Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase

We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser⁷¹⁹, NarG-His¹¹⁶³, and NarG-His¹¹⁸⁴); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His¹⁰⁹² and NarG-His¹⁰⁹⁸). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔE_m values of −88 and −36 mV, respectively). Ala variants of His¹⁰⁹² and His¹⁰⁹⁸ also elicit large ΔE_m values of −143 and −101 mV, respectively. An Arg variant of His¹⁰⁹² elicits a small ΔE_m of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum E_m value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis.     

Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α₂β₂ heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αβ subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5′-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.     

Biooxidation of monoterpenes with bacterial monooxygenases

Biotechnological monoterpene oxidation has a considerable economic potential as an alternative route to natural monoterpenoid compounds with desirable organoleptic and pharmaceutical properties. Bacterial cytochrome P450 monooxygenases (CYPs) constitute ideal catalysts for monoterpene oxidation due to their pronounced selectivities, comparably high activities and ease of recombinant expression. Research activities of the recent decades resulted in the identification and characterization of many monoterpene oxidizing bacterial CYPs, often together with their electron transfer partners. To the authors’ knowledge, no industrial process of bacterial monoterpene oxidation has been established up to date. However, the last decade has seen movement away from small scale test tube sized reactions to research activities focusing on more sophisticated processes in larger volumes and in bioreactors. These research activities successfully combined improvements on all levels of a biotransformation process. Activity, selectivity and stability of bacterial CYPs were enhanced by rational protein design, substrate and product toxicity was counteracted with the development of feeding strategies and in situ product removal techniques. The disadvantage of costly cofactors was bypassed by the application of cofactor regeneration systems and by electrochemical substitution of cofactors.     

Optimization of redox reactions employing whole cell biocatalysis

The ability of Saccharomyces cerevisiae to catalyse the reduction reaction of carboxylic acids into alcohols is described. Earlier reports have led to the characterization of the reduction of carbonyl groups into alcohols mediated by the enzyme alcohol dehydrogenase. We investigated the ability of this organism to catalyse the said conversion using the carboxylic acids, acetic acid and butyric acid. In the absence of any previous characterization, whole cell catalysis proved effective. The uptake of these acids from the medium was estimated using a plate assay method involving litmus-agar. The plate assay was found to be a convenient and extremely adaptable method for quantitation of acids in organic as well as aqueous medium. The comparison of existing paradigms in pure protein catalysis with whole cells catalysis proved anomalous. We report that it is solvent toxicity rather than hydrophobic index that correlates with the activity observed in non-aqueous conditions for whole cell biocatalysis. Reduction of acetic acid as well as butyric acid occurred, with efficiency of reaction with butyric acid being marginally higher. The reduction therefore occurs for both the short chain carboxylic acids used in this study. We therefore illustrate the reduction route of acids into alcohols and propose a model two-step pathway for the reaction. Process optimization may be further attempted to enhance the presently moderate reaction efficiencies. Steps made in the direction by studying the pH dependency and use of sacrificial substrate have yielded encouraging results.     

Organic-inorganic hybrid materials constructed from copper-organoimine subunits and molybdoarsonate clusters

The hydrothermal reactions of MoO₃, As₂O₅, Cu(CH₃CO₂)₂ · H₂O and an appropriate organonitrogen ligand in the presence of HF as mineralizer yield a series of bimetallic oxides of the Cu/Mo/O/As system. The compounds [{Cu₂(4,7-phen)(4,7-phenH)₂}Mo₁₂AsO₄₀] · 2.66H₂O (1 · 2.66H₂O) and [{Cu₃(qtpyr)₂}Mo₁₂AsO₄₀] · 0.4H₂O (2 · 0.4H₂O) (qtpyr = 2,4′:5′, 3″:4″,2?-quaterpyridine) are two-dimensional phases constructed from Keggin clusters linked through binuclear {Cu₂(4,7-phen)(4,7-Hphen)₂}²⁺ units in metal organic networks in 2. In contrast, the structure of [{Cu₂(2,4′-Hbpy)₄}Mo₁₈As₂O₆₂] · 2H₂O (3 · 2H₂O) is one-dimensional, consisting of Dawson clusters linked through binuclear {Cu₂(Hbpy)₄}⁶⁺ subunits. In the case of the compounds [{Cu(5,5′-dimethyl-2,2′-bpy)}₂Mo₂O₄F₂(AsO₄)₂] (4) and [{Cu(phen)}₂Mo₂O₄F₂(AsO₄)₂] (5), the fluoride mineralizer has been incorporated into the structure to give one-dimensional phases constructed from oxyfluoride {Mo₂O₄F₂(AsO₄)₂}²⁻clusters bridged through {Cu(organonitrogen)}²⁺ units.     

Substrate specificity and evolutionary implications of a NifDK enzyme carrying NifB-co at its active site

The in vitro reconstitution of molybdenum nitrogenase was manipulated to generate a chimeric enzyme in which the active site iron-molybdenum cofactor (FeMo-co) is replaced by NifB-co. The NifDK/NifB-co enzyme was unable to reduce N₂ to NH₃, while exhibiting residual C₂H₄ and considerable H₂ production activities. Production of H₂ by NifDK/NifB-co was stimulated by N₂ and was dependent on NifH and ATP hydrolysis. Thus, NifDK/NifB-co is a useful tool to gain insights into the catalytic mechanism of nitrogenase. Furthermore, phylogenetic analysis of D and K homologs indicates that several early emerging lineages, which contain NifB, NifH and NifDK encoding genes but which lack other genes required for processing NifB-co into FeMo-co, might encode an enzyme with similar catalytic properties to NifDK/NifB-co.     

The crystal structure of the apoenzyme of the iron-sulphur cluster-free hydrogenase

The iron-sulphur cluster-free hydrogenase (Hmd, EC 1.12.98.2) from methanogenic archaea is a novel type of hydrogenase that tightly binds an iron-containing cofactor. The iron is coordinated by two CO molecules, one sulphur and a pyridone derivative, which is linked via a phosphodiester bond to a guanosine base. We report here on the crystal structure of the Hmd apoenzyme from Methanocaldococcus jannaschii at 1.75 A and from Methanopyrus kandleri at 2.4 A resolution. Homodimeric Hmd reveals a unique architecture composed of one central and two identical peripheral globular units. The central unit is composed of the intertwined C-terminal segments of both subunits, forming a novel intersubunit fold. The two peripheral units consist of the N-terminal domain of each subunit. The Rossmann fold-like structure of the N-terminal domain contains a mononucleotide-binding site, which could harbour the GMP moiety of the cofactor. Another binding site for the iron-containing cofactor is most probably Cys176, which is located at the bottom of a deep intersubunit cleft and which has been shown to be essential for enzyme activity. Adjacent to the iron of the cofactor modelled as a ligand to Cys176, an extended U-shaped extra electron density, interpreted as a polyethyleneglycol fragment, suggests a binding site for the substrate methenyltetrahydromethanopterin.     

Evidence for a synergistic salt-protein interaction -- complex patterns of activation vs. inhibition of nitrogenase by salt

The molybdenum nitrogenase enzyme system, comprised of the MoFe protein and the Fe protein, catalyzes the reduction of atmospheric N(2) to NH(3). Interactions between these two proteins and between Fe protein and nucleotides (MgADP and MgATP) are crucial to catalysis. It is well established that salts are inhibitors of nitrogenase catalysis that target these interactions. However, the implications of salt effects are often overlooked. We have reexamined salt effects in light of a comprehensive framework for nitrogenase interactions to offer an in-depth analysis of the sources of salt inhibition and underlying apparent cooperativity. More importantly, we have identified patterns of salt activation of nitrogenase that correspond to at least two mechanisms. One of these mechanisms is that charge screening of MoFe protein-Fe protein interactions in the nitrogenase complex accelerates the rate of nitrogenase complex dissociation, which is the rate-limiting step of catalysis. This kind of salt activation operates under conditions of high catalytic activity and low salt concentrations that may resemble those found in vivo. While simple kinetic arguments are strong evidence for this kind of salt activation, further confirmation was sought by demonstrating that tight complexes that have previously displayed little or no activity due to the inability of Fe protein to dissociate from the complex are activated by the presence of salt. This occurs for the combination Azotobacter vinelandii MoFe protein with: (a) the L127Delta Fe protein; and (b) Clostridium pasteurianum Fe protein. The curvature of activation vs. salt implies a synergistic salt-protein interaction.     

Arabidopsis AtIscA-I is affected by deficiency of Fe-S cluster biosynthetic scaffold AtCnfU-V

IscA has been proposed to be a scaffold protein of the iron-sulfur cluster biosynthetic machinery. We have identified the IscA homolog to be localized to plastids, termed AtIscA-I, in Arabidopsis thaliana. The AtIscA-I protein was apparently constitutively expressed in all tissues analyzed in Arabidopsis. The AtIscA-I protein exists in the stroma as a soluble protein which tends to form a homo-dimer and can host a [2Fe-2S]-like cluster. Complete loss of the protein from plastids did not cause any significant defect either in normal plant growth or in biogenesis of major iron-sulfur proteins, indicating this protein is not essential or redundant for these functions. In contrast, loss of one of the three plastid-localized CnfU scaffold proteins, AtCnfU-V, caused significant reduction in the level of AtIscA-I. These data suggest that efficient biogenesis of AtIscA-I scaffold requires function of another essential scaffold protein CnfU.     

Overcoming the thermodynamic limitation in asymmetric hydrogen transfer reactions catalyzed by whole cells

Whole lyophilized cells of an Escherichia coli overexpressing the alcohol dehydrogenase (ADH-'A') from Rhodococcus ruber DSM 44541 were used for the asymmetric reduction of ketones to secondary alcohols. The recycling of the required nicotinamide cofactor (NADH) was achieved in a coupled-substrate process. In the course of the reaction the ketone is reduced to the alcohol and the hydrogen donor 2-propanol is oxidized to acetone by one enzyme. This leads to a thermodynamic equilibrium between all four components determining the maximum achievable conversion. To overcome this limitation an in situ product removal technique (ISPR) for the application with whole cells was developed. In this method the most volatile compound is separated from the reaction vessel by an air flow resulting in a shift of the equilibrium towards the desired secondary alcohol. The so-called stripping process represents a simple and efficient method to overcome the thermodynamic limitation in biocatalytic reactions. Employing this method, the conversion of selected biotransformations was increased up to completeness.