Amide bond formation is a basal transformation in synthetic chemistry and the pharmaceutical industry that is traditionally performed under harsh conditions, using excess amounts of amine and relying on coupling agents. Biocatalysis shows great potential in contributing to milder and more sustainable amide bond formation in water, in particular using the emerging family of amide bond synthetase (ABS) enzymes. Here, we use molecular dynamics, biocatalysis, and enzyme engineering to study amide bond formation in extant and ancestral ABS from Marinactinospora thermotolerans (McbA). Our results show that while being more thermostable, the C-terminal domain that delivers the amine substrate to the adenylated acid intermediate is more flexible in ancestral McbA, presumably leading to an extended amine scope as observed experimentally from a small panel of aliphatic and aromatic substrates. An engineered ancestor of McbA harboring a single mutation that presumptively represent a catalytic shift residue when going from ancestral to modern biocatalyst, show two to ten-fold improved conversions over its ancestral template while maintaining high thermostability, highlighting ancestral sequence reconstruction as a potent method in protein engineering. Kinetic experiments showed that the engineered ancestral enzyme had 2-fold higher apparent kcat values in amide formation compared to extant enzyme, concomitant with relaxed substrate inhibition and loss-of-dependency on magnesium. Finally, we optimize ATP recycling utilizing a single polyphosphate kinase to showcase how engineered ancestral McbA together with reaction optimization is amenable for pharmacophore synthesis at a preparative scale.