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V K Shah

Publications and source records attributed to V K Shah.

At least 19 recordsLinked to original sources
PubMed2001

Accumulation of 55Fe-labeled precursors of the iron-molybdenum cofactor of nitrogenase on NifH and NifX of Azotobacter vinelandii.

Iron-molybdenum cofactor (FeMo-co) biosynthesis involves the participation of several proteins. We have used (55)Fe-labeled NifB-co, the specific iron and sulfur donor to FeMo-co, to investigate the accumulation of protein-bound precursors of FeMo-co. The (55)Fe label from radiolabeled NifB-co became associated with two major protein bands when the in vitro FeMo-co synthesis reaction was carried out with the extract of an Azotobacter vinelandii mutant lacking apodinitrogenase. One of the bands, termed (55)Fe-labeled upper band, was purified and shown to be NifH by immunoblot analysis. The (55)Fe-labeled lower band was identified as NifX by N-terminal sequencing. NifX purified from an A. vinelandii nifB strain showed a different electrophoretic mobility on anoxic native gels than did NifX with the FeMo-co precursor bound.

Azotobacter vinelandii↗
PubMed2000

Requirement of homocitrate for the transfer of a 49V-labeled precursor of the iron-vanadium cofactor from VnfX to nif-apodinitrogenase.

A vanadium- and iron-containing cluster has been shown previously to accumulate on VnfX in the Azotobacter vinelandii mutant strain CA11.1 (DeltanifHDKvnfDGK::spc). In the present study, we show the homocitrate-dependent transfer of (49)V label from VnfX to nif-apodinitrogenase in vitro. This transfer of radiolabel correlates with acquisition of acetylene reduction activity. Acetylene is reduced both to ethylene and ethane by the hybrid holodinitrogenase so formed, a feature characteristic of alternative nitrogenases. Structural analogues of homocitrate prevent the acetylene reduction ability of the resulting dinitrogenase. Addition of NifB cofactor (-co) or a source of vanadium (Na(3)VO(4) or VCl(3)) does not increase nitrogenase activity. Our results suggest that there is in vitro incorporation of homocitrate into the V-Fe-S cluster associated with VnfX and that the completed cluster can be inserted into nif-apodinitrogenase. The homocitrate incorporation reaction and the insertion of the cluster into nif-apodinitrogenase (alpha(2)beta(2)gamma(2)) do not require MgATP. Attempts to achieve FeV-co synthesis using extracts of other FeV-co-negative mutants were unsuccessful, showing that earlier steps in FeV-co synthesis, such as the steps requiring VnfNE or VnfH, do not occur in vitro.

Azotobacter vinelandii↗
PubMed1999

Inhibition of iron-molybdenum cofactor biosynthesis by L127Delta NifH and evidence for a complex formation between L127Delta NifH and NifNE.

Besides serving as the obligate electron donor to dinitrogenase during nitrogenase turnover, dinitrogenase reductase (NifH) is required for the biosynthesis of the iron-molybdenum cofactor (FeMo-co) and for the maturation of alpha(2)beta(2) apo-dinitrogenase (apo-dinitrogenase maturation). In an attempt to understand the role of NifH in FeMo-co biosynthesis, a site-specific altered form of NifH in which leucine at position 127 has been deleted, L127Delta, was employed in in vitro FeMo-co synthesis assays. This altered form of NifH has been shown to inhibit substrate reduction by the wild-type nitrogenase complex, forming a tight protein complex with dinitrogenase. The L127Delta NifH was found to inhibit in vitro FeMo-co synthesis by wild-type NifH as detected by the gamma gel shift assay. Increasing the concentration of NifNE and NifB-cofactor (NifB-co) relieved the inhibition of FeMo-co synthesis by L127Delta NifH. The formation of a complex of L127Delta NifH with NifNE was investigated by gel filtration chromatography. We herein report the formation of a complex between L127Delta NifH and NifNE in the presence of NifB-co. This work presents evidence for one of the possible roles for NifH in FeMo-co biosynthesis, i.e. the interaction of NifH with a NifNE.NifB-co complex.

Azotobacter vinelandii↗
PubMed1999

In vitro biosynthesis of iron-molybdenum cofactor and maturation of the nif-encoded apodinitrogenase. Effect of substitution for NifH with site-specifically altered forms of NifH.

NifH has three different roles in the nitrogenase enzyme system. Apart from serving as the physiological electron donor to dinitrogenase, NifH is involved in iron-molybdenum cofactor (FeMo-co) biosynthesis and in maturation of the FeMo-co-deficient form of apodinitrogenase to a FeMo-co-activable form (apodinitrogenase maturation). The exact roles of NifH in these processes are not well understood. In the present study, the features of NifH required for the aforementioned processes have been investigated by the use of site-specifically altered forms of the enzyme. The ability of six altered forms of NifH inactive in substrate reduction (K15R, D39N, D43N, L127Delta, D129E, and F135Y) to function in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions was investigated. We report that the ability of NifH to bind and not hydrolyze MgATP is required for it to function in these processes. We also present evidence that the ability of NifH to function in these processes is not dictated by the properties known to be required for its function in electron transfer to dinitrogenase. Evidence toward the existence of separate, overlapping sites on NifH for each of its functions (substrate reduction, FeMo-co biosynthesis, and apodinitrogenase maturation) is presented.

Adenosine Triphosphate↗
PubMed1999

A vanadium and iron cluster accumulates on VnfX during iron-vanadium-cofactor synthesis for the vanadium nitrogenase in Azotobacter vinelandii.

The vnf-encoded nitrogenase from Azotobacter vinelandii contains an iron-vanadium cofactor (FeV-co) in its active site. Little is known about the synthesis pathway of FeV-co, other than that some of the gene products required are also involved in the synthesis of the iron-molybdenum cofactor (FeMo-co) of the widely studied molybdenum-dinitrogenase. We have found that VnfX, the gene product of one of the genes contained in the vnf-regulon, accumulates iron and vanadium in a novel V-Fe cluster during synthesis of FeV-co. The electron paramagnetic resonance (EPR) and metal analyses of the V-Fe cluster accumulated on VnfX are consistent with a VFe7-8Sx precursor of FeV-co. The EPR spectrum of VnfX with the V-Fe cluster bound strongly resembles that of isolated FeV-co and a model VFe3S4 compound. The V-Fe cluster accumulating on VnfX does not contain homocitrate. No accumulation of V-Fe cluster on VnfX was observed in strains with deletions in genes known to be involved in the early steps of FeV-co synthesis, suggesting that it corresponds to a precursor of FeV-co. VnfX purified from a nifB strain incapable of FeV-co synthesis has a different electrophoretic mobility in native anoxic gels than does VnfX, which has the V-Fe cluster bound. NifB-co, the Fe and S precursor of FeMo-co (and presumably FeV-co), binds to VnfX purified from the nifB strain, producing a shift in its electrophoretic mobility on anoxic native gels. The data suggest that a precursor of FeV-co that contains vanadium and iron accumulates on VnfX, and thus, VnfX is involved in the synthesis of FeV-co.

Azotobacter vinelandii↗
PubMed1999

Incorporation of molybdenum into the iron-molybdenum cofactor of nitrogenase.

The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase was investigated using 99Mo to follow the incorporation of Mo into precursors. 99Mo label accumulates on dinitrogenase only when all known components of the FeMo-co synthesis system, NifH, NifNE, NifB-cofactor, homocitrate, MgATP, and reductant, are present. Furthermore, 99Mo label accumulates only on the gamma protein, which has been shown to serve as a chaperone/insertase for the maturation of apodinitrogenase when all known components are present. It appears that only completed FeMo-co can accumulate on the gamma protein. Very little FeMo-co synthesis was observed when all known components are used in purified forms, indicating that additional factors are required for optimal FeMo-co synthesis. 99Mo did not accumulate on NifNE under any conditions tested, suggesting that Mo enters the pathway at some other step, although it remains possible that a Mo-containing precursor of FeMo-co that is not sufficiently stable to persist during gel electrophoresis occurs but is not observed. 99Mo accumulates on several unidentified species, which may be the additional components required for FeMo-co synthesis. The molybdenum storage protein was observed and the accumulation of 99Mo on this protein required nucleotide.

Adenosine Triphosphate↗
PubMed1999

Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase.

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42. 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.

Azotobacter vinelandii↗
PubMed1997

ApoNifH functions in iron-molybdenum cofactor synthesis and apodinitrogenase maturation.

NifH (dinitrogenase reductase) has three important roles in the nitrogenase enzyme system. In addition to its role as the obligate electron donor to dinitrogenase, NifH is required for the iron-molybdenum cofactor (FeMo-co) synthesis and apodinitrogenase maturation. We have investigated the requirement of the Fe-S cluster of NifH for these processes by preparing apoNifH. The 4Fe-4S cluster of NifH was removed by chelation of the cluster with alpha, alpha'-bipyridyl. The resulting apoNifH was tested in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions and was found to function in both these processes. Thus, the presence of a redox active 4Fe-4S cluster in NifH is not required for its function in FeMo-co synthesis and in apodinitrogenase maturation. This, in turn, implies that the role of NifH in these processes is not one of electron transfer or of iron or sulfur donation.

Adenosine Triphosphate↗
PubMed1997

In vitro synthesis of the iron-molybdenum cofactor and maturation of the nif-encoded apodinitrogenase. Effect of substitution of VNFH for NIFH.

NIFH (the nifH gene product) has several functions in the nitrogenase enzyme system. In addition to reducing dinitrogenase during nitrogenase turnover, NIFH functions in the biosynthesis of the iron-molybdenum cofactor (FeMo-co), and in the processing of alpha2beta2 apodinitrogenase 1 (a catalytically inactive form of dinitrogenase 1 that lacks the FeMo-co) to the FeMo-co-activatable alpha2beta2gamma2 form. The molybdenum-independent nitrogenase 2 (vnf-encoded) has a distinct dinitrogenase reductase protein, VNFH. We investigated the ability of VNFH to function in the in vitro biosynthesis of FeMo-co and in the maturation of apodinitrogenase 1. VNFH can replace NIFH in both the biosynthesis of FeMo-co and in the maturation of apodinitrogenase 1. These results suggest that the dinitrogenase reductase proteins do not specify the heterometal incorporated into the cofactors of the respective nitrogenase enzymes. The specificity for the incorporation of molybdenum into FeMo-co was also examined using the in vitro FeMo-co synthesis assay system.

Apoproteins↗
PubMed1997

Characterization of VNFG, the delta subunit of the vnf-encoded apodinitrogenase from Azotobacter vinelandii. Implications for its role in the formation of functional dinitrogenase 2.

The vnf-encoded apodinitrogenase (apodinitrogenase 2) from Azotobacter vinelandii is an alpha2beta2delta2 hexamer. The delta subunit (the VNFG protein) has been characterized in order to further delineate its function in the nitrogenase 2 enzyme system. Two species of VNFG were observed in cell-free extracts resolved on anoxic native gels; one is composed of VNFG associated with the VNFDK polypeptides, and the other is a homodimer of the VNFG protein. Both species of VNFG are observed in extracts of A. vinelandii strains that accumulate dinitrogenase 2, whereas extracts of strains impaired in the biosynthetic pathway of the iron-vanadium cofactor (FeV-co) that accumulate apodinitrogenase 2 (a catalytically inactive form of dinitrogenase 2 that lacks FeV-co) exhibit only the VNFG dimer on native gels. FeV-co and nucleotide are required for the stable association of VNFG with the VNFDK polypeptides; this stable association can be correlated with the formation of active dinitrogenase 2. The iron-molybdenum cofactor was unable to replace FeV-co in promoting the stable association of VNFG with VNFDK. FeV-co specifically associates with the VNFG dimer in vitro to form a complex of unknown stoichiometry; combination of this VNFG-FeV-co species with apodinitrogenase 2 results in its reconstitution to dinitrogenase 2. The results presented here suggest that VNFG is required for processing apodinitrogenase 2 to functional dinitrogenase 2.

Animals↗
PubMed1997

How to bring surgery to remote tribal areas.

In a mission hospital located in a tribal area in India we found that surgical patients were not coming to the hospital due to a variety of reasons. Table 1 summarizes the main problems and how they were overcome. The effectiveness of our efforts was reflected in the increase in the number of operations after these measures were carried out (1022 before and 1865 after).

Community Participation↗
PubMed1996

Purification and characterization of the vnf-encoded apodinitrogenase from Azotobacter vinelandii.

The vnf-encoded apodinitrogenase (apodinitrogenase 2) has been purified from Azotobacter vinelandii strain CA117.30 (DeltanifKDB), and is an alpha2beta2delta2 hexamer. Apodinitrogenase 2 can be activated in vitro by the addition of the iron-vanadium cofactor (FeV-co) to form holodinitrogenase 2, which functions in C2H2, H+, and N2 reduction. Under certain conditions, the alpha2beta2delta2 hexamer dissociates to yield the free delta subunit (the VNFG protein) and a form of apodinitrogenase 2 that exhibits no C2H2, H+, or N2 reduction activities in the in vitro FeV-co activation assay; however, these activities can be restored upon addition of VNFG to the FeV-co activation assay system. No other vnf-, nif-, or non-nif-encoded proteins were able to replace the function of VNFG in the in vitro processing of alpha2beta2 apodinitrogenase 2 (in the presence of FeV-co) to a form capable of substrate reduction. Apodinitrogenase 2 is also activable in vitro by the iron-molybdenum cofactor to form a hybrid enzyme with unique properties, most notably the inability to reduce N2 and insensitivity to CO inhibition of C2H2 reduction.

Azotobacter vinelandii↗
PubMed1996

The requirement of reductant for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase.

A source of reductant is routinely added to the in vitro iron-molybdenum cofactor (FeMo-co) synthesis assay, although a requirement for reductant has not been established. This report demonstrates that the addition of reductant to the in vitro FeMo-co synthesis system is not required when Azotobacter vinelandii cell-free extract is prepared in buffer that lacks added reductant. The addition of reductant is required, however, if the A. vinelandii cell-free extract is chemically oxidized prior to addition to the assay. These results might suggest that extracts of A. vinelandii contain a physiological source of reductant that functions in the in vitro synthesis of FeMo-co. It is possible that the proteins required for FeMo-co biosynthesis (e.g. NIFNE and dinitrogenase reductase) are at the appropriate redox state to function in the in vitro reaction in the extract that is free of added reductant but not in the chemically oxidized extract. It is also possible that dinitrogenase reductase and/or NIFNE (both Fe-S proteins required for FeMo-co synthesis) might catalyze the reductant-dependent reaction for FeMo-co synthesis. Dithionite, Ti(III) citrate, and NADH are able to serve as the source of reductant for in vitro FeMo-co biosynthesis.

Azotobacter vinelandii↗
PubMed1996

Purification and characterization of the alternative nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum.

The alternative nitrogenase from a nifH mutant of the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized. The dinitrogenase protein (ANF1) contains three subunits in an apparent alpha2beta2gamma2 structure and contains Fe but no Mo or V. A factor capable of activating apo-dinitrogenase (lacking the FeMo cofactor) from Azotobacter vinelandii was extracted from the alternative dinitrogenase protein with N-methylformamide. The electron paramagnetic resonance (EPR) signal of the dinitrogenase protein is not characteristic of the EPR signals of molybdenum- or vanadium-containing dinitrogenases. The alternative dinitrogenase reductase (ANF2) was purified as an alpha2 dimer containing an Fe4S4 cluster and exhibited an EPR spectrum characteristic of dinitrogenase reductases. The enzyme complex reduces protons to H2 very well but reduces N2 to ammonium poorly. Acetylene is reduced to a mixture of ethylene and ethane.

Acetylene↗
PubMed1995

Incorporation of iron and sulfur from NifB cofactor into the iron-molybdenum cofactor of dinitrogenase.

NifB-co is an iron- and sulfur-containing precursor to the iron-molybdenum cofactor (FeMo-co) of dinitrogenase. The synthesis of NifB-co requires at least the product of the nifB gene. Incorporation of 55Fe and 35S from NifB-co into FeMo-co was observed only when all components of the in vitro FeMo-co synthesis system were present. Incorporation of iron and sulfur from NifB-co into dinitrogenase was not observed in control experiments in which the apodinitrogenase (lacking FeMo-co) was initially activated with purified, unlabeled FeMo-co or in assays where NifB-co was oxygen-inactivated prior to addition to the synthesis system. These data clearly demonstrate that iron and sulfur from active NifB-co are specifically incorporated into FeMo-co of dinitrogenase and provide direct biochemical identification of an iron-sulfur precursor of FeMo-co. Under different in vitro FeMo-co synthesis conditions, iron and sulfur from NifB-co were associated with at least two other proteins (NIFNE and gamma) that are involved in the formation of active dinitrogenase. The results presented here suggest that multiple FeMo-co processing steps might occur on NIFNE and that FeMo-co synthesis is most likely completed prior to the association of FeMo-co with gamma.

Azotobacter vinelandii↗
PubMed1995

Characteristics of NIFNE in Azotobacter vinelandii strains. Implications for the synthesis of the iron-molybdenum cofactor of dinitrogenase.

The products of the nifN and nifE genes of Azotobacter vinelandii function as a 200-kDa alpha 2 beta 2 tetramer (NIFNE) in the synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase, the enzyme system required for biological nitrogen fixation. NIFNE was purified using a modification of the published protocol. Immunoblot analysis of anoxic native gels indicated that distinct forms of NIFNE accumulate in strains deficient in either NIFB (delta nifB::kan delta nifDK) or NIFH (delta nifHDK). During the purification of NIFNE from the delta nifHDK mutant, its mobility in these gels changed, becoming similar to that of NIFNE from the delta nifB::kan delta nifDK mutant. While NIFB activity initially co-purified with the NIFNE activity from the delta nifHDK mutant, further purification of NIFNE activity resulted in the loss of the co-purifying NIFB activity; this loss correlated with the change in NIFNE mobility on native gels. These results suggest that the form of NIFNE accumulated in the delta nifHDK mutant is associated with NIFB activity in crude extract but loses this association during NIFNE purification. Addition of the purified metabolic product of NIFB, termed NifB-co, to either NIFNE purified from the delta nifHDK strain or to the NIFNE in crude extract of the delta nifB::kan delta nifDK strain caused a change in the mobility of NIFNE on anoxic native gels to that of the form accumulated in a delta nifHDK mutant. These results support a model where both NifB-co and dinitrogenase reductase participate in FeMo-co synthesis through NIFNE, which serves as a scaffold for this process.

Azotobacter vinelandii↗
PubMed1994

In vitro synthesis of the iron-molybdenum cofactor of nitrogenase. Purification and characterization of NifB cofactor, the product of NIFB protein.

The requirement of NIFB activity for the biosynthesis of iron-molybdenum cofactor (FeMo-co) can be satisfied by the addition of the low molecular weight product of NIFB, termed NifB cofactor (NifB-co). NifB-co has been purified to homogeneity by a unique one-step method. Addition of NifB-co into the FeMo-co synthesis system generated nitrogenase activity of 27-32 nmol of ethylene formed/min/nmol of iron. Iron is the only metal detected in the NifB-co. NifB-co-dependent in vitro FeMo-co synthesis is absolutely dependent on the presence of molybdate, homocitrate and active NIFNE protein in the reaction mixture. The cofactor appears to be a small Fe-S cluster synthesized by NIFB, as a precursor of FeMo-co. NifB-co did not display any EPR signal at 4 K in 0-4000 gauss range. A solution of NifB-co is greenish-brown in color, similar to FeMo-co. NifB-co exhibits a broad absorbance between 400 and 700 nm with no distinctive peaks or shoulders. NifB-co is stable to repeated freeze-thaw cycles and is also stable in N-methylformamide, the solvent used for the isolation of FeMo-co. The NifB-co is stable to a 5-min heat treatment at 60 degrees C. The cofactor is extremely O2-labile, with half-life of less then 15 s in air.

Bacterial Proteins↗