Monoglycylase which modifies both tubulin and non-tubulin proteins, adding a single glycine on the gamma-carboxyl groups of specific glutamate residues to generate monoglycine side chains within the C-terminal tail of target proteins (PubMed:19524510, PubMed:28576883). Not involved in elongation step of the polyglycylation reaction (PubMed:19524510). Preferentially monoglycylates alpha-tubulin over beta-tubulin (PubMed:19524510). Together with TTLL3, mediates microtubule glycylation of primary and motile cilia, which is essential for their stability and maintenance (PubMed:19524510, PubMed:23897886, PubMed:25180231). Together with TTLL3, glycylates sperm flagella which regulates axonemal dynein motor activity, thereby controlling flagellar beat, directional sperm swimming and male fertility (PubMed:33414192). Monoglycylates non-tubulin proteins such as ANP32A, ANP32B, SET, NCL and NAP1 (PubMed:19524510, PubMed:19427864). ATP + glycine + L-glutamyl-[protein] = ADP + glycyl-L-glutamyl-[protein] + H(+) + phosphate Highly expressed in testis (PubMed:17499049, PubMed:25180231). Expressed in brain, heart, kidney, liver, lung, muscle, spleen and trachea (PubMed:17499049, PubMed:25180231, PubMed:33414192). Expressed in sperm flagellum (PubMed:33414192). In the brain, specifically expressed in ependymal cilia (PubMed:23897886). Two conserved structural elements specific among monoglycylases, IS1 and IS2, are involved in glycyl chains initiation. Two conserved structural interfaces likely constitute the binding platforms for tubulin tail and microtubule. Arg-403 is the main determinant for regioselectivity, which segregates between initiases and elongases in all tubulin--tyrosine ligase family. A glutamine residue at this position is found in elongases TTLL6, TTLL9, TTLL11, TTLL13, TTLL10 and favors glutamate-chain elongation, whereas an arginine residue is found in initiases TTLL2, TTLL4, TTLL5, TTLL3, TTLL8 and favors initiation. Simultaneous TTLL3 and TTLL8 knockout mice are subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. TTLL3 and TTLL8 monoglycylase-mediated glycylation of tubulin was initially reported to play a role in ependymal motile ciliary maintenance (PubMed:23897886). However, contradictory results were later observed (PubMed:33414192).