acting as the nucleophile and gly-met providing stabilization of the transition state by forming an "oxyanion hole". the active site amino acids are almost entirely the same as dnr p, and the mechanism is almost certainly identical. although dox a is shown next in the biosynthetic scheme (figure 2), dnr k, carminomycin 4-o-methyltransferase is able to o-methylate the 4-hydroxyl group of any of the glycosides in figure 2. a 2.35 å resolution crystal structure of the enzyme with bound products has recently been solved. the orientation of the products is consistent with a sn2 mechanism of methyl transfer. site-directed mutagenesis of the potential acid/base residues in the active site did not affect catalysis leading to the conclusion that dnr k most likely acts as an entropic enzyme in that rate enhancement is mainly due to orientational and proximity effects. this is in contrast to most other o-methyltransferases where acid/base catalysis has been demonstrated to be an essential contribution to rate enhancement. dox a catalyzes three successive oxidations in streptomyces peucetius. deficient dxr production is not primarily due to low levels of or malfunctioning dox a, but because there are many products diverted away from the pathway shown in figure 2. each of the glycosides is a potential target of shunt enzymes, not shown, some of which are products of the dnr gene cluster. mutations of these enzymes does significantly boost dxr production. in addition, dox a has a very low kcat/km value for c-14 oxidation (130/m) compared to c-13 oxidation (up to 22,000/m for some substrates). genetic manipulation to overexpress dox a has also increased yields, particularly if the genes for the shunt enzymes are inactivated simultaneously. dox a is a cytochrome p-450 monooxygenase that has broad substrate specificity, catalyzing anthracycline hydroxylation at c-13 and c-14 ( figure 2). the enzyme has an absolute requirement for molecular oxygen and nadph. initially, two successive oxidations are done at c-13, followed by a single oxidation of c-14 that converts daunorubicin to doxorubicin.