Amanda A. Brindley

Biochemical and Structural Insights into Bacterial Organelle Form and Biogenesis

Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required.

Three-Dimensional Structure of the Rhodobacter sphaeroides RC- LH1-PufX Complex: Dimerization and Quinone Channels Promoted by PufX

Reaction center-light harvesting 1 (RC-LH1) complexes are the fundamental units of bacterial photosynthesis, which use solar energy to power the reduction of quinone to quinol prior to the formation of the proton gradient that drives ATP synthesis. The dimeric RC-LH1-PufX complex of Rhodobacter sphaeroides is composed of 64 polypeptides and 128 cofactors, including 56 LH1 bacteriochlorophyll a (BChl a) molecules that surround and donate energy to the two RCs. The 3D structure was determined to 8 Å by X-ray crystallography, and a model was built with constraints provided by electron microscopy (EM), nuclear magnetic resonance (NMR), mass spectrometry (MS), and site-directed mutagenesis. Each half of the dimer complex consists of a RC surrounded by an array of 14 LH1 αβ subunits, with two BChls sandwiched between each αβ pair of transmembrane helices. The N- and C-terminal extrinsic domains of PufX promote dimerization by interacting with the corresponding domains of an LH1 β polypeptide from the other half of the RC-LH1-PufX complex. Close contacts between PufX, an LH1 αβ subunit, and the cytoplasmic domain of the RC-H subunit prevent the LH1 complex from encircling the RC and create a channel connecting the RC QB site to an opening in the LH1 ring, allowing Q/QH₂ exchange with the external quinone pool. We also identified a channel that connects the two halves of the dimer, potentially forming a long-range pathway for quinone migration along rows of RC-LH1-PufX complexes in the membrane. The structure of the RC-LH1-PufX complex explains the crucial role played by PufX in dimer formation, and it shows how quinone traffic traverses the LH1 complex as it shuttles between the RC and the cytochrome bc₁ complex.

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides.

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll-protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc1 whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc1-RC-LH1-PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.

The Structure of Saccharomyces cerevisiae Met8p, a Bifunctional Dehydrogenase and Ferrochelatase

Sirohaem is a tetrapyrrole‐derived prosthetic group that is required for the essential assimilation of sulfur and nitrogen into all living systems as part of the sulfite and nitrite reductase systems. The final two steps in the biosynthesis of sirohaem involve a β‐NAD+‐dependent dehydrogenation of precorrin‐2 to generate sirohydrochlorin followed by ferrochelation to yield sirohaem. In Saccharomyces cerevisiae, Met8p is a bifunctional enzyme that carries out both of these reactions. Here, we report the 2.2 Å resolution crystal structure of Met8p, which adopts a novel fold that bears no resemblance to the previously determined structures of cobalt‐ or ferro‐chelatases. Analysis of mutant proteins suggests that both catalytic activities share a single active site, and that Asp141 plays an essential role in both dehydrogenase and chelatase processes.

Enzyme Sequence and Its Relationship to Hyperbaric Stability of Artificial and Natural Fish Lactate Dehydrogenases

The cDNAs of lactate dehydrogenase b (LDH-b) from both deep-sea and shallow living fish species, Corphaenoides armatus and Gadus morhua respectively, have been isolated, sequenced and their encoded products overproduced as recombinant enzymes in E. coli. The proteins were characterised in terms of their kinetic and physical properties and their ability to withstand high pressures. Although the two proteins are very similar in terms of their primary structure, only 21 differences at the amino acid level exist between them, the enzyme from the deep-sea species has a significantly increased tolerance to pressure and a higher thermostability. It was possible to investigate whether the changes in the N-terminal or C-terminal regions played a greater role in barophilic adaptation by the construction of two chimeric enzymes by use of a common restriction site within the cDNAs. One of these hybrids was found to have even greater pressure stability than the recombinant enzyme from the deep-living fish species. It was possible to conclude that the major adaptive changes to pressure tolerance must be located in the N-terminal region of the protein. The types of changes that are found and their spatial location within the protein structure are discussed. An analysis of the kinetic parameters of the enzymes suggests that there is clearly a trade off between Km and kcat values, which likely reflects the necessity of the deep-sea enzyme to operate at low temperatures.

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors

The structures of 5‐aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4‐oxosebacic acid and 4,7‐dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P‐side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A‐ and P‐side substrates). The most intriguing result is the novel finding that 4,7‐dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P‐side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A‐side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A‐ and P‐sites is proposed.

Functional characterization of the early steps of tetrapyrrole biosynthesis and modification in Desulfovibrio vulgaris Hildenborough.

The biosynthesis of the tetrapyrrole framework has been investigated in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough by characterization of the enzymes required for the transformation of aminolaevulinic acid into sirohydrochlorin. PBG (porphobilinogen) synthase (HemB) was found to be a zinc-dependent enzyme that exists in its native state as a homohexamer. PBG deaminase (HemC) was shown to contain the dipyrromethane cofactor. Uroporphyrinogen III synthase is found fused with a uroporphyrinogen III methyltransferase (HemD-CobA). Both activities could be demonstrated in this amalgamated protein and the individual enzyme activities were separated by dissecting the relevant gene to allow the production of two distinct proteins. A gene annotated in the genome as a bifunctional precorrin-2 dehydrogenase/sirohydrochlorin ferrochelatase was in fact shown to act only as a dehydrogenase and is simply capable of synthesizing sirohydrochlorin rather than sirohaem. Genome analysis also reveals a lack of any uroporphyrinogen III decarboxylase, an enzyme necessary for the classical route to haem synthesis. However, the genome does encode some predicted haem d1 biosynthetic enzymes even though the bacterium does not contain the cd1 nitrite reductase. We suggest that sirohydrochlorin acts as a substrate for haem synthesis using a novel pathway that involves homologues of the d1 biogenesis system. This explains why the uroporphyrinogen III synthase is found fused with the methyltransferase, bypassing the need for uroporphyrinogen III decarboxylase activity.

NirJ, a radical SAM family member of the d1 heme biogenesis cluster

NirJ is involved in the transformation of precorrin‐2 into heme d 1, although its precise role in the process has not been established. The purified protein was found to contain a 4Fe–4S centre, in line with the prediction that it belongs to the radical SAM class of enzymes. This was further confirmed by binding of S‐adenosyl‐l‐methionine (SAM) to dithionite‐reduced NirJ, which resulted in a decrease in the signal intensity and in a shift to higher field of the [4Fe–4S]1+ EPR signal. Significantly, though, this approach also led to the appearance of a small but reproducible organic radical signal that was associated with about 2% of the NirJ molecules and was affected by the incorporation of SAM deuterated at the 5′ adenosyl group.

d1 haem biogenesis – assessing the roles of three nir gene products

The synthesis of the modified tetrapyrrole known as d1 haem requires several dedicated proteins which are coded for by a set of genes that are often found adjacent to the structural gene, nirS, for cytochrome cd1 nitrite reductase. NirE, the product of the first gene in the nir biogenesis operon, was anticipated to catalyse the conversion of uroporphyrinogen III into precorrin‐2; this was confirmed, but it was shown that this enzyme is less sensitive to product inhibition than similar enzymes that function in other biosynthetic pathways. Sequence analysis suggesting that one of these proteins, NirN, is a c‐type cytochrome, and has similarity to the part of cytochrome cd1 that binds d1, was validated by recombinant production and characterization of NirN. A NirN–d1 haem complex was demonstrated to release the cofactor to a semi‐apo form of cytochrome cd1 from which d1 was extracted, suggesting a role for NirN in the assembly of cytochrome cd1 (NirS). However, inactivation of nirN surprisingly led to only a marginal attenuation of growth of Paracoccus pantotrophus under anaerobic denitrifying conditions. As predicted, NirC is a c‐type cytochrome; it was shown in vitro to be an electron donor to the NirN–d1 complex.

Elucidation of Substrate Specificity in the Cobalamin (Vitamin B12) Biosynthetic Methyltransferases STRUCTURE AND FUNCTION OF THE C20 METHYLTRANSFERASE (CbiL) FROM METHANOTHERMOBACTER THERMAUTOTROPHICUS

Ring contraction during cobalamin (vitamin B12) biosynthesis requires a seemingly futile methylation of the C20 position of the tetrapyrrole framework. Along the anaerobic route, this reaction is catalyzed by CbiL, which transfers a methyl group from S-adenosyl-l-methionine to cobalt factor II to generate cobalt factor III. CbiL belongs to the class III methyltransferases and displays similarity to other cobalamin biosynthetic methyltransferases that are responsible for the regiospecific methylation of a number of positions on the tetrapyrrole molecular canvas. In an attempt to understand how CbiL selectively methylates the C20 position, a detailed structure function analysis of the enzyme has been undertaken. In this paper, we demonstrate that the enzyme methylates the C20 position, that its preferred substrate is cobalt factor II, and that the metal ion does not undergo any oxidation change during the course of the reaction. The enzyme was crystallized, and its structure was determined by x-ray crystallography, revealing that the 26-kDa protein has a similar overall topology to other class III enzymes. This helped in the identification of some key amino acid residues (Asp104, Lys176, and Tyr220). Analysis of mutant variants of these groups has allowed us to suggest potential roles that these side chains may play in substrate binding and catalysis. EPR analysis of binary and ternary complexes indicate that the protein donates a fifth ligand to the cobalt ion via a gated mechanism to prevent transfer of the methyl group to water. The chemical logic underpinning the methylation is discussed.