Albomycins are peptidyl thionucleoside natural products that display antimicrobial activity against clinically important pathogens. Their structures are characterized by a thioheptose with atypical stereo chemistry including a D-xylofuranose ring modified with a 6-amino acid moiety. Herein it is demonstrated that AbmH is a pyridoxal 5'-phosphate (PLP)-dependent transaldolase that catalyzes a threo-selective aldol-type reaction to generate the thioheptose core with a D-ribofuranose ring and an L-amino acid moiety. The conversion of L-to 6-amino acid configuration is catalyzed by the PLP-dependent epimerase AbmD. The D-ribo to D-xylo conversion of the thiofuranose ring appears according to gene deletion experiments to be mediated by AbmJ, which is annotated as a radical S-adenosyl-L-methionine (SAM) enzyme. These studies establish several key steps in the assembly of the thioheptose core during the biosynthesis of albomycins.