region in NafY based on structural similarity (12).…”

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confidence: 99%

A Sterile α-Motif Domain in NafY Targets Apo-NifDK for Iron-Molybdenum Cofactor Delivery via a Tethered Domain

Hernández

Phillips

Erbil

et al. 2011

Journal of Biological Chemistry

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“…Two roles for NafY have been proposed: (a) stabilization of P-clustercontaining apo-NifDK protein in a conformation amenable to FeMo-co insertion, and (b) insertion of FeMo-co into apo-NifDK (33, 69). Consistently, purified NafY protein binds tightly either apo-NifDK or FeMo-co (70). NafY is a 26-kDa monomeric protein composed of two domains that can be separately expressed and purified.…”

Section: The Metallocluster Carrier Proteins Nifx and Nafymentioning

confidence: 97%

“…The three-dimensional structure of the FeMo-co binding domain of NafY has been solved and folds differently than the FeMo-co binding site in NifDK (20) (Figure 8). Site-directed mutagenesis studies on NafY suggested the His 121 residue as a likely ligand to FeMo-co (70). This residue is part of a His-Phe-Gly sequence conserved in NifX, NifB, and VnfX, all of which bind either NifBco or FeMo-co.…”

Section: The Metallocluster Carrier Proteins Nifx and Nafymentioning

confidence: 99%

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Rubio

Ludden

2008

Annu. Rev. Microbiol.

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384

311

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The iron-molybdenum cofactor (FeMo-co), located at the active site of the molybdenum nitrogenase, is one of the most complex metal cofactors known to date. During the past several years, an intensive effort has been made to purify the proteins involved in FeMo-co synthesis and incorporation into nitrogenase. This effort is starting to provide insights into the structures of the FeMo-co biosynthetic intermediates and into the biochemical details of FeMo-co synthesis. 93

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“…Its 14 kDa C-terminal domain binds FeMo-co without the participation of any intermediates and promotes its insertion into apo-NifDK. In particular, mutational analysis indicated that a conserved His 121 of NafY is directly involved in FeMo-co binding (78).…”

Section: Metallocluster Carrier Proteins: Nifx and Nafymentioning

confidence: 99%

Caracterización de una nueva proteína hipertermófila NifB y su papel en el mecanismo de síntesis de NifB-co, precursor del grupo metálico de FeMo-co

Scandurra¹

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© This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognize that its copyright rest with the author and that no quotation from the thesis, or any information derived therefrom may be published without the author's prior, written consent. II III AcknowledgmentsAs each journey, also this one has an end (Thanks goodness). Moreover I find fun and witty to compare my PhD experience as one of "Goldoni's novel", divided in two principal acts, where unlikely characters develop a plot that at first time seems overwhelmed, but at the end is concluded with a brilliant ending.In the first act, I started as "neophyte" my work experience in the laboratory with Emilio, another PhD student. Regarding our work relationship, I always thought that we were the antithesis of a "noble principle " (acid and base, electron and proton, communism and capitalism, ying and yang). Despite we argued constantly and we had many personal clashes, I have always admired his relentless tenacity, which until now remains incomparable. In the same time another two key actors have played a fundamental role at this time. The first was Carlos, a veteran post doc, with an unbridled passion for sailing and for the sea life in general. Perhaps for this passion, when we were working together, Ialways compared our lab during the endless experiments imposed by the ruthless protein purification IV protocols of NifB as a "Spanish galleon" in the middle of a storm and Carlos was the impetuous "boatswain" that shouts the orders to the crew. At the beginning I misunderstood this attitude, but nowadays I have realized that without such determination, we never have come out from the stormy tempest of such complex experiments. The second actor was Cesar, a post doc with many experiences on his shoulders and a strong capacity to be empathetic with the people around him. He always was close to me and he helped me out on my first steps in the molecular biology world.But it was in the second act, when the principal commedians of this story, group of people that maybe influenced more my scientific and personal formation, entered in the scenario. This was the "Landsknecht post docs team", so called because they were "mercenary researchers" coming from the most exotic countries (Morocco, India, US and France). In this group, there was Nasser, a perspicacious guy, from whom I learned to be attentive to the details and cautious in my decisions;Hemant, sunny and cheerful, always ready to comfort me with his maxims of Indian wisdom; Ryan, my mythical friend, pensive and precise scientist, used to communicate an idea with few good words.His ability to remain shiny and cold in the most complicated experiments has always reminded me Clint Eastwood in the movie "Inspector Callaghan". Last but perhaps most important, Simon. This "big boy", coming from the French mountains, despite his young age, from the first time has impressed me for his deep scientific maturity, combined with the passion and curiosity of a children. Not only he ...

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Purification and Characterization of NafY (Apodinitrogenase γ Subunit) from Azotobacter vinelandii

Cited by 34 publications

References 36 publications

A Sterile α-Motif Domain in NafY Targets Apo-NifDK for Iron-Molybdenum Cofactor Delivery via a Tethered Domain

A Sterile α-Motif Domain in NafY Targets Apo-NifDK for Iron-Molybdenum Cofactor Delivery via a Tethered Domain

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Caracterización de una nueva proteína hipertermófila NifB y su papel en el mecanismo de síntesis de NifB-co, precursor del grupo metálico de FeMo-co

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