Interaction of the molecular chaperone HtpG with uroporphyrinogen decarboxylase in the cyanobacterium Synechococcus elongatus PCC 7942

Uroporphyrinogen decarboxylase (HemE) is important due to its location at the first branch-point in tetrapyrrole biosynthesis. We detected a complex formation between full-length polypeptides of HtpG and HemE by biochemical studies in vivo and in vitro. The interaction suppressed the enzyme activity, suggesting a regulatory role of HtpG in tetrapyrrole biosynthesis.     

Studies on the role of HtpG in the tetrapyrrole biosynthesis pathway of the cyanobacterium Synechococcus elongatus PCC 7942

In cyanobacterium Synechococcus elongatus PCC 7942, we observed that htpG-overexpression caused remarkable growth inhibition. In addition, subcellular fractionation experiments showed that HtpG was localized in the membrane fraction. To understand its function in cyanobacteria, we carried out yeast two-hybrid screening to identify specific proteins interacting with HtpG, and found out, HemE, uroporphyrinogen decarboxylase. When compared to the wild-type strain, the htpG-null mutant and -overexpressing strains exhibited higher and lower cytosolic HemE activity, based on the coproporphyrin production, respectively. These results strongly suggest that HtpG is involved in the regulation of tetrapyrrole biosynthesis through interacting with HemE protein.     

Cytokinin effects on tetrapyrrole biosynthesis and photosynthetic activity in barley seedlings

Cytokinin promotes morphological and physiological processes including the tetrapyrrole biosynthetic pathway during plant development. Only a few steps of chlorophyll (Chl) biosynthesis, exerting the phytohormonal influence, have been individually examined. We performed a comprehensive survey of cytokinin action on the regulation of tetrapyrrole biosynthesis with etiolated and greening barley seedlings. Protein contents, enzyme activities and tetrapyrrole metabolites were analyzed for highly regulated metabolic steps including those of 5-aminolevulinic acid (ALA) biosynthesis and enzymes at the branch point for protoporphyrin IX distribution to Chl and heme. Although levels of the two enzymes of ALA synthesis, glutamyl-tRNA reductase and glutamate 1-semialdehyde aminotransferase, were elevated in dark grown kinetin-treated barley seedlings, the ALA synthesis rate was only significantly enhanced when plant were exposed to light. While cytokinin do not stimulatorily affect Fe-chelatase activity and heme content, it promotes activities of the first enzymes in the Mg branch, Mg protoporphyrin IX chelatase and Mg protoporphyrin IX methyltransferase, in etiolated seedlings up to the first 5 h of light exposure in comparison to control. This elevated activities result in stimulated Chl biosynthesis, which again parallels with enhanced photosynthetic activities indicated by the photosynthetic parameters F _V/F _M, J _CO2max and J _CO2 in the kinetin-treated greening seedlings during the first hours of illumination. Thus, cytokinin-driven acceleration of the tetrapyrrole metabolism supports functioning and assembly of the photosynthetic complexes in developing chloroplasts.     

Recent overview of the Mg branch of the tetrapyrrole biosynthesis leading to chlorophylls

In plants, chlorophylls (chlorophyll a and chlorophyll b) are the most abundant tetrapyrrole molecules and are essential for photosynthesis. The first committed step of chlorophyll biosynthesis is the insertion of Mg²⁺ into protoporphyrin IX, and thus subsequent steps of the biosynthesis are called the Mg branch. As the Mg branch in higher plants is complex, it was not until the last decade—after many years of intensive research—that most of the genes encoding the enzymes for the pathway were identified. Biochemical and molecular genetic analyses have certainly modified the classic metabolic map of tetrapyrrole biosynthesis, and only recently have the molecular mechanisms of regulatory pathways governing chlorophyll metabolism been elucidated. As a result, novel functions of tetrapyrroles and biosynthetic enzymes have been proposed. In this review, I summarize the recent findings on enzymes involved in the Mg branch, mainly in higher plants.     

HtpG,the prokaryotic homologue of Hsp90, stabilizes a phycobilisome protein in the cyanobacterium Synechococcus elongatus PCC 7942

HtpG, a homologue of HSP90, is essential for thermotolerance in cyanobacteria. It is not known how it plays this important role. We obtained evidence that HtpG interacts with linker polypeptides of phycobilisome in the cyanobacterium Synechococcus elongatus PCC 7942. In an htpG mutant, the 30 kDa rod linker polypeptide was reduced. In vitro studies with purified HtpG and phycobilisome showed that HtpG interacts with the linker polypeptide as well as other linker polypeptides to suppress their thermal aggregation with a stoichiometry of one linker polypeptide/HtpG dimer. We constructed various domain‐truncated derivatives of HtpG to identify putative chaperone sites at which HtpG binds linker polypeptides. The middle domain and the N‐terminal domain, although less efficiently, prevented the aggregation of denatured polypeptides, while the C‐terminal domain did not. Truncation of the C‐terminal domain that is involved in the dimerization of HtpG led to decrease in the anti‐aggregation activity, while fusion of the N‐terminal domain to the middle domain lowered the activity. In vitro studies with HtpG and the isolated 30 kDa rod linker polypeptide provided basically similar results to those with HtpG and phycobilisome. ADP inhibited the anti‐aggregation activity, indicating that a compact ADP conformational state provides weaker aggregation protection compared with the others.     

Redox status affects the catalytic activity of glutamyl-tRNA synthetase

Glutamyl-tRNA synthetases (GluRS) provide Glu-tRNA for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. Many organisms contain multiple GluRSs, but whether these duplications solely broaden tRNA specificity or also play additional roles in tetrapyrrole biosynthesis is not known. Previous studies have shown that GluRS1, one of two GluRSs from the extremophile Acidithiobacillus ferrooxidans, is inactivated when intracellular heme is elevated suggesting a specific role for GluRS1 in the regulation of tetrapyrrole biosynthesis. We now show that, in vitro, GluRS1 activity is reversibly inactivated upon oxidation by hemin and hydrogen peroxide. The targets for oxidation-based inhibition were found to be cysteines from a SWIM zinc-binding motif located in the tRNA acceptor helix-binding domain. tRNA^Glu was able to protect GluRS1 against oxidative inactivation by hemin plus hydrogen peroxide. The sensitivity to oxidation of A. ferrooxidans GluRS1 might provide a means to regulate tetrapyrrole and protein biosynthesis in response to extreme changes in both the redox and heme status of the cell via a single enzyme.     

Role for the Cyanobacterial HtpG in Protection from Oxidative Stress

The heat shock protein HtpG, which is a homolog of HSP90, is essential for basal and acquired thermotolerances in cyanobacteria. Recently we demonstrated that HtpG was involved in the acclimation to low temperatures in cyanobacteria. In this study, we elucidated a role of HtpG in the cyanobacterium Synechococcus sp. PCC 7942, in the acclimation to oxidative stress that was caused by high irradiance and/or methyl viologen. The inactivation of the htpG gene resulted in a decrease in the survival rate and an increase in the photoinhibition of photosystem II when cells in a liquid medium were incubated under high light conditions. The htpG mutant was highly sensitive to methyl viologen when it was grown on an agar plate. High irradiance and/or methyl viologen greatly increased the expression of the htpG gene as well as the groEL gene in the wild-type strain. Taken together, our results suggest that HtpG may play a role by itself or with other molecular chaperones in the acclimation to oxidative stress. Received: 1 April 2002 / Accepted: 4 May 2002     

Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

In the cyanobacterium Synechocystis sp. PCC 6803 five open reading frames (scpA–scpE) have been identified that code for single-helix proteins resembling helices I and III of chlorophyll a/b-binding (Cab) antenna proteins from higher plants. They have been named SCPs (small Cab-like proteins). Deletion of a single scp gene in a wild-type or in a photosystem I-less (PS I-less) strain has little effect. However, the effects of functional deletion of scpB or scpE were remarkable under conditions where chlorophyll availability was limited. When cells of a strain lacking PS I and chlL (coding for a polypeptide needed for light-independent protochlorophyllide reduction) were grown in darkness, the phycobilin and protochlorophyllide levels decreased upon deletion of scpB or scpE and the protoheme level was reduced in the strain lacking scpE. Addition of -aminolevulinic acid (ALA) in darkness drastically increased the level of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester in the PS I-less/chlL ^–/scpE ^– strain, whereas PChlide accumulated in the PS I-less/chlL ^–/scpB ^– strain. In the PS I-less/chlL ^– control strain ALA supplementation did not lead to large changes in the levels of tetrapyrrole biosynthesis intermediates. We propose that ScpE and ScpB regulate tetrapyrrole biosynthesis as a function of pigment availability. This regulation occurs primarily at an early step of tetrapyrrole biosynthesis, prior to ALA. In view of the conserved nature of chlorophyll-binding sites in these proteins, it seems likely that regulation by SCPs occurs as a function of chlorophyll availability, with SCPs activating chlorophyll biosynthesis steps when they do not have pigments bound.     

Amitrole treatment of etiolated barley seedlings leads to deregulation of tetrapyrrole synthesis and to reduced expression of Lhc and RbcS genes

The effect of amitrole, known as an inhibitor of carotenoid biosynthesis, upon tetrapyrrole biosynthesis and its regulation has been studied. Etiolated barley (Hordeum vulgare L.) seedlings, grown in 125 μM amitrole, accumulated high levels of 5-aminolevulinate, Mg-protoporphyrin, Mg-protoporphyrin monomethyl ester, and protochlorophyllide. The amitrole-treated seedlings did not form paracrystalline prolamellar bodies, and the induction of Lhc and RbcS gene expression was reduced by non-photooxidative, low-intensity light. None of these events was observed upon treatment of the seedlings with 100 μM norflurazon, another inhibitor of carotenoid biosynthesis. The effect of amitrole cannot be explained solely by interaction with a presumed feedback inhibition of 5-aminolevulinate synthesis since incubation with amitrole and 5-aminolevulinate indicated that deregulation also occurs at later steps of tetrapyrrole biosynthesis. A possible relationship between this deregulation and ultrastructural changes is discussed. In connection with previously published data, we discuss Mg-protoporphyrin and its monomethyl ester as possible candidates for a “plastid signal” that operates as a negative factor, reducing the expression of Lhc and RbcS genes in this higher plant. Received: 27 June 2000 / Accepted: 12 October 2000     

Coproporphyrin III excretion identifies the anaerobic coproporphyrinogen III oxidase HemN as a copper target in the Cu+‐ATPase mutant copA− of Rubrivivax gelatinosus

Two genes encoding structurally similar Copper P_1B‐type ATPases can be identified in several genomes. Notwithstanding the high sequence and structural similarities these ATPases held, it has been suggested that they fulfil distinct physiological roles. In deed, we have shown that the Cu⁺‐ATPase CtpA is required only for the activity of cuproproteins in the purple bacterium Rubrivivax gelatinosus; herein, we show that CopA is not directly required for cytochrome c oxidase but is vital for copper tolerance. Interestingly, excess copper in the copA^? mutant resulted in a substantial decrease of the cytochrome c oxidase and the photosystem under microaerobic and anaerobic conditions together with the extrusion of coproporphyrin III. The data indicated that copper targeted the tetrapyrrole biosynthesis pathway at the level of the coproporphyrinogen III oxidase HemN and thereby affects the oxidase and the photosystem. This is the first in vivo demonstration that copper, like oxygen, affects tetrapyrrole biosynthesis presumably at the level of the SAM and [4Fe‐4S] containing HemN enzyme. In light of these results and similar findings in Escherichia coli, the potential role of copper ions in the evolution of [4Fe‐4S] enzymes and the Cu⁺‐ATPases is discussed.     

HtpG Plays a Role in Cold Acclimation in Cyanobacteria

The heat shock protein HtpG is homologous to members of the Hsp90 protein family of eukaryotes and is essential for basal and acquired thermotolerances in cyanobacteria. In this study we have examined the role of HtpG in the cyanobacterium, Synechococcus sp. PCC 7942, in the acclimation to low temperatures. The inactivation of the htpG gene resulted in severe inhibition of cell growth and of the photosynthetic activity when the htpG mutant was shifted to 16°C from 30°C. Wild-type cells were able to resume growth without a lag period when shifted to 30°C after 5 days at 16°C, while the mutant displayed a detectable lag. The HtpG protein was induced in the wild-type cells at 16°C. Electrophoresis in the absence of sodium dodecyl sulfate (SDS) showed that a novel, high-molecular-weight complex containing GroEL and DnaK accumulated at 16°C, but the accumulation was strongly inhibited in the htpG mutant. Our results demonstrate that the HtpG protein contributes significantly to the ability of cyanobacteria to acclimate to low temperatures. Received: 16 July 2001/Accepted: 15 August 2001     

Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium

Cobalamin (vitamin B₁₂) production in Bacillus megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome-based overexpression of genes involved in vitamin B₁₂ biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysG^AcbiYbtuR, and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding coproporphyrinogen III oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B₁₂ riboswitch upstream of the cbiXJCDETLFGAcysG^AcbiYbtuR operon and the recombinant production of three different vitamin B₁₂ binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B₁₂-dependent feedback inhibition. All these strategies increased cobalamin production in B. megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B₁₂ concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.     

The Bacillus subtilis htpG gene is not involved in thermal stress management

To study the influence of the htpG gene on thermal stress management in Bacillus subtilis, two different kinds of htpG mutation were constructed. In one case, the gene was inactivated by insertion of a cat cassette in to the coding region; htpG was thus found to be non-essential. In the second case, the htpG gene was fused to a xylose-dependent promoter, allowing expression of the gene to be controlled. In the absence of HtpG protein, recovery of cells from a heat shock at 53° C was retarded, and this delay could be eliminated by overproduction of HtpG. While htpG is not involved in the development of induced thermotolerance, DnaK and GroE proteins are absolutely required. Overproduction of class I heat-shock proteins prior to shifting cells to a lethal temperature is important but not sufficient for the development of intrinsic thermotolerance. It could be shown that the HtpG protein does not act as a cellular thermometer in B. subtilis. Received: 2 December 1998 / Accepted: 28 January 1999     

Crystal Structure of the Vitamin B12 Biosynthetic Cobaltochelatase, CbiXS, from Archaeoglobus Fulgidus

The Archaeoglobus fulgidus gene af0721 encodes CbiX^S, a small cobaltochelatase associated with the anaerobic biosynthesis of vitamin B₁₂ (cobalamin). The protein was shown to have activity both in vivo and in vitro, catalyzing the insertion of Co²⁺ into sirohydrochlorin. The structure of CbiX^S was determined in two different crystal forms and was shown to consist of a central mixed β-sheet flanked by four α-helices, one of which originates in the C-terminus of a neighboring molecule. CbiX^S is about half the size of other Class II tetrapyrrole chelatases. The overall topography of CbiX^S exhibits substantial resemblance to both the N- and C-terminal regions of several members of the Class II metal chelatases involved in tetrapyrrole biosynthesis. Two histidines (His10 and His74), are in similar positions as the catalytic histidine residues in the anaerobic cobaltochelatase CbiK (His145 and His207). In light of the hypothesis that suggests the larger chelatases evolved via gene duplication and fusion from a CbiX^S-like enzyme, the structure of AF0721 may represent that of an “ancestral” precursor of class II metal chelatases.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Cellular levels of heme affect the activity of dimeric glutamyl-tRNA reductase

Glutamyl-tRNA reductase (GluTR) is the first enzyme committed to tetrapyrrole biosynthesis by the C₅-pathway. This enzyme transforms glutamyl-tRNA into glutamate-1-semi-aldehyde, which is then transformed into 5-amino levulinic acid by the glutamate-1-semi-aldehyde 2,1-aminomutase. Binding of heme to GluTR seems to be relevant to regulate the enzyme function. Recombinant GluTR from Acidithiobacillus ferrooxidans an acidophilic bacterium that participates in bioleaching of minerals was expressed in Escherichia coli and purified as a soluble protein containing type b heme. Upon control of the cellular content of heme in E. coli, GluTR with different levels of bound heme was obtained. An inverse correlation between the activity of the enzyme and the level of bound heme to GluTR suggested a control of the enzyme activity by heme. Heme bound preferentially to dimeric GluTR. An intact dimerization domain was essential for the enzyme to be fully active. We propose that the cellular levels of heme might regulate the activity of GluTR and ultimately its own biosynthesis.     

Xanthomonas albilineans HtpG is required for biosynthesis of the antibiotic and phytotoxin albicidin

Xanthomonas albilineans, the causal agent of leaf scald disease of sugarcane, produces a highly potent polyketide-peptide antibiotic and phytotoxin called albicidin. Previous studies established the involvement of a large cluster of genes in the biosynthesis of this toxin. We report here the sub-cloning and sequencing of an additional gene outside of the main cluster and essential for albicidin biosynthesis. This gene encodes a 634-amino-acid protein that shows high identity with the Escherichia coli heat shock protein HtpG. Complementation studies of X. albilineans Tox- mutants confirmed the requirement of htpG for albicidin biosynthesis and revealed functional interchangeability between E. coli and X. albilineans htpG genes. HtpG was co-localised with albicidin in the cellular membrane, i.e., the cellular fraction where the toxin is most probably biosynthesised. Here we show the requirement of an HtpG protein for the biosynthesis of a polyketide-peptide antibiotic.