Metalloproteins containing iron and tungsten: Biocatalytic links between organic and inorganic redox chemistry

Applied Science

Microbial Metalloproteomics

Metalloproteomics is a rapidly developing field of science that involves the comprehensive analysis of all metal-containing or metal-binding proteins in a biological sample. The purpose of this review is to offer a comprehensive overview of the research involving approaches that can be categorized as inductively coupled plasma (ICP)-MS based methods, X-ray absorption/fluorescence, radionuclide based methods and bioinformatics. Important discoveries in microbial proteomics will be reviewed, as well as the outlook to new emerging approaches and research areas.BT/BiotechnologyApplied Science

Steady-state kinetics of the tungsten containing aldehyde: Ferredoxin oxidoreductases from the hyperthermophilic archaeon Pyrococcus furiosus

The tungsten containing Aldehyde:ferredoxin oxidoreductases (AOR) offer interesting opportunities for biocatalytic approaches towards aldehyde oxidation and carboxylic acid reduction. The hyperthermophilic archaeon Pyrococcus furiosus encodes five different AOR family members: glyceraldehyde-3-phosphate oxidoreductase (GAPOR), aldehyde oxidoreductase (AOR), and formaldehyde oxidoreductase (FOR), WOR4 and WOR5. GAPOR functions as a glycolytic enzyme and is highly specific for the substrate glyceraldehyde-3-phosphate (GAP). AOR, FOR and WOR5 have a broad substrate spectrum, and for WOR4 no substrate has been identified to date. As ambiguous kinetic parameters have been reported for different AOR family enzymes the steady state kinetics under different physiologically relevant conditions was explored. The GAPOR substrate GAP was found to degrade at 60 °C by non-enzymatic elimination of the phosphate group to methylglyoxal with a half-life t1/2 = 6.5 min. Methylglyoxal is not a substrate or inhibitor of GAPOR. D-GAP was identified as the only substrate oxidized by GAPOR, and the kinetics of the enzyme was unaffected by the presence of L-GAP, which makes GAPOR the first enantioselective enzyme of the AOR family. The steady-state kinetics of GAPOR showed partial substrate inhibition, which assumes the GAP inhibited form of the enzyme retains some activity. This inhibition was found to be alleviated completely by a 1 M NaCl resulting in increased enzyme activity at high substrate concentrations. GAPOR activity was strongly pH dependent, with the optimum at pH 9. At pH 9, the substrate is a divalent anion and, therefore, positively charged amino acid residues are likely to be involved in the binding of the substrate. FOR exhibited a significant primary kinetic isotope effect of the apparent Vmax for the deuterated substrate, formaldehyde-d2, which shows that the rate-determining step involves a C[sbnd]H bond break from the aldehyde. The implications of these results for the reaction mechanism of tungsten-containing AORs, are discussed.Accepted Author ManuscriptBT/Biocatalysi

Isothermal Titration Calorimetry in Biocatalysis

Isothermal titration calorimetry (ITC) is a popular chemical analysis technique that can be used to measure macromolecular interactions and chemical and physical processes. ITC involves the measurement of heat flow to and from a measurement cell after each injection during a titration experiment. ITC has been useful to measure the thermodynamics of macromolecular interactions such as protein-ligand or protein-protein binding affinity and also chemical processes such as enzyme catalyzed reactions. The use of ITC in biocatalysis has a number of advantages as ITC enables the measurement of enzyme kinetic parameters in a direct manner and, in principle, can be used for most enzymes and substrates. ITC approaches have been developed to measure reversible and irreversible enzyme inhibition, the effects of molecular crowding on enzyme activity, the activity of immobilized enzymes and the conversion of complex polymeric substrates. A disadvantage is that in order to obtain accurate kinetic parameters special care has to be taken in proper experimental design and data interpretation, which unfortunately is not always the case in reported studies. Furthermore, special caution is necessary when ITC experiments are performed that include solvents, reducing agents and may have side reactions. An important bottleneck in the use of calorimetry to measure enzyme activity is the relatively low throughput, which may be solved in the future by sensitive chip based microfluidic enzyme calorimetric devices.BT/Biocatalysi

A thermostable hybrid cluster protein from Pyrococcus furiosus: Effects of the loss of a three helix bundle subdomain

Pyrococcus furiosus hybrid cluster protein (HCP) was expressed in Escherichia coli, purified, and characterized. This is the first archaeal and thermostable HCP to be isolated. Compared with the protein sequences of previously characterized HCPs from mesophiles, the protein sequence of P. furiosus HCP exhibits a deletion of approximately 13 kDa as a single amino acid stretch just after the N-terminal cysteine motif, characteristic for class-III HCPs from (hyper)thermophilic archaea and bacteria. The protein was expressed as a thermostable, soluble homodimeric protein. Hydroxylamine reductase activity of P. furiosus HCP showed a K m value of 0.40 mM and a k cat value of 3.8 s?1 at 70 °C and pH 9.0. Electron paramagnetic resonance spectroscopy showed evidence for the presence of a spin-admixed, S = 3/2 [4Fe–4S]+ cubane cluster and of the hybrid cluster. The cubane cluster of P. furiosus HCP is presumably coordinated by a CXXC–X7–C–X5–C motif close to the N-terminus, which is similar to the CXXC–X8–C–X5–C motif of the Desulfovibrio desulfuricans and Desulfovibrio vulgaris HCPs. Amino acid sequence alignment and homology modeling of P. furiosus HCP reveal that the deletion results in a loss of one of the two three-helix bundles of domain 1. Clearly the loss of one of the three-helix bundles of domain 1 does not diminish the hydroxylamine reduction activity and the incorporation of the iron–sulfur clusters.BiotechnologyApplied Science

Novel oleate hydratases and potential biotechnological applications

Oleate hydratase catalyses the addition of water to the CC double bond of oleic acid to produce (R)-10-hydroxystearic acid. The enzyme requires an FAD cofactor that functions to optimise the active site structure. A wide range of unsaturated fatty acids can be hydrated at the C10 and in some cases the C13 position. The substrate scope can be expanded using ‘decoy’ small carboxylic acids to convert small chain alkenes to secondary alcohols, albeit at low conversion rates. Systematic protein engineering and directed evolution to widen the substrate scope and increase the conversion rate is possible, supported by new high throughput screening assays that have been developed. Multi-enzyme cascades allow the formation of a wide range of products including keto-fatty acids, secondary alcohols, secondary amines and α,ω-dicarboxylic acids. Key points: • Phylogenetically distinct oleate hydratases may exhibit mechanistic differences. • Protein engineering to improve productivity and substrate scope is possible. • Multi-enzymatic cascades greatly widen the product portfolio.BT/Biocatalysi

Rhodococcus as A Versatile Biocatalyst in Organic Synthesis

The application of purified enzymes as well as whole-cell biocatalysts in synthetic organic chemistry is becoming more and more popular, and both academia and industry are keen on finding and developing novel enzymes capable of performing otherwise impossible or challenging reactions. The diverse genus Rhodococcus offers a multitude of promising enzymes, which therefore makes it one of the key bacterial hosts in many areas of research. This review focused on the broad utilization potential of the genus Rhodococcus in organic chemistry, thereby particularly highlighting the specific enzyme classes exploited and the reactions they catalyze. Additionally, close attention was paid to the substrate scope that each enzyme class covers. Overall, a comprehensive overview of the applicability of the genus Rhodococcus is provided, which puts this versatile microorganism in the spotlight of further research.BT/Biocatalysi

The workings of ferritin: a crossroad of opinions

Biochemistry of the essential element iron is complicated by radical chemistry associated with Fe(II) ions and by the extremely low solubility of the Fe(III) ion in near-neutral water. To mitigate these problems cells from all domains of life synthesize the protein ferritin to take up and oxidize Fe(II) and to form a soluble storage of Fe(III) from which iron can be made available for physiology. A long history of studies on ferritin has not yet resulted in a generally accepted mechanism of action of this enzyme. In fact strong disagreement exists between extant ideas on several key steps in the workings of ferritin. The scope of this review is to explain the experimental background of these controversies and to indicate directions towards their possible resolution.BT/Biocatalysi

Self-assembly is prerequisite for catalysis of Fe(II) oxidation by catalytically active subunits of ferritin

BT/BiotechnologyApplied Science

Immobilisation and flow chemistry: tools for implementing biocatalysis

The merger of enzyme immobilisation and flow chemistry has attracted the attention of the scientific community during recent years. Immobilisation enhances enzyme stability and enables recycling, flow chemistry allows process intensification. Their combination is desirable for the development of more efficient and environmentally friendly biocatalytic processes. In this feature article, we aim to point out important metrics for successful enzyme immobilisation and for reporting flow biocatalytic processes. Relevant examples of immobilised enzymes used in flow systems in organic, biphasic and aqueous systems are discussed. Finally, we describe recent developments to address the cofactor recycling hurdle.BT/Biocatalysi