The following are the supplementary data

The following are the supplementary data related to this article.
Introduction The Nuclear Hormone Receptor (NHR) superfamily of intracellular ligand-dependent transcription factors has historically been a rich source of targets for drug development, and compounds which selectively modulate gene-transcription through activation of these receptors have the potential to provide more favorable clinical profiles than those of the native Enasidenib [1], [2], [3], [4]. In this regard, clinical use of the endogenous agonists of the Androgen Receptor (AR), testosterone and dihydrotestosterone, as well as related steroidal agents have demonstrated excellent anabolic efficacy in limited clinical trials. However, their use has been limited due to drawbacks associated with both the route of administration and concerns due to side-effects and toxicity of these steroidal agonists. In order to circumvent these liabilities, several groups have been actively engaged in the discovery and development of Selective Androgen Receptor Modulators (SARMs) [5], [6], [7], [8], [9]. The most advanced of these compounds has entered clinical trials for treatment of a variety of disorders, including muscle wasting from HIV, cancer chemotherapy, chronic renal failure, male hypogonadism, benign prostatic hyperplasia, functional decline in the aging male, as well as for osteoporosis and sexual dysfunction in both men and women [10], [11], [12], [13], [14], [15], [16]. SARMs as a class hold significant potential for achieving the beneficial anabolic and cognitive enhancing effects of classical pure agonist compounds, without the associated side-effects, by virtue of multiple mechanisms mediating gene- and tissue-selective action. Further exploration of structure–activity relationships (SAR) within BMS\'s previously described novel series of SARMs resulted in the discovery of a number of diverse scaffolds conferring potent and tissue-selective agonist activity both in vitro and in vivo [17], [18], [19], [20]. Subtle changes in ligand structure were found to induce profound pharmacology differences when studied in whole-cells and in rodents. BMS-564929 is a potential Selective Androgen Receptor Modulator under development as an anticancer agent. The synthesis of BMS-564929 [20], [21] requires a key chiral intermediate, cis-3-hydroxy-l-proline. This paper describes the construction, cloning and heterologous expression of the l-proline-3-hydroxylase in Escherichia coli and the development of a process for the conversion of l-proline to cis-3-hydroxy-l-proline (Scheme 1).
Materials and methods
Results and discussion Iron (II)/2-oxoglutarate (2-OG)-dependent oxygenases [28], [29] catalyze oxidative reactions of a range of metabolic processes including the hydroxylation of l-proline and l-lysine residues during the post-translational modification of collagen. Mori et al. [22] purified proline 3-hydroxylase from Streptomyces sp. strain TH1, and its structural gene was cloned. The detected DNA sequence of the cloned fragment revealed a 870-bp open reading frame (ORF 3), encoding a protein of 290 amino acids with a calculated molecular weight of 33,158 [22]. The enzyme hydroxylated free l-proline to cis-3-hydroxy-l-proline and showed properties of a 2-oxoglutarate-dependent dioxygenase. Proline trans-4-hydroxylase (P-4-H) from the etamycin producer Streptomyces griseoviridus was identified and shown that the purified enzyme to be a 2-OG-dependent oxygenase [24]. Proline trans-4-hydroxylase and proline cis-3-hydroxylase (P-3-H) have been isolated from Streptomyces and Bacillus sp. [30], [31], respectively. The former is used in a recombinant process for the commercial preparation of trans-4-hydroxyproline, which is a starting material for the synthesis of pharmaceuticals [24]. Proline 3-hydroxylase has been purified from Streptomyces sp. strain TH1. Its structural gene has been cloned.