Gideon Grogan

Identification of a new class of cytochrome P450 from a Rhodococcus sp.

A degenerate set of PCR primers were used to clone a gene encoding a cytochrome P450 (the P450RhF gene) from Rhodococcus sp. strain NCIMB 9784 which is of unique primary structural organization. Surprisingly, analysis of the translation product revealed that the P450 is fused to a reductase domain at the C terminus which displays sequence conservation for dioxygenase reductase proteins. The reductase partner comprises flavin mononucleotide- and NADH-binding motifs and a [2Fe2S] ferredoxin-like center. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a soluble form. A recombinant strain of E. coli was able to mediate the O dealkylation of 7-ethoxycoumarin in good yield, despite the absence of any recombinant redox proteins. This unprecedented finding leads us to propose that P450RhF represents the first example of a new class of cytochromes P450 in which the reducing equivalents are supplied by a novel reductase in a fused arrangement.

Cytochromes P450: exploiting diversity and enabling application as biocatalysts.

The remarkable chemical reactivity and substrate range displayed by cytochromes P450 (P450s) renders them attractive as potential catalysts for a host of challenging chemical reactions in industry. The opportunities afforded by these biocatalysts are increased by the availability of greater diversity provided by the genomic resource and the variant libraries of well-known P450s produced by rational and random engineering techniques. The exploitation of this enormous diversity will require novel tools in screening, to identify enzyme reactions of interest, and also in the enabling of these valuable activities through protein engineering and bioprocess optimisation.

Ligation independent cloning (LIC) as a rapid route to families of recombinant biocatalysts from sequenced prokaryotic genomes

A technique is presented for the high throughput generation of families of recombinant biocatalysts sourced from prokaryotic genomes, providing rapid access to the naturally evolved diversity of enzyme specificity for biocatalyst discovery. The method exploits a novel ligation independent cloning strategy, based on the locally engineered vector pET-YSBLIC and has been used for the rapid generation of a suite of expression plasmids containing genes encoding a family of six Baeyer-Villiger monooxygenases (BVMOs) from Mycobacterium tuberculosis H37Rv (MTb). The six resultant recombinant strains of E. coli B834 (DE3) expressing the genes were assayed for oxygenating activity in respect of the target reaction; the resolution of bicyclo[3.2.0]hept-2-en-6-one. The analysis of biotransformations catalysed by growing cells of E. coli was complicated by the production of indole in the reaction mixtures, possibly resulting from the in vivo activity of E. coli tryptophanase. Four of the recombinant strains expressing different BVMOs catalysed the oxidation of one or more of four screening substrates, well above controls that had been transformed with the re-ligated parent vector. One of the recombinant strains, E. coli B834 (DE3) pDB5, expressing the Rv3049c gene from MTb, was found to effectively resolve the target substrate, yielding a 19% yield of (1R, 5S)-(+)-bicyclo[3.2.0]hept-2-en-6-one with >95% enantiomeric excess in a 4 L fermentation reaction.

Emergent mechanistic diversity of enzyme-catalysed β-diketone cleavage

The enzymatic cleavage of C–C bonds in β-diketones is, comparatively, a little studied biochemical process, but one that has important relevance to human metabolism, bioremediation and preparative biocatalysis. In recent studies, four types of enzymes have come to light that cleave C–C bonds in the β-diketone functionality using different chemical mechanisms. OPH [oxidized poly(vinyl alcohol) hydrolase from Pseudomonas sp. strain VM15C], which cleaves nonane-4,6-dione to butyrate and pentan-2-one is a serine-triad hydrolase. Dke1 (diketone-cleaving enzyme from Acinetobacter johnsonii) is a dioxygenase, cleaving acetylacetone to methylglyoxal and acetate. Fumarylacetoacetate hydrolase cleaves fumarylacetoacetate to fumarate and acetoacetate using a water molecule, activated by a catalytic His/Asp dyad, aided by a calcium ion that both chelates the enol acid form of the substrate and indirectly positions the water for nucleophilic attack at a carbonyl group. 6-Oxocamphor hydrolase cleaves nonenolizable cyclic β-diketones and is a homologue of the crotonase superfamily, employing a catalytic His/Asp dyad to activate a water molecule for nucleophilic attack at a carbonyl group on one prochiral face of the diketone substrate, effecting desymmetrizations of symmetrical substrates.

The biocatalytic reactions of Beauveria spp.

Fungi of the Beauveria spp., typified by Beauveria bassiana ATCC 7159, are among the most frequently used whole cell biocatalysts. They have been reported to catalyse many different reactions, including oxidative, reductive and hydrolytic transformations, of a wide range of substrates. This review covers the range and application of biocatalytic reactions of Beauveria spp., with emphasis on the scope and utility of Beauveria-catalysed reactions for preparative biotransformations.

Structure and Activity of Nadph-Dependent Reductase Q1Eqe0 from Streptomyces Kanamyceticus, which Catalyses the R-Selective Reduction of an Imine Substrate.

NADPH‐dependent oxidoreductase Q1EQE0 from Streptomyces kanamyceticus catalyzes the asymmetric reduction of the prochiral monocyclic imine 2‐methyl‐1‐pyrroline to the chiral amine (R)‐2‐methylpyrrolidine with >99 % ee, and is thus of interest as a potential biocatalyst for the production of optically active amines. The structures of Q1EQE0 in native form, and in complex with the nicotinamide cofactor NADPH have been solved and refined to a resolution of 2.7 Å. Q1EQE0 functions as a dimer in which the monomer consists of an N‐terminal Rossman‐fold motif attached to a helical C‐terminal domain through a helix of 28 amino acids. The dimer is formed through reciprocal domain sharing in which the C‐terminal domains are swapped, with a substrate‐binding cleft formed between the N‐terminal subunit of monomer A and the C‐terminal subunit of monomer B. The structure is related to those of known β‐hydroxyacid dehydrogenases, except that the essential lysine, which serves as an acid/base in the (de)protonation of the nascent alcohol in those enzymes, is replaced by an aspartate residue, Asp187 in Q1EQE0. Mutation of Asp187 to either asparagine or alanine resulted in an inactive enzyme.

An (R)‐Imine Reductase Biocatalyst for the Asymmetric Reduction of Cyclic Imines

Although the range of biocatalysts available for the synthesis of enantiomerically pure chiral amines continues to expand, few existing methods provide access to secondary amines. To address this shortcoming, we have over‐expressed the gene for an (R)‐imine reductase [(R)‐IRED] from Streptomyces sp. GF3587 in Escherichia coli to create a recombinant whole‐cell biocatalyst for the asymmetric reduction of prochiral imines. The (R)‐IRED was screened against a panel of cyclic imines and two iminium ions and was shown to possess high catalytic activity and enantioselectivity. Preparative‐scale synthesis of the alkaloid (R)‐coniine (90 % yield; 99 % ee) from the imine precursor was performed on a gram‐scale. A homology model of the enzyme active site, based on the structure of a closely related (R)‐IRED from Streptomyces kanamyceticus, was constructed and used to identify potential amino acids as targets for mutagenesis.

Insights into Sequence–Activity Relationships amongst Baeyer–Villiger Monooxygenases as Revealed by the Intragenomic Complement of Enzymes from Rhodococcus jostii RHA1

The Rhodococcus jostii RHA1 genome encodes a number of enzymes that can be exploited as biocatalysts. Study of the substrate spectrum and enantioselectivity of Baeyer–Villiger monooxygenases from R. jostii allowed the identification of short amino acid sequences specific to groups displaying certain catalytic characteristics. The gel illustrates the substrate acceptance spectra and selectivities of the different proteins.

InspIRED by Nature: NADPH‐Dependent Imine Reductases (IREDs) as Catalysts for the Preparation of Chiral Amines

Imine reductases (IREDs) are NADPH-dependent oxidoreductases that catalyse the asymmetric reduction of cyclic prochiral imines to amines, with excellent stereoselectivity. Since their discovery, stereocomplementary IREDs have been applied to the production of both (S) and (R) cyclic secondary amines, and the expansion in gene sequences recently identified has hinted at new substrate ranges that extend into acyclic imines and even suggest the possibility of asymmetric reductive amination from suitable ketone and amine precursors. Structural studies of various IREDs are beginning to reveal the complexities inherent in determining substrate range, stereoselectivity and mechanism in these enzymes, which represent a valuable emerging addition to the toolbox of available biocatalysts for chiral amine production.

Biological Baeyer–Villiger oxidation of some monocyclic and bicyclic ketones using monooxygenases from Acinetobacter calcoaceticus NCIMB 9871 and Pseudomonas putida NCIMB 10007

A. calcoaceticus NCIMB 9871 and Ps. putida NCIMB 10007 [grown on (+)-camphor] have been utilized as biocatalysts in Baeyer–Villiger oxidations. The former microorganism oxidized the racemic ketone 6 non-selectively but transformed the dihalogeno ketone (±)-8 into optically active lactone 10 and recovered ketone. Ps. putida NCIMB 10007 oxidized the two enantiomers of the ketone 6 at different rates while both enantiomers of ketone (±)-1 were converted into lactones, one enantiomer giving 3-oxabicyclooctenone preferentially, while the other enantiomer gave 2-oxabicyclooctenone. Ps. putida NCIMB 10007 contains two quite different types of monooxygenase enzyme, one using NADH as cofactor (labelled MO1) the other employing NADPH as cofactor (labelled MO2). Monooxygenase MO1 proved to be a selective efficient biocatalyst for the oxidation of bicyclic ketones such as 1 and 6 while monooxygenase MO2 is a useful catalyst for the oxidation of cyclopentanones 15–17. Cofactor recycling was effected using dehydrogenase enzymes in preparative-scale experiments.