The HBx protein from hepatitis B virus coordinates a redox-active Fe-S cluster.

The viral protein HBx is the key regulatory factor of the hepatitis B virus (HBV) and the main etiology for HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma. Historically, HBx has defied biochemical and structural characterization, deterring efforts to understand its molecular mechanisms. Here we show that soluble HBx fused to solubility tags copurifies with either a [2Fe-2S] or a [4Fe-4S] cluster, a feature that is shared among five HBV genotypes. We show that the O2-stable [2Fe-2S] cluster form converts to an O2-sensitive [4Fe-4S] state when reacted with chemical reductants, a transformation that is best described by a reductive coupling mechanism reminiscent of Fe-S cluster scaffold proteins. In addition, the Fe-S cluster conversions are partially reversible in successive reduction-oxidation cycles, with cluster loss mainly occurring during (re)oxidation. The considerably negative reduction potential of the [4Fe-4S]2+/1+ couple (-520 mV) suggests that electron transfer may not be likely in the cell. Collectively, our findings identify HBx as an Fe-S protein with striking similarities to Fe-S scaffold proteins both in cluster type and reductive transformation. An Fe-S cluster in HBx offers new insights into its previously unknown molecular properties and sets the stage for deciphering the roles of HBx-associated iron (mis)regulation and reactive oxygen species in the context of liver tumorigenesis.

Vibrio cholerae V-cGAP3 Is an HD-GYP Phosphodiesterase with a Metal Tunable Substrate Selectivity.

Cyclic dinucleotides (CDNs) are signaling molecules involved in the immune response and virulence factor production. CDN cellular levels are fine-tuned by metal-dependent phosphodiesterases (PDEs), among which HD-GYPs make up a subclass of the larger HD-domain protein superfamily. The human pathogen Vibrio cholerae (Vc) encodes nine HD-GYPs, one of which is V-cGAP3 (or VCA0931). V-cGAP3 acts on c-di-GMP and 3'3'c-GAMP, and this activity is related to bacterial infectivity. However, the extant chemical makeup of the V-cGAP3 cofactor and steady state parameters have not been established. Employing electron paramagnetic resonance and Mössbauer spectroscopy in tandem with elemental analyses and activity assays, we demonstrate that V-cGAP3 coordinates different dimetal cofactors with variable activities. MnII and FeII afford c-di-GMP hydrolysis with the highest observed rates, while c-GAMP hydrolysis is selectively dependent on Mn. V-cGAP3 has a single functional domain, and this simple architecture allows us to examine the roles of characteristic conserved residues in catalysis. Substitution of the adjacent to the active site GYP residue triad and the specifically conserved in HD-domain PDEs fifth histidine ligand (i.e., H371 in V-cGAP3) with alanines severely compromises CDN hydrolysis but only modestly affects cofactor incorporation. Our data are consistent with V-cGAP3 being the major regulator of 3'3'c-GAMP hydrolysis in Vc and delineate the importance of specific residues in tuning activity in HD-GYPs in general. We propose that HD-GYPs exhibit diversity in their metallocofactors and substrates, which may serve to increase their functional potential in regulatory pathways or allow for PDE activity upon adaptation of the parent organism to diverse environmental niches.

Metal Dependence and Functional Diversity of Type I Cas3 Nucleases.

Type I CRISPR-Cas systems provide prokaryotes with protection from parasitic genetic elements by cleaving foreign DNA. In addition, they impact bacterial physiology by regulating pathogenicity and virulence, making them key players in adaptability and evolution. The signature nuclease Cas3 is a phosphodiesterase belonging to the HD-domain metalloprotein superfamily. By directing specific metal incorporation, we map a promiscuous metal ion cofactor profile for Cas3 from Thermobifida fusca (Tf). Tf Cas3 affords significant ssDNA cleavage with four homo-dimetal centers (Fe2+, Co2+, Mn2+, and Ni2+), while the diferrous form is the most active and likely biologically relevant in vivo. Electron paramagnetic resonance (EPR) spectroscopy and Mössbauer spectroscopy show that the diiron cofactor can access three redox forms, while the diferrous form can be readily obtained with mild reductants. We further employ EPR and Mössbauer on Fe-enriched proteins to establish that Cas3″ enzymes harbor a dinuclear cofactor, which was not previously confirmed. We demonstrate that the ancillary His ligand is critical for efficient ssDNA cleavage but not for diiron assembly or small molecule hydrolysis. We further explore the ability of Cas3 to hydrolyze cyclic mononucleotides and show that Tf Cas3 hydrolyzes 2'3'-cAMP with catalytic efficiency comparable to that of the conserved virulence factor A (CvfA), an HD-domain protein hydrolyzing 2'3'-cylic phosphodiester bonds at RNA 3'-termini. Because this CvfA activity is linked to virulence regulation, Cas3 may also utilize 2'3'-cAMP hydrolysis as a possible molecular route to control virulence.

Domain Fusion of Two Oxygenases Affords Organophosphonate Degradation in Pathogenic Fungi.

Proteins of the HD-domain superfamily employ a conserved histidine-aspartate (HD) dyad to coordinate diverse metallocofactors. While most known HD-domain proteins are phosphohydrolases, new additions to this superfamily have emerged such as oxygenases and lyases, expanding their functional repertoire. To date, three HD-domain oxygenases have been identified, all of which employ a mixed-valent FeIIFeIII cofactor to activate their substrates and utilize molecular oxygen to afford cleavage of C-C or C-P bonds via a diferric superoxo intermediate. Phylogenetic analysis reveals an uncharacterized multidomain protein in the pathogenic soil fungus Fonsecaea multimorphosa, herein designated PhoF. PhoF consists of an N-terminal FeII/α-ketoglutarate-dependent domain resembling that of PhnY and a C-terminal HD-domain like that of PhnZ. PhnY and PhnZ are part of an organophosphonate degradation pathway in which PhnY hydroxylates 2-aminoethylphosphonic acid, and PhnZ cleaves the C-P bond of the hydroxylated product yielding phosphate and glycine. Employing electron paramagnetic resonance and Mössbauer spectroscopies in tandem with activity assays, we determined that PhoF carries out the O2-dependent degradation of two aminophosphonates, demonstrating an expanded catalytic efficiency with respect to the individual, but mechanistically coupled PhnY and PhnZ. Our results recognize PhoF as a new example of an HD-domain oxygenase and show that domain fusion of an organophosphonate degradation pathway may be a strategy for disease-causing fungi to acquire increased functional versatility, potentially important for their survival.

HD-[HD-GYP] Phosphodiesterases: Activities and Evolutionary Diversification within the HD-GYP Family.

Cyclic dinucleotides are signaling molecules that modulate many processes, including immune response and virulence factor production. Their cellular levels in bacteria are fine-tuned by metal-dependent phosphodiesterases, namely, the EAL and HD-GYP proteins, with HD-GYPs belonging to the larger HD domain superfamily. In this study, we first focus on the catalytic properties and the range of metal ions and substrates of the HD-[HD-GYP] subfamily, consisting of two HD domains. We identified SO3491 as a homologue of VCA0681 and the second example of an HD-[HD-GYP]. Both proteins hydrolyze c-di-GMP and 3'3'c-GAMP and coordinate various metal ions, but only Fe and to a lesser extent Co support hydrolysis. The proteins are active only in the diferrous form and not in the one-electron more oxidized FeIIFeIII state. Although the C-terminal HD-GYP domain is essential for activity, the role of the N-terminal HD domain remains unknown. We show that the N-terminal site is important for protein stability, influences the individual apparent kcat and KM (but not kcat/KM), and cannot bind c-di-GMP, thus precluding its involvement in cyclic dinucleotide sensing. We proceeded to perform phylogenetic analyses to examine the distribution and functional relationships of the HD-[HD-GYP]s to the rest of the HD-GYPs. The phylogeny provides a correlation map that draws a link between the evolutionary and functional diversification of HD-GYPs, serving as a template for predicting the chemical nature of the metallocofactor, level of activity, and reaction outcome.

A New Microbial Pathway for Organophosphonate Degradation Catalyzed by Two Previously Misannotated Non-Heme-Iron Oxygenases.

The assignment of biochemical functions to hypothetical proteins is challenged by functional diversification within many protein structural superfamilies. This diversification, which is particularly common for metalloenzymes, renders functional annotations that are founded solely on sequence and domain similarities unreliable and often erroneous. Definitive biochemical characterization to delineate functional subgroups within these superfamilies will aid in improving bioinformatic approaches for functional annotation. We describe here the structural and functional characterization of two non-heme-iron oxygenases, TmpA and TmpB, which are encoded by a genomically clustered pair of genes found in more than 350 species of bacteria. TmpA and TmpB are functional homologues of a pair of enzymes (PhnY and PhnZ) that degrade 2-aminoethylphosphonate but instead act on its naturally occurring, quaternary ammonium analogue, 2-(trimethylammonio)ethylphosphonate (TMAEP). TmpA, an iron(II)- and 2-(oxo)glutarate-dependent oxygenase misannotated as a γ-butyrobetaine (γbb) hydroxylase, shows no activity toward γbb but efficiently hydroxylates TMAEP. The product, ( R)-1-hydroxy-2-(trimethylammonio)ethylphosphonate [( R)-OH-TMAEP], then serves as the substrate for the second enzyme, TmpB. By contrast to its purported phosphohydrolytic activity, TmpB is an HD-domain oxygenase that uses a mixed-valent diiron cofactor to enact oxidative cleavage of the C-P bond of its substrate, yielding glycine betaine and phosphate. The high specificities of TmpA and TmpB for their N-trimethylated substrates suggest that they have evolved specifically to degrade TMAEP, which was not previously known to be subject to microbial catabolism. This study thus adds to the growing list of known pathways through which microbes break down organophosphonates to harvest phosphorus, carbon, and nitrogen in nutrient-limited niches.

Structures of Class Id Ribonucleotide Reductase Catalytic Subunits Reveal a Minimal Architecture for Deoxynucleotide Biosynthesis.

Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate ß subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a MnII2 cluster by free superoxide to yield a metal-based MnIIIMnIV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae ( Fj) and Actinobacillus ureae ( Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive ß subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.

Spectroscopic and Electrochemical Characterization of the Mycofactocin Biosynthetic Protein, MftC, Provides Insight into Its Redox Flipping Mechanism.

Mycofactocin is a putative redox cofactor and is classified as a ribosomally synthesized and post-translationally modified peptide (RiPP). Some RiPP natural products, including mycofactocin, rely on a radical S-adenosylmethionine (RS, SAM) protein to modify the precursor peptide. Mycofactocin maturase, MftC, is a unique RS protein that catalyzes the oxidative decarboxylation and C-C bond formation on the precursor peptide MftA. However, the number, chemical nature, and catalytic roles for the MftC [Fe-S] clusters remain unknown. Here, we report that MftC binds a RS [4Fe-4S] cluster and two auxiliary [4Fe-4S] clusters that are required for MftA modification. Furthermore, electron paramagnetic resonance spectra of MftC suggest that SAM and MftA affect the environments of the RS and Aux I cluster, whereas the Aux II cluster is unaffected by the substrates. Lastly, reduction potential assignments of individual [4Fe-4S] clusters by protein film voltammetry show that their potentials are within 100 mV of each other.

Structural Basis for Superoxide Activation of Flavobacterium johnsoniae Class I Ribonucleotide Reductase and for Radical Initiation by Its Dimanganese Cofactor.

A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae ( Fj) differs fundamentally from known (subclass a-c) class I RNRs, warranting its assignment to a new subclass, Id. Its ß subunit shares with Ib counterparts the requirements for manganese(II) and superoxide (O2-) for activation, but it does not require the O2--supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj ß has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj ß directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive "met" (III/III) forms seen with I(a-c) ßs. This tendency makes Fj ß unusually robust, as the II/II form can readily be reactivated. The structure of the protein rationalizes its distinctive traits. A distortion in a core helix of the ferritin-like architecture renders the active site unusually open, introduces a cavity near the cofactor, and positions a subclass-d-specific Lys residue to shepherd O2- to the Mn2II/II cluster. Relative to the positions of the radical tyrosines in the Ia/b proteins, the unreactive Tyr 104 of Fj ß is held away from the cofactor by a hydrogen bond with a subclass-d-specific Thr residue. Structural comparisons, considered with its uniquely simple mode of activation, suggest that the Id protein might most closely resemble the primordial RNR-ß.

NADH reduction of nitroaromatics as a probe for residual ferric form high-spin in a cytochrome P450.

The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2) ferric iron is a well-established phenomenon in the cytochrome P450 (CYP) superfamily, although its origins are still a subject of discussion. A series of mutations that strongly perturb the spin state equilibrium in the camphor hydroxylase CYP101A1 were recently described (Colthart et al., Sci. Rep. 6, 22035 (2016)). Wild type CYP101A1 as well as some CYP101A1 mutants are herein shown to be capable of catalyzing the reduction of nitroacetophenones by NADH to the corresponding anilino compounds (nitroreductase or NRase activity). The distinguishing characteristic between those mutants that catalyze the reduction and those that cannot appears to be the extent to which residual high spin form exists in the absence of the native substrate d-camphor, with those showing the largest spin state shifts upon camphor binding also exhibiting NRase activity. Optical and EPR spectroscopy was used to further examine these phenomena. These results suggest that reduction of nitroaromatics may provide a useful probe of residual high spin states in the CYP superfamily. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Characterization of Lipoyl Synthase from Mycobacterium tuberculosis.

The prevalence of multiple and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is on the rise, necessitating the identification of new targets to combat an organism that has infected one-third of the world's population, according to the World Health Organization. The biosynthesis of the lipoyl cofactor is one possible target, given its critical importance in cellular metabolism and the apparent lack of functional salvage pathways in Mtb that are found in humans and many other organisms. The lipoyl cofactor is synthesized de novo in two committed steps, involving the LipB-catalyzed transfer of an octanoyl chain derived from fatty acid biosynthesis to a lipoyl carrier protein and the LipA-catalyzed insertion of sulfur atoms at C6 and C8 of the octanoyl chain. A number of in vitro studies of lipoyl synthases from Escherichia coli, Sulfolobus solfataricus, and Thermosynechococcus elongatus have been conducted, but the enzyme from Mtb has not been characterized. Herein, we show that LipA from Mtb contains two [4Fe-4S] clusters and converts an octanoyl peptide substrate to the corresponding lipoyl peptide product via the same C6-monothiolated intermediate as that observed in the E. coli LipA reaction. In addition, we show that LipA from Mtb forms a complex with the H protein of the glycine cleavage system and that the strength of association is dependent on the presence of S-adenosyl-l-methionine. We also show that LipA from Mtb can complement a lipA mutant of E. coli, demonstrating the commonalities of the two enzymes. Lastly, we show that the substrate for LipA, which normally acts on a post-translationally modified protein, can be reduced to carboxybenzyl-octanoyllysine.

Mössbauer spectroscopy of Fe/S proteins.

Iron-sulfur (Fe/S) clusters are structurally and functionally diverse cofactors that are found in all domains of life. (57)Fe Mössbauer spectroscopy is a technique that provides information about the chemical nature of all chemically distinct Fe species contained in a sample, such as Fe oxidation and spin state, nuclearity of a cluster with more than one metal ion, electron spin ground state of the cluster, and delocalization properties in mixed-valent clusters. Moreover, the technique allows for quantitation of all Fe species, when it is used in conjunction with electron paramagnetic resonance (EPR) spectroscopy and analytical methods. (57)Fe-Mössbauer spectroscopy played a pivotal role in unraveling the electronic structures of the "well-established" [2Fe-2S](2+/+), [3Fe-4S](1+/0), and [4Fe-4S](3+/2+/1+/0) clusters and -more-recently- was used to characterize novel Fe/S clustsers, including the [4Fe-3S] cluster of the O2-tolerant hydrogenase from Aquifex aeolicus and the 3Fe-cluster intermediate observed during the reaction of lipoyl synthase, a member of the radical SAM enzyme superfamily.

The Carbon Monoxide Dehydrogenase from Desulfovibrio vulgaris.

Ni-containing Carbon Monoxide Dehydrogenases (CODHs) catalyze the reversible conversion between CO and CO2and are involved in energy conservation and carbon fixation. These homodimeric enzymes house two NiFeS active sites (C-clusters) and three accessory [4Fe-4S] clusters. The Desulfovibrio vulgaris (Dv) genome contains a two-gene CODH operon coding for a CODH (cooS) and a maturation protein (cooC) involved in nickel insertion in the active site. According to the literature, the question of the precise function of CooC as a chaperone folding the C-cluster in a form which accommodates free nickel or as a mere nickel donor is not resolved. Here, we report the biochemical and spectroscopic characterization of two recombinant forms of the CODH, produced in the absence and in the presence of CooC, designated CooS and CooS(C), respectively. CooS contains no nickel and cannot be activated, supporting the idea that the role of CooC is to fold the C-cluster so that it can bind nickel. As expected, CooS(C) is Ni-loaded, reversibly converts CO and CO2, displays the typical Cred1 and Cred2 EPR signatures of the C-cluster and activates in the presence of methyl viologen and CO in an autocatalytic process. However, Ni-loaded CooS(C) reaches maximum activity only upon reductive treatment in the presence of exogenous nickel, a phenomenon that had not been observed before. Surprisingly, the enzyme displays the Cred1 and Cred2 signatures whether it has been activated or not, showing that this activation process of the Ni-loaded Dv CODH is not associated with structural changes at the active site.

Organometallic Complex Formed by an Unconventional Radical S-Adenosylmethionine Enzyme.

Pyrococcus horikoshii Dph2 (PhDph2) is an unusual radical S-adenosylmethionine (SAM) enzyme involved in the first step of diphthamide biosynthesis. It catalyzes the reaction by cleaving SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. To probe the reaction mechanism, we synthesized a SAM analogue (SAMCA), in which the ACP group of SAM is replaced with a 3-carboxyallyl group. SAMCA is cleaved by PhDph2, yielding a paramagnetic (S = 1/2) species, which is assigned to a complex formed between the reaction product, α-sulfinyl-3-butenoic acid, and the [4Fe-4S] cluster. Electron-nuclear double resonance (ENDOR) measurements with (13)C and (2)H isotopically labeled SAMCA support a π-complex between the C═C double bond of α-sulfinyl-3-butenoic acid and the unique iron of the [4Fe-4S] cluster. This is the first example of a radical SAM-related [4Fe-4S](+) cluster forming an organometallic complex with an alkene, shedding additional light on the mechanism of PhDph2 and expanding our current notions for the reactivity of [4Fe-4S] clusters in radical SAM enzymes.

Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster.

Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We determined the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resolution, providing the first structure of a BMC shell protein containing a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a reduction potential of -370 mV vs the standard hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical S-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells.

Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases.

The founding members of the HD-domain protein superfamily are phosphohydrolases, and newly discovered members are generally annotated as such. However, myo-inositol oxygenase (MIOX) exemplifies a second, very different function that has evolved within the common scaffold of this superfamily. A recently discovered HD protein, PhnZ, catalyzes conversion of 2-amino-1-hydroxyethylphosphonate to glycine and phosphate, culminating a bacterial pathway for the utilization of environmentally abundant 2-aminoethylphosphonate. Using Mössbauer and EPR spectroscopies, X-ray crystallography, and activity measurements, we show here that, like MIOX, PhnZ employs a mixed-valent Fe(II)/Fe(III) cofactor for the O2-dependent oxidative cleavage of its substrate. Phylogenetic analysis suggests that many more HD proteins may catalyze yet-unknown oxygenation reactions using this hitherto exceptional Fe(II)/Fe(III) cofactor. The results demonstrate that the catalytic repertoire of the HD superfamily extends well beyond phosphohydrolysis and suggest that the mechanism used by MIOX and PhnZ may be a common strategy for oxidative C-X bond cleavage.

Electronic structure of the unique [4Fe-3S] cluster in O2-tolerant hydrogenases characterized by 57Fe Mossbauer and EPR spectroscopy.

Iron-sulfur clusters are ubiquitous electron transfer cofactors in hydrogenases. Their types and redox properties are important for H(2) catalysis, but, recently, their role in a protection mechanism against oxidative inactivation has also been recognized for a [4Fe-3S] cluster in O(2)-tolerant group 1 [NiFe] hydrogenases. This cluster, which is uniquely coordinated by six cysteines, is situated in the proximity of the catalytic [NiFe] site and exhibits unusual redox versatility. The [4Fe-3S] cluster in hydrogenase (Hase) I from Aquifex aeolicus performs two redox transitions within a very small potential range, forming a superoxidized state above +200 mV vs. standard hydrogen electrode (SHE). Crystallographic data has revealed that this state is stabilized by the coordination of one of the iron atoms to a backbone nitrogen. Thus, the proximal [4Fe-3S] cluster undergoes redox-dependent changes to serve multiple purposes beyond classical electron transfer. In this paper, we present field-dependent (57)Fe-Mössbauer and EPR data for Hase I, which, in conjunction with spectroscopically calibrated density functional theory (DFT) calculations, reveal the distribution of Fe valences and spin-coupling schemes for the iron-sulfur clusters. The data demonstrate that the electronic structure of the [4Fe-3S] core in its three oxidation states closely resembles that of corresponding conventional [4Fe-4S] cubanes, albeit with distinct differences for some individual iron sites. The medial and distal iron-sulfur clusters have similar electronic properties as the corresponding cofactors in standard hydrogenases, although their redox potentials are higher.

Efficient delivery of long-chain fatty aldehydes from the Nostoc punctiforme acyl-acyl carrier protein reductase to its cognate aldehyde-deformylating oxygenase.

A two-step pathway consisting of an acyl-acyl carrier protein (ACP) reductase (AAR) and an aldehyde-deformylating oxygenase (ADO) allows various cyanobacteria to convert long-chain fatty acids into hydrocarbons. AAR catalyzes the two-electron, NADPH-dependent reduction of a fatty acid attached to ACP via a thioester linkage to the corresponding fatty aldehyde, while ADO transforms the fatty aldehyde to a Cn-1 hydrocarbon and C1-derived formate. Considering that heptadec(a/e)ne is the most prevalent hydrocarbon produced by cyanobacterial ADOs, the insolubility of its precursor, octadec(a/e)nal, poses a conundrum with respect to its acquisition by ADO. Herein, we report that AAR from the cyanobacterium Nostoc punctiforme is activated almost 20-fold by potassium and other monovalent cations of similar ionic radius, and that AAR and ADO form a tight isolable complex with a Kd of 3 ± 0.3 µM. In addition, we show that when the aldehyde substrate is supplied to ADO by AAR, efficient in vitro turnover is observed in the absence of solubilizing agents. Similarly to studies by Lin et al. with AAR from Synechococcus elongatus [Lin et al. (2013) FEBS J. 280, 4773-4781], we show that catalysis by AAR proceeds via formation of a covalent intermediate involving a cysteine residue that we have identified as Cys294. Moreover, AAR specifically transfers the pro-R hydride of NADPH to the Cys294-thioester intermediate to afford its aldehyde product. Our results suggest that the interaction between AAR and ADO facilitates either direct transfer of the aldehyde product of AAR to ADO or formation of the aldehyde product in a microenvironment allowing for its efficient uptake by ADO.

ChlR protein of Synechococcus sp. PCC 7002 is a transcription activator that uses an oxygen-sensitive [4Fe-4S] cluster to control genes involved in pigment biosynthesis.

Synechococcus sp. PCC 7002 and many other cyanobacteria have two genes that encode key enzymes involved in chlorophyll a, biliverdin, and heme biosynthesis: acsFI/acsFII, ho1/ho2, and hemF/hemN. Under atmospheric O2 levels, AcsFI synthesizes 3,8-divinyl protochlorophyllide from Mg-protoporphyrin IX monomethyl ester, Ho1 oxidatively cleaves heme to form biliverdin, and HemF oxidizes coproporphyrinogen III to protoporphyrinogen IX. Under microoxic conditions, another set of genes directs the synthesis of alternative enzymes AcsFII, Ho2, and HemN. In Synechococcus sp. PCC 7002, open reading frame SynPCC7002_A1993 encodes a MarR family transcriptional regulator, which is located immediately upstream from the operon comprising acsFII, ho2, hemN, and desF (the latter encodes a putative fatty acid desaturase). Deletion and complementation analyses showed that this gene, denoted chlR, is a transcriptional activator that is essential for transcription of the acsFII-ho2-hemN-desF operon under microoxic conditions. Global transcriptome analyses showed that ChlR controls the expression of only these four genes. Co-expression of chlR with a yfp reporter gene under the control of the acsFII promoter from Synechocystis sp. PCC 6803 in Escherichia coli demonstrated that no other cyanobacterium-specific components are required for proper functioning of this regulatory circuit. A combination of analytical methods and Mössbauer and EPR spectroscopies showed that reconstituted, recombinant ChlR forms homodimers that harbor one oxygen-sensitive [4Fe-4S] cluster. We conclude that ChlR is a transcriptional activator that uses a [4Fe-4S] cluster to sense O2 levels and thereby control the expression of the acsFII-ho2-hemN-desF operon.

Rapid Reduction of the Diferric-Peroxyhemiacetal Intermediate in Aldehyde-Deformylating Oxygenase by a Cyanobacterial Ferredoxin: Evidence for a Free-Radical Mechanism.

Aldehyde-deformylating oxygenase (ADO) is a ferritin-like nonheme-diiron enzyme that catalyzes the last step in a pathway through which fatty acids are converted into hydrocarbons in cyanobacteria. ADO catalyzes conversion of a fatty aldehyde to the corresponding alk(a/e)ne and formate, consuming four electrons and one molecule of O2 per turnover and incorporating one atom from O2 into the formate coproduct. The source of the reducing equivalents in vivo has not been definitively established, but a cyanobacterial [2Fe-2S] ferredoxin (PetF), reduced by ferredoxin-NADP(+) reductase (FNR) using NADPH, has been implicated. We show that both the diferric form of Nostoc punctiforme ADO and its (putative) diferric-peroxyhemiacetal intermediate are reduced much more rapidly by Synechocystis sp. PCC6803 PetF than by the previously employed chemical reductant, 1-methoxy-5-methylphenazinium methyl sulfate. The yield of formate and alkane per reduced PetF approaches its theoretical upper limit when reduction of the intermediate is carried out in the presence of FNR. Reduction of the intermediate by either system leads to accumulation of a substrate-derived peroxyl radical as a result of off-pathway trapping of the C2-alkyl radical intermediate by excess O2, which consequently diminishes the yield of the hydrocarbon product. A sulfinyl radical located on residue Cys71 also accumulates with short-chain aldehydes. The detection of these radicals under turnover conditions provides the most direct evidence to date for a free-radical mechanism. Additionally, our results expose an inefficiency of the enzyme in processing its radical intermediate, presenting a target for optimization of bioprocesses exploiting this hydrocarbon-production pathway.