Stefan Leitgeb

Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+

Aldo‐keto reductases of family 2 employ single site replacement Lys → Arg to switch their cosubstrate preference from NADPH to NADH. X‐ray crystal structures of Lys‐274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+ were determined at a resolution of 2.4 and 2.3 Å, respectively. Due to steric conflicts in the NADP+‐bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water‐mediated interactions between Glu‐227, Lys‐274 and the cofactor 2′‐phosphate and 3′‐hydroxy groups. Because anchoring contacts of its Glu‐227 are lost, the coenzyme‐enfolding loop that becomes ordered upon binding of NAD(P)+ in the wild‐type remains partly disordered in the NADP+‐bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild‐type.

Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis

Little is known about how substrates bind to CtXR (Candida tenuis xylose reductase; AKR2B5) and other members of the AKR (aldo-keto reductase) protein superfamily. Modelling of xylose into the active site of CtXR suggested that Trp23, Asp50 and Asn309 are the main components of pentose-specific substrate-binding recognition. Kinetic consequences of site-directed substitutions of these residues are reported. The mutants W23F and W23Y catalysed NADH-dependent reduction of xylose with only 4 and 1% of the wild-type efficiency (kcat/K(m)) respectively, but improved the wild-type selectivity for utilization of ketones, relative to xylose, by factors of 156 and 471 respectively. Comparison of multiple sequence alignment with reported specificities of AKR members emphasizes a conserved role of Trp23 in determining aldehyde-versus-ketone substrate selectivity. D50A showed 31 and 18% of the wild-type catalytic-centre activities for xylose reduction and xylitol oxidation respectively, consistent with a decrease in the rates of the chemical steps caused by the mutation, but no change in the apparent substrate binding constants and the pattern of substrate specificities. The 30-fold preference of the wild-type for D-galactose compared with 2-deoxy-D-galactose was lost completely in N309A and N309D mutants. Comparison of the 2.4 A (1 A=0.1 nm) X-ray crystal structure of mutant N309D bound to NAD+ with the previous structure of the wild-type holoenzyme reveals no major structural perturbations. The results suggest that replacement of Asn309 with alanine or aspartic acid disrupts the function of the original side chain in donating a hydrogen atom for bonding with the substrate C-2(R) hydroxy group, thus causing a loss of transition-state stabilization energy of 8-9 kJ/mol.

Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii

beta-diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1protomer adopts a single-domain beta-barrel fold characteristic of the cupin superfamily of proteins and features a mononuclear non-haem Fe2+ centre where a triad of histidine residues, His-62, His-64 and His-104, co-ordinate the catalytic metal. To provide structure-function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp binding site for non-haem Fe2+,we replaced each histidine residue individually with glutamate and asparagine and compared binding of Fe2+ and four non-native catalytically inactive metals with purified apo-forms of wild-type and mutant enzymes. Results from anaerobic equilibrium microdialysis (Fe2+) and fluorescence titration (Fe2+, Cu2+, Ni2+, Mn2+ and Zn2+) experiments revealed the presence of two broadly specific metal-binding sites in native Dke1 that bind Fe2+ with a dissociation constant (Kd) of 5 microM (site I) and approximately 0.3 mM (site II). Each mutation, except for the substitution of asparagine for His-104, disrupted binding of Fe2+, but not that of the other bivalent metal ions, at site I,while leaving metal binding at site II largely unaffected. Dke1 mutants harbouring glutamate substitutions were completely inactive and not functionally complemented by external Fe2+.The Fe2+ catalytic centre activity (kcat) of mutants with asparagine substitution of His-62 and His-104 was decreased 140- and 220-fold respectively, compared with the kcat value of 8.5 s(-1) for the wild-type enzyme in the reaction with pentane-2,4-dione.The H64N mutant was not catalytically competent, except in the presence of external Fe2+ (1 mM) which elicited about 1/1000 of wild-type activity. Therefore co-ordination of Fe2+ by Dke1 requires an uncharged metallocentre, and three histidine ligands are needed for the assembly of a fully functional catalytic site. Oxidative inactivation of Dke1 was shown to involve conversion of enzyme-bound Fe2+ into Fe3+, which is then released from the metal centre.

Structural and functional comparison of 2-His- 1-carboxylate and 3-His metallocentres in non-haem iron(II)-dependent enzymes

The canonical structural motif for co-ordination of non-haem ferrous iron in metal-dependent oxygenases is a facial triad of two histidine residues and one aspartate or glutamate residue. This so-called 2-His-1-carboxylate metallocentre is often accommodated in a double-stranded beta-helix fold with the iron-co-ordinating residues located in the rigid core structure of the protein. At the sequence level, the metal ligands are arranged in a HXD/E...H motif (where the distance between the conserved histidine residues is variable). Interestingly, cysteine dioxygenase, among a growing number of other iron(II) oxygenases, has the carboxylate residue replaced by another histidine. In the present review, we compare the properties of 3-His and 2-His-1-carboxylate sites based on current evidence from high-resolution crystal structures, spectroscopic characterization of the metal centres and results from mutagenesis studies. Although the overall conformation of the two metal sites is quite similar, the carboxylate residue seems to accommodate a slightly closer co-ordination distance than the counterpart histidine. The ability of the 2-His-1-carboxylate site to fit a site-directed substitution by an alternatively co-ordinating or non-co-ordinating residue with retention of metal-binding capacity and catalytic function varies among different enzymes. However, replacement by histidine disrupted the activity in the three iron(II) oxygenases examined so far.

Characterization of a Laboratory-Scale Container for Freezing Protein Solutions with Detailed Evaluation of a Freezing Process Simulation

A 300-mL stainless steel freeze container was constructed to enable QbD (Quality by Design)-compliant investigations and the optimization of freezing and thawing (F/T) processes of protein pharmaceuticals at moderate volumes. A characterization of the freezing performance was conducted with respect to freezing kinetics, temperature profiling, cryoconcentration, and stability of the frozen protein. Computational fluid dynamic (CFD) simulations of temperature and phase transition were established to facilitate process scaling and process analytics as well as customization of future freeze containers. Protein cryoconcentration was determined from ice-core samples using bovine serum albumin. Activity, aggregation, and structural perturbation were studied in frozen rabbit muscle l-lactic dehydrogenase (LDH) solution. CFD simulations provided good qualitative and quantitative agreement with highly resolved experimental measurements of temperature and phase transition, allowing also the estimation of spatial cryoconcentration patterns. LDH exhibited stability against freezing in the laboratory-scale system, suggesting a protective effect of cryoconcentration at certain conditions. The combination of the laboratory-scale freeze container with accurate CFD modeling will allow deeper investigations of F/T processes at advanced scale and thus represents an important step towards a better process understanding.

In Situ Protein Secondary Structure Determination in Ice: Raman Spectroscopy-Based Process Analytical Tool for Frozen Storage of Biopharmaceuticals

A Raman spectroscopy-based method for in situ monitoring of secondary structural composition of proteins during frozen and thawed storage was developed. A set of reference proteins with different α-helix and β-sheet compositions was used for calibration and validation in a chemometric approach. Reference secondary structures were quantified with circular dichroism spectroscopy in the liquid state. Partial least squares regression models were established that enable estimation of secondary structure content from Raman spectra. Quantitative secondary structure determination in ice was accomplished for the first time and correlation with existing (qualitative) protein structural data from the frozen state was achieved. The method can be used in the presence of common stabilizing agents and is applicable in an industrial freezer setup. Raman spectroscopy represents a powerful, noninvasive, and flexibly applicable tool for protein stability monitoring during frozen storage.

Enzyme Catalytic Promiscuity: The Nonheme Fe2+ Center of β-Diketone-Cleaving Dioxygenase Dke1 Promotes Hydrolysis of Activated Esters

Natural enzymes are often described as highly efficient and finely tuned catalysts for specific chemical transformations of biological relevance. Some of these enzymes, however, are promiscuous in a sense that they catalyze secondary reactions, chemically distinct from the reaction promoted in the conversion of the physiological substrate. Catalytic promiscuity has drawn mechanistic attention as it necessitates that different reaction coordinates be accommodated by a single enzyme active site. Furthermore, it presents an often untapped source of potentially useful enzymatic functionality. Activesite redesign, by using minimal structural modifications of the original catalytic center, has been exploited successfully with some enzymes to revive latent alternative reactivity present in the native form. b-Diketone-cleaving enzyme Dke1 is a nonheme Fe -dependent dioxygenase from Acinetobacter johnsonii that converts 2,4-pentanedione and O2 into methyglyoxal and acetate (Scheme 1). The metal cofactor of Dke1 is coordinated by the triad His62, His64, and His104 (Figure 1) and is absolutely required for oxygenase activity. Other metal ions, like Zn , Ni or Cu , that compete with Fe for binding to the 3-His site are completely inactive in the enzymatic reaction with O2. [5] We show here that the catalytic center of Dke1 in its native Fe + form catalyses the hydrolysis of 4-nitrophenylesters of short-chain alkanoic acids, which is an accidental type of catalytic promiscuity. A non-native Zn form of the enzyme was likewise active as esterase—about ten-times more so than the Fe + enzyme. Substitution of His104 by Glu, which destroys the oxygenase activity, was fully compatible with the function of a Fe /Zn + esterase. The type of catalytic promiscuity (oxygenase!esterase) seen for Dke1 is novel, and with the exception of methionine aminopeptidase, for which a role for Fe + as cofactor has been proposed, hydrolytic activity of a catalytic Fe + site appears to have limited precedence in reported enzymes (see later). When a purified preparation of Dke1 (5 mm Fe + sites) was incubated in the presence of 4-nitrophenylpropionate (pNPP; 3 mm in 20 mm Tris-Cl buffer, pH 7.5; 25 8C) formation of 4-nitrophenol occurred at a spectrophotometric rate (405 nm) that was 40-fold faster than the rate of the uncatalyzed hydrolysis of the ester substrate under otherwise identical conditions lacking the enzyme. Apo-Dke1, from which Fe + had been re-

Engineering of Aerococcus viridans L-lactate oxidase for site-specific PEGylation: characterization and selective bioorthogonal modification of a S218C mutant.

A defined bioconjugate of Aerococcus viridans L-lactate oxidase and poly(ethylene glycol) 5000 was prepared and characterized in its structural and functional properties in comparison to the unmodified enzyme. Because the L-lactate oxidase in the native form does not contain cysteines, we introduced a new site for chemical modification via thiol chemistry by substituting the presumably surface-exposed serine-218, a nonconserved residue in the amino acid sequence, with cysteine. The resulting S218C mutant was isolated from Escherichia coli and shown in kinetic assays to be similarly (i.e., about half as) active as the native enzyme, thus validating the structure-guided design of the mutation. Using maleimide-activated methoxypoly(ethylene glycol) 5000 in about 10-fold molar excess over protein, the S218C mutant was converted in high yield (94%) into PEGylated derivative, while the native enzyme was totally unreactive under equivalent conditions. PEGylation caused only a relatively small decrease (30%) in the specific activity of the S218C mutant, and it did not change the protein stability. PEGylation went along with enhancement of the apparent size of the homotetrameric L-lactate oxidase in gel permeation chromatography, from 170 kDa to 250 kDa. The protein hydrodynamic diameter determined by dynamic light scattering increased from 11.9 nm in unmodified S218C mutant to 16.4 nm in the PEGylated form. Site-selective PEGylation of the mutated L-lactate oxidase, using orthogonal maleimide-thiol coupling, could therefore facilitate incorporation of the enzyme into biosensors currently employed for determination of blood L-lactate levels, and it could also support different applications of the enzyme in applied biocatalysis.

Non-native aggregation of recombinant human granulocyte-colony stimulating factor under simulated process stress conditions

Effective inhibition of protein aggregation is a major goal in biopharmaceutical production processes optimized for product quality. To examine the characteristics of process-stress-dependent aggregation of human granulocyte colony-stimulating factor (G-CSF), we applied controlled stirring and bubble aeration to a recombinant non-glycosylated preparation of the protein produced in Escherichia coli. We characterized the resulting denaturation in a time-resolved manner using probes for G-CSF conformation and size in both solution and the precipitate. G-CSF was precipitated rapidly from solutions that were aerated or stirred; only small amounts of soluble aggregates were found. Exposed hydrophobic surfaces were a characteristic of both soluble and insoluble G-CSF aggregates. Using confocal laser scanning microscopy, the aggregates presented mainly a circular shape. Their size varied according to incubation time and stress applied. The native intramolecular disulfide bonds in the insoluble G-CSF aggregates were largely disrupted as shown by mass spectrometry. New disulfide bonds formed during aggregation. All involved Cys(18) , which is the only free cysteine in G-CSF; one of them had an intermolecular Cys(18(A)) -Cys(18(B)) crosslink. Stabilization strategies can involve external addition of thiols and extensive reduction of surface exposition during processing.

The Ala95‐to‐Gly substitution in Aerococcus viridans l‐lactate oxidase revisited – structural consequences at the catalytic site and effect on reactivity with O2 and other electron acceptors

Aerococcus viridansl‐lactate oxidase (avLOX) is a biotechnologically important flavoenzyme that catalyzes the conversion of l‐lactate and O2 into pyruvate and H2O2. The enzymatic reaction underlies different biosensor applications of avLOX for blood l‐lactate determination. The ability of avLOX to replace O2 with other electron acceptors such as 2,6‐dichlorophenol‐indophenol (DCIP) allows the possiblity of analytical and practical applications. The A95G variant of avLOX was previously shown to exhibit lowered reactivity with O2 compared to wild‐type enzyme and therefore was employed in a detailed investigation with respect to the specificity for different electron acceptor substrates. From stopped‐flow experiments performed at 20 °C (pH 6.5), we determined that the A95G variant (fully reduced by l‐lactate) was approximately three‐fold more reactive towards DCIP (1.0 ± 0.1 × 106 M−1·s−1) than O2, whereas avLOX wild‐type under the same conditions was 14‐fold more reactive towards O2 (1.8 ± 0.1 × 106 m−1·s−1) than DCIP. Substituted 1,4‐benzoquinones were up to five‐fold better electron acceptors for reaction with l‐lactate‐reduced A95G variant than wild‐type. A 1.65‐Å crystal structure of oxidized A95G variant bound with pyruvate was determined and revealed that the steric volume created by removal of the methyl side chain of Ala95 and a slight additional shift in the main chain at position Gly95 together enable the accomodation of a new active‐site water molecule within hydrogen‐bond distance to the N5 of the FMN cofactor. The increased steric volume available in the active site allows the A95G variant to exhibit a similar trend with the related glycolate oxidase in electron acceptor substrate specificities, despite the latter containing an alanine at the analogous position.