Env7 is a vacuole membrane-localized protein kinase that is orthologous to the human serine/threonine protein kinase, STK16. It is evolutionarily well-conserved throughout Eukarya, and it has one ortholog in Bacteria. Phylogenetic analyses of sequences homologous to Env7 revealed clades that are inconsistent with established eukaryotic phylogeny, suggesting that both horizontal and vertical gene transmission are responsible for their conservation. Conserved amino acid residues and motifs that are potentially important to Env7's catalytic activity, localization, and interactions with other proteins were also identified and assessed. Additionally, one such conserved motif—the glycine-rich loop—was mutated in an effort to affect ATP binding in Env7. The phenotype resulting from this mutation was a slightly increased number of mutant cells exhibiting multi-lobed vacuoles under normal conditions.