Biotransformation of alkaloids.

Biotransformations of alkaloids over the last decade have continued to encompass a wide variety of substrates and enzymes. The elucidation of novel alkaloid biosynthetic and catabolic pathways will continue to furnish new biocatalysts for the synthetic organic chemist. Furthermore, an improved understanding of the genetic and biochemical basis of metabolic pathways will also permit the engineering of pathways in plants and other heterologous hosts for the production of therapeutically important alkaloids. The combination of increasing commercial interest and advances in molecular biology will facilitate the availability of robust biocatalysts which are a prerequsite to achieve economically feasible processes for the production of alkaloid-based therapeutics.

Degradation of explosives by nitrate ester reductases.

Explosive-contaminated land poses a hazard both to the environment and to human health. Microbial enzymes, either in their native or heterologous hosts, are a powerful and low-cost tool for eliminating this environmental hazard. As many explosives have only been present in the environment for 10 years, and with similar molecules not known in Nature, the origin of enzymes specialized for the breakdown of explosives is of particular interest. Screening of environmental isolates resulted in the discovery of flavoproteins capable of denitrating the explosives pentaerythritol tetranitrate (PETN) and glycerol trinitrate. These nitrate ester reductases are related in sequence and structure to Old Yellow Enzyme from Saccharomyces carlsbergenisis. All the members of this family have alpha/beta barrel structures and FMN as a prosthetic group, and reduce various electrophilic substrates. The nitrate ester reductases are, however, unusual in that they display activity towards the highly recalcitrant, aromatic explosive 2,4,6-trinitrotoluene, via a reductive pathway resulting in nitrogen liberation. We have embarked on a detailed study of the structure and mechanism of PETN reductase from a strain of Enterobacter cloacae. Work is focused currently on relating structure and function within this growing family of enzymes, with a view to engineering novel enzymes exhibiting useful characteristics.

Engineering novel biocatalytic routes for production of semisynthetic opiate drugs.

The morphine alkaloids and their semisynthetic derivatives provide a diverse range of important pharmaceutical drugs. Current production of semisynthetic opiate drugs is by chemical means from naturally occurring morphine, codeine and thebaine. Although various microbial transformations of morphine alkaloids have been identified since the 1960s, more recently there has been considerable effort devoted to engineering biocatalytic routes for producing these important compounds. Such biocatalytic routes are attractive, as they would provide an alternative to the chemical production processes which suffer from limited supply of precursors, often low yields and toxic wastes. The biotransformation of morphine and codeine to the potent analgesic hydromorphone and the mild analgesic/antitussive hydrocodone, respectively, by recombinant Escherichia coli has been demonstrated and the problems encountered when engineering such a system will be discussed.

Cofactor regeneration by a soluble pyridine nucleotide transhydrogenase for biological production of hydromorphone.

We have applied the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP(+)-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.

Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation.

Morphine dehydrogenase (MDH) of Pseudomonas putida M10 catalyses the NADP(+)-dependent oxidation of morphine and codeine to morphinone and codeinone. This enzyme forms the basis of a sensitive detection and assay method for heroin metabolites and a biotransformation process for production of hydromorphone and hydrocodone. To improve these processes we have undertaken a thorough examination of the kinetic mechanism of MDH. Sequence comparisons indicated that MDH belongs within the aldose reductase enzyme family. MDH was shown to be specific for the pro-R hydrogen of NADPH. In steady-state kinetic studies, product inhibition patterns suggested that MDH follows a Theorell-Chance mechanism for codeinone reduction at pH 7, and a non-Theorell-Chance sequential ordered mechanism for codeine oxidation at pH 9.5. Residues corresponding to the catalytically important Tyr-48, Lys-77 and Asp-43 of aldose reductase were modified by site-directed mutagenesis, resulting in substantial loss of activity consistent with a catalytic role for these residues. Loss of activity of MDH in the presence of the reaction product morphinone was found to be due to the formation of a covalent adduct with Cys-80; alteration of Cys-80 to serine resulted in an enzyme with greatly enhanced stability.

Transformations of codeine to important semisynthetic opiate derivatives by Pseudomonas putida m10.

A biotransformation mixture which contained codeine and washed cells of Pseudomonas putida M10 gave rise to a number of transformation products that are of clinical importance which included hydrocodone, dihydrocodeine and 14beta-hydroxycodeine. Incubations with the same organism and codeinone gave rise to 14beta-hydroxycodeinone and 14beta-hydroxycodeine. Cell-free extracts and membrane fractions of P. putida M10 were shown to catalyse the 14beta-hydroxylation of codeinone. In addition, the potent analgesic oxycodone was shown to be produced from 14beta-hydroxycodeinone.

Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli.

The structural gene for heroin esterase was cloned from Rhodococcus sp. strain H1 and expressed in Escherichia coli BL21(DE3). The purified enzyme was found to be a tetramer with an M(r) of 137,000 and an apparent K(m) of 0.88 mM for 6-acetylmorphine. The G-x-S-x-G motif was observed in the deduced amino acid sequence, suggesting that the enzyme is a serin esterase.

Biological production of semisynthetic opiates using genetically engineered bacteria.

Semisynthetic derivatives of morphine and related alkaloids are in widespread clinical use. Due to the complexity of these molecules, however, chemical transformations are difficult to achieve in high yields. We recently identified the powerful analgesic hydromorphone as an intermediate in the metabolism of morphine by Pseudomonas putida M10. Here we describe the construction of recombinant strains of Escherichia coli that express morphine dehydrogenase and morphinone reductase. These strains are capable of efficiently transforming the naturally occurring alkaloids morphine and codeine to hydromorphone and the antitussive hydrocodone, respectively. Our results demonstrate the potential for recombinant DNA technology to provide biological routes for the synthesis of known and novel semisynthetic opiate drugs.