Probing the structural plasticity of an archaeal primordial cobaltochelatase CbiX(S).

Insertion of metal ions into tetrapyrrole macrocycles is catalyzed by a diverse group of enzymes called chelatases. Structures are known for several chelatases catalyzing metal insertion into protoporphyrin IX or sirohydrochlorin. Despite a lack of significant amino acid sequence similarity, these ferro- and cobaltochelatases share a high degree of structural similarity. Cobaltochelatase CbiK and ferrochelatase HemH are bilobial enzymes with two alpha/beta domains, which were suggested to origin from a common ancestral protein via gene duplication. Small, single-domain chelatases (CbiX(S)) were recently described in archaea and are believed to represent primordial chelatases. Here, we tested the structural plasticity of an archaeal cobaltochelatase CbiX(S) by rearranging its structure with a novel method producing random in-frame deletions, duplications and insertions. A number of functional chelatase variants with insertion of duplicated sequence stretches, encompassing from one to nine secondary structural elements, were obtained. CbiX(S) was found to tolerate large sequence rearrangements in four out of the nine loop regions of the protein, indicating a high degree of structural plasticity. The predicted topologies of two variants (M51 and M518) are strikingly similar to CbiK and HemH, suggesting that we recreated duplication events that are believed to have created the bilobial chelatases.

The putative lipase, AF1763, from Archaeoglobus fulgidusis is a carboxylesterase with a very high pH optimum.

The open reading frame AF1763, annotated as a putative lipase gene (lipA) of the hyperthermophilic archaeon, Archaeoglobus fulgidus DSM 4304, was cloned and over-expressed in E. coli. A sequence analysis of LipA and the investigation of a truncated enzyme implied a special function of the C-terminal part of LipA. The substrate spectrum of the enzyme suggested that LipA is a carboxylesterase rather than a canonical lipase. The enzyme showed optimal activity at 70 degrees C and between pH 10 and 11, which is among the most alkaline pH range detected for hydrolases.

Directed evolution of Escherichia coli farnesyl diphosphate synthase (IspA) reveals novel structural determinants of chain length specificity.

Directed evolution of farnesyl diphosphate (FPP, C15) synthase (IspA) of Escherichia coli was carried out by error-prone PCR with a color complementation screen utilizing C40 carotenoid pathway enzymes. This allowed IspA mutants with enhanced production of the C40 carotenoid precursor geranylgeranyl diphosphate (GGPP, C20) to be readily identified. Analysis of these mutants was carried out in order to better understand the mechanisms of product chain length specificity in this enzyme. The 12 evolved clones having enhanced C20 GGPP production have characteristic mutations in the conserved regions of prenyl diphosphate synthases (designated regions I through VII). Some of these mutations (I76T, Y79S, Y79H, C75Y, H83Y, and H83Q) are found near or before the conserved first aspartate rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl synthases. Molecular modeling suggested a mechanism for chain length determination for these mutations including substitutions at the 1st and 9th amino acids upstream of the FARM that have not been reported previously. In addition, a mutation on a helix adjacent to the FARM within the substrate-binding pocket (D115G) suggests a novel mechanism for chain length determination. One mutant IspA clone carries a mutation of C155G at the 2nd amino acid upstream of conserved region IV (GQxxDL), which was recently found to be an important region controlling the chain elongation of a Type III GGPP synthase. One IspA clone carries mutations (T234A and T249I) near the conserved second aspartate rich motif (SARM). As a verification of the in vivo activity of the mutant clones (represented as C40 carotenoid formation), we confirmed the product distribution of wild-type and mutant IspA using an in vitro assay.

Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution.

Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity. Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate-rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases. One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and Escherichia coli C15 farnesyl diphosphate synthase. Finally, one mutant carrying four mutations (V84I, H88R, I177 M and M191V) is of interest. Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids.

A high-throughput screen for porphyrin metal chelatases: application to the directed evolution of ferrochelatases for metalloporphyrin biosynthesis.

Porphyrins are of particular interest in a variety of applications ranging from biocatalysis and chemical synthesis to biosensor and electronic technologies as well as cancer treatment. Recently, we have developed a versatile system for the high-level production of porphyrins in engineered E. coli cells with the aim of diversifying substitution patterns and accessing porphyrin systems not readily available through chemical synthesis. However, this approach failed to produce significant amounts of the metalloporphyrin in vivo from overproduced protoporphyrin due to insufficient metal insertion. Therefore, we systematically assessed the activity of the B. subtilis ferrochelatase in vivo and in vitro. A true high-throughput-screening approach based on catalytic in vivo ferrochelatase activity was developed by using fluorescence-activated cell sorting (FACS). This assay was used to screen a library of 2.4 x 10(6) ferrochelatase mutants expressed in protoporphyrin-overproducing recombinant E. coli cells. Several selected protein variants were purified, and their improved catalytic activity was confirmed in vitro. In addition to ferrochelatase activity, metal transport into E. coli was identified as another limitation for in vivo heme overproduction. Overexpression of the metal transporter zupT as part of the assembled pathway increased the overall metalloporphyrin production twofold. This report represents the most exhaustive in vitro evolution study of a ferrochelatase and demonstrates the effectiveness of our novel high-throughput-screening system for directed evolution of ferrochelatases based on their catalytic activity.

Dealing with complexity: evolutionary engineering and genome shuffling.

Comparative analysis of the growing number of microbial genome sequences has shown a high plasticity of genomes and several mechanisms for the adaptation of microbial cells to changing environmental conditions have been discovered. By contrast, the underlying metabolic networks of microorganisms are under strict control and relatively rigid, which poses a significant challenge for rational metabolic engineering approaches. Recursive shuffling of whole genomes has recently been demonstrated as an effective new evolutionary whole-cell engineering approach for the rapid improvement of industrially important microbial phenotypes.

High-level production of porphyrins in metabolically engineered Escherichia coli: systematic extension of a pathway assembled from overexpressed genes involved in heme biosynthesis.

Due to their spectroscopic properties porphyrins are of special interest for a variety of applications, ranging from drug development or targeting to material sciences and chemical and biological sensors. Since chemical syntheses are limited in terms of regio- and stereoselective functionalization of porphyrins, a biosynthetic approach with tailored enzyme catalysts offers a promising alternative. In this paper, we describe assembly of the entire heme biosynthetic pathway in a three-plasmid system and overexpression of the corresponding genes with Escherichia coli as a host. Without further optimization, this approach yielded remarkable porphyrin production levels, up to 90 micro mol/liter, which is close to industrial vitamin B(12) production levels. Different combinations of the genes were used to produce all major porphyrins that occur as intermediates in heme biosynthesis. All these porphyrin intermediates were obtained in high yields. The product spectrum was analyzed and quantified by using high-performance liquid chromatography. Intriguingly, although protoporphyrin IX could be produced at high levels, overexpressed Bacillus subtilis ferrochelatase could not convert this substrate appreciably into heme. However, further investigation clearly revealed a high level of expression of the ferrochelatase and a high level of activity in vitro. These results may indicate that heme has a regulatory impact on the iron uptake of E. coli or that the ferrochelatase is inactive in vivo due to an incompatible enzyme interaction.