The ability to engineer proteins by directed evolution requires functional expression of the target polypeptide in a recombinant host suitable for construction and screening libraries of enzyme variants. Bacteria and yeast are preferred, but eukaryotic proteins often fail to express in active form in these cells. We have attempted to resolve this problem by identifying mutations in the target gene that facilitate its functional expression in a given recombinant host. Here we examined expression of HRP in Saccharomyces cerevisiae. Through three rounds of directed evolution by random point mutagenesis and screening, we obtained a 40-fold increase in total HRP activity in the S.cerevisiae culture supernatant compared with wild-type, as measured on ABTS ¿2, 2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (260 units/l/OD(600)). Genes from wild-type and two high-activity clones were expressed in Pichia pastoris, where the total ABTS activity reached 600 units/l/OD(600) in shake flasks. The mutants show up to 5.4-fold higher specific activity towards ABTS and 2.3-fold higher specific activity towards guaiacol.

Biotechnology applications of horseradish peroxidase (HRP) would benefit from access to tailor-made variants with greater specific activity, lower K(m) for peroxide, and higher thermostability. Starting with a mutant that is functionally expressed in Saccharomyces cerevisiae, we used random mutagenesis, recombination, and screening to identify HRP-C mutants that are more active and stable to incubation in hydrogen peroxide at 50 degrees C. A single mutation (N175S) in the HRP active site was found to improve thermal stability. Introducing this mutation into an HRP variant evolved for higher activity yielded HRP 13A7-N175S, whose half-life at 60 degrees C and pH 7.0 is three times that of wild-type (recombinant) HRP and a commercially available HRP preparation from Sigma (St. Louis, MO). The variant is also more stable in the presence of H(2)O(2), SDS, salts (NaCl and urea), and at different pH values. Furthermore, this variant is more active towards a variety of small organic substrates frequently used in diagnostic applications. Site-directed mutagenesis to replace each of the four methionine residues in HRP (M83, M181, M281, M284) with isoleucine revealed no mutation that significantly increased the enzyme's stability to hydrogen peroxide.

An Acinetobacter sp. genetic screen was used to probe structure-function relationships in vanillate demethylase, a two-component monooxygenase. Mutants with null, leaky, and heat-sensitive phenotypes were isolated. Missense mutations tended to be clustered in specific regions, most of which make known contributions to catalytic activity. The vanillate analogs m-anisate, m-toluate, and 4-hydroxy-3,5-dimethylbenzoate are substrates of the enzyme and weakly inhibit the metabolism of vanillate by wild-type Acinetobacter bacteria. PCR mutagenesis of vanAB, followed by selection for strains unable to metabolize vanillate, yielded mutant organisms in which vanillate metabolism is more strongly inhibited by the vanillate analogs. Thus, the procedure opens for investigation amino acid residues that may contribute to the binding of either vanillate or its chemical analogs to wild-type and mutant vanillate demethylases. Selection of phenotypic revertants following PCR mutagenesis gave an indication of the extent to which amino acid substitutions can be tolerated at specified positions. In some cases, only true reversion to the original amino acid was observed. In other examples, a range of amino acid substitutions was tolerated. In one instance, phenotypic reversion failed to produce a protein with the original wild-type sequence. In this example, constraints favoring certain nucleotide substitutions appear to be imposed at the DNA level.Vanillate demethylase is a two-component enzyme classified as a IA oxygenase (25, 28). It comprises a reductase containing both a flavin and a [2Fe-2S] redox center and an oxygenase containing, in addition to a substrate-binding site, an ironbinding site and a Rieske-type [2Fe-2S] cluster. Little is known about how structure influences function in vanillate demethylase. Demethylases involved in the metabolism of p-anisate in Pseudomonas putida (1) and vanillate in P. testosteroni (3,35) and P. fluorescens (5) are known to be air sensitive and unstable. The vanillate demethylases from P. testosteroni and P. fluorescens are mixed-function oxygenases and have a wide substrate specificity: m-anisate, p-anisate, m-toluate, 3,4,5-trimethoxybenzoate, and 3,4-dimethoxybenzoate were oxidized by vanillate-induced cells (5, 36). As described here, the Acinetobacter vanillate demethylase also possesses a broad substrate range.Inferences can be drawn about the mechanism of vanillate demethylase from results obtained with the evolutionarily related enzyme phthalate dioxygenase (6). In this enzyme, electrons for hydroxylation flow from NADH to flavin mononucleotide to [2Fe-2S] in the reductase and from the Rieske-type [2Fe-2S] center to the Fe 2ϩ site in the oxygenase, where oxygen binding and hydroxylation occur (9,10,33,40). As recently shown for the naphthalene dioxygenase, another member of this group of aromatic dioxygenases, Fe1 of the Rieske [2Fe-2S] center is coordinated by two cysteinyl residues and Fe2 is coordinated by two histidyl residues (14,15,18). The iron atom at the active site is coord...

A Burkholderia strain (JT 1500), able to use 2-naphthoate as the sole source of carbon, was isolated from soil. On the basis of growth characteristics, oxygen uptake experiments, enzyme assays, and detection of intermediates, a degradation pathway of 2-naphthoate is proposed. The features of this pathway are convergent with those for phenanthrene. We propose a pathway for the conversion of 2-naphthoate to 1 mol (each) of pyruvate, succinate, and acetyl coenzyme A and 2 mol of CO 2 . During growth in the presence of 2-naphthoate, six metabolites were detected by thin-layer chromatography, high-performance liquid chromatography, and spectroscopy. 1-Hydroxy-2-naphthoate accumulated in the culture broth during growth on 2-naphthoate. Also, the formation of 2-carboxybenzalpyruvate, phthalaldehydate, phthalate, protocatechuate, and ␤-carboxy-cis,cismuconic acid was demonstrated. (1R,2S)-cis-1,2-Dihydro-1,2-dihydroxy-2-naphthoate was thus considered an intermediate between 2-naphthoate and 1-hydroxy-2-naphthoate, but it was not transformed by whole cells or their extracts. We conclude that this diol is not responsible for the formation of 1-hydroxy-2-naphthoate from 2-naphthoate but that one of the other three diastereomers is not eliminated as a potential intermediate for a dehydration reaction.Fused carbo-and heterocyclic ring systems are a class of aromatic compounds which are of great concern because of their toxicity, carcinogenicity, and resistance to biodegradation (7,8,28). As products of combustion and industrial synthesis and as components of fossil fuels, aromatic ring systems are ubiquitous environmental pollutants and are abundant near urban and industrial centers.In bacterial systems, naphthoic acids were shown to accumulate in the medium when some strains of Pseudomonas putida were grown in the presence of 1-and 2-methylnaphthalene (20) or 2,6-dimethylnaphthalene (21). Involvement of several hydroxylated naphthoic acids as intermediates in the bacterial degradation of anthracene and phenanthrene is well documented (7,14). However, there are a few reports on the complete bacterial metabolism of naphthoic acids. Recently Phale et al. (29) described a pathway for biodegradation of 1-naphthoic acid by Pseudomonas maltophilia CSV89. They proposed a pathway including 1,2-dihydroxy-8-carboxynaphthalene, 3-formylsalicyclic acid, and salicylic acid as intermediates.This report deals with the bacterial oxidation of 2-naphthoate (1) by a Burkholderia sp. The pathway is convergent with those for phenanthrene, described elsewhere (12,13,18,32,33). These proposed pathways involve the formation, accumulation, and utilization of 1-hydroxy-2-naphthoate (compound 2 [2] [ Fig. 1] (Fig. 1). This report provides evidence for a 13-step catabolic route of 2-naphthoate by Burkholderia sp. strain JT 1500 as shown in Fig. 1, part of which we described before (22). MATERIALS AND METHODSBacteria and culture conditions. A Burkholderia strain, JT 1500, isolated from Miami soil and able to grow on 2-naphthoate as the sole source of ca...