The antifungal and antimicrobial kutznerides, hexadepsipeptides composed of one a-hydroxy acid and five nonproteinogenic amino acids, are remarkable examples of the structural diversity found in nonribosomally produced natural products. They contain D-3-hydroxyglutamic acid, which is found in the threo and erythro isomers in mature kutznerides. In this study, two putative nonheme iron oxygenase enzymes, KtzO and KtzP, were recombinantly expressed, characterized biochemically in vitro, and found to stereospecifically hydroxylate the beta-position of glutamic acid. KtzO generates threo-L-hydroxyglutamic acid and KtzP catalyzes the formation of the erythro-isomer bound to the peptidyl carrier protein of the third module of the nonribosomal peptide synthetase KtzH. This module has a truncated adenylation domain and is unable to activate and incorporate glutamic acid. The lack of a functional adenylation domain in the third KtzH module is compensated in trans by the stand-alone adenylation domain KtzN, which activates and transfers glutamic acid onto the carrier of KtzH in the presence of the truncated adenylation domain and either KtzO or KtzP. A method that employs nonhydrolyzable coenzyme A analogs was developed and used to determine the kinetic parameters for KtzO- and KtzP-catalyzed hydroxylation of glutamic acid bound to the carrier protein. A detailed mechanism for the in trans compensation of the truncated adenylation domain and the stereospecific hydroxyglutamic acid generation and incorporation is presented. These insights may guide the use of KtzO/KtzP and KtzN or other in trans modification/restoration tools in biocombinatorial engineering approaches.