Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation

Abstract

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.

Cofactor Regeneration by a Soluble Pyridine Nucleotide Transhydrogenase for Biological Production of Hydromorphone

“…Furthermore, MDH became inactivated by morphinone through the formation of a covalent adduct with Cys80, and it was found that changing this residue to a serine greatly enhanced the stability of MDH (25). A mutation, K244M, leading to an approximately 10-fold-lessactive MDH was combined with the C80S mutation, to give morA C80S ⅐ K244M (25).…”

“…A 1.2-kb PstI fragment bearing the mutant morA gene complete with its upstream ribosome binding site and promoter sequences was excised from pMDH1.7 (25) and ligated into PstI-digested ps 1EMBL (21). This resulted in the construct pMORA4C80S ⅐ K244M, which contained suitable restriction sites for further subcloning.…”

“…A second construct was made where the morA gene was replaced by morA C80S ⅐ K244M (25), which encodes a less active and more stable MDH. A 1.2-kb PstI fragment bearing the mutant morA gene complete with its upstream ribosome binding site and promoter sequences was excised from pMDH1.7 (25) and ligated into PstI-digested ps 1EMBL (21).…”

Crystal Structure and Comparative Functional Analyses of a Mycobacterium Aldo-Keto Reductase

“…It is this proR hydride that is transferred to the substrate, as has been shown by studies with labelled cofactor. 28 Sequence similarity of AKR5H1 to that of other mycobacterial AKRs and putative orthologues from the Corynebacterineae In order to gain insight into the functional role of AKR5H1, we compared the sequence of AKR5H1 to those of its orthologues from the closely related mycobacterial species M. tuberculosis and Mycobacterium leprae and from other representatives of the suborder Corynebacterineae, Nocardia farcinica and Corynebacterium glutamicum (Fig. 5).…”

Biotransformation of codeine to 14-OH-codeine derivatives by Rhizobium radiobacter R89-1

“…Bacterial transformations of the morphine have also been reported with the strains Arthrobacter sp. [14], Pseudomonas testosteroni [15], Pseudomonas putida M10 [16,17], Bacillus sp. [18], Mycobacterium neoaurum MTP650 [19], and Streptomyces griseus NRRL B8090 [20].…”