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.

Functional characterization of an orphan cupin protein from Burkholderia xenovorans reveals a mononuclear nonheme Fe2+-dependent oxygenase that cleaves beta-diketones.

Cupins constitute a large and widespread superfamily of beta-barrel proteins in which a mononuclear metal site is both a conserved feature of the structure and a source of functional diversity. Metal-binding residues are contributed from two core motifs that provide the signature for the superfamily. On the basis of conservation of this two-motif structure, we have identified an ORF in the genome of Burkholderia xenovorans that encodes a novel cupin protein (Bxe_A2876) of unknown function. Recombinant Bxe_A2876, as isolated from Escherichia coli cell extract, was a homotetramer in solution, and showed mixed fractional occupancy of its 16.1 kDa subunit with metal ligands (0.06 copper; 0.11 iron; 0.17 zinc). Our quest for possible catalytic functions of Bxe_A2876 focused on Cu2+ and Fe2+ oxygenase activities known from related cupin enzymes. Fe2+ elicited enzymatic catalysis of O2-dependent conversion of various beta-diketone substrates via a nucleophilic mechanism of carbon-carbon bond cleavage. Data from X-ray absorption spectroscopy (XAS) support a five-coordinate or six-coordinate Fe2+ center where the metal is bound by three imidazole nitrogen atoms at 1.98 A. Results of structure modeling studies suggest that His60, His62 and His102 are the coordinating residues. In the 'best-fit' model, one or two oxygens from water and a carboxylate oxygen (presumably from Glu96) are further ligands of Fe2+ at estimated distances of 2.04 A and 2.08 A, respectively. The three-histidine Fe2+ site of Bxe_A2876 is compared to the mononuclear nonheme Fe2+ centers of the structurally related cysteine dioxygenase and acireductone dioxygenase, which also use a facial triad of histidines for binding of their metal cofactor but promote entirely different substrate transformations.

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.

The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography.

CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2'-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme-NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274-->Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R-N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 degrees C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 A resolution.