Non-ribosomal peptide synthetase (NRPS)-encoding products and their biosynthetic logics in Fusarium.

Fungal non-ribosomal peptide synthetase (NRPS)-encoding products play a paramount role in new drug discovery. Fusarium, one of the most common filamentous fungi, is well-known for its biosynthetic potential of NRPS-type compounds with diverse structural motifs and various biological properties. With the continuous improvement and extensive application of bioinformatic tools (e.g., anti-SMASH, NCBI, UniProt), more and more biosynthetic gene clusters (BGCs) of secondary metabolites (SMs) have been identified in Fusarium strains. However, the biosynthetic logics of these SMs have not yet been well investigated till now. With the aim to increase our knowledge of the biosynthetic logics of NPRS-encoding products in Fusarium, this review firstly provides an overview of research advances in elucidating their biosynthetic pathways.

Poly-γ-glutamylation of biomolecules.

Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F420. Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F420 by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L-glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.

Heterologous Biosynthesis of the Sterol O-Acyltransferase Inhibitor Helvamide Unveils an α-Ketoglutarate-Dependent Cross-Linking Oxygenase.

We have identified the biosynthetic gene cluster (hvm) for the sterol O-acyltransferase inhibitor helvamide (1) from the genome of Aspergillus rugulosus MST-FP2007. Heterologous expression of hvm in A. nidulans produced a previously unreported analog helvamide B (5). An α-ketoglutarate-dependent oxygenase Hvm1 was shown to catalyze intramolecular cyclization of 1 to yield 5. The biosynthetic branch to the related hancockiamides and helvamides was found to be controlled by the substrate selectivity of monomodular nonribosomal peptide synthetases.

Controlling Substrate- and Stereospecificity of Condensation Domains in Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases (NRPSs) are sophisticated molecular machines that biosynthesize peptide drugs. In attempts to generate new bioactive compounds, some parts of NRPSs have been successfully manipulated, but especially the influence of condensation (C-)domains on substrate specificity remains enigmatic and poorly controlled. To understand the influence of C-domains on substrate preference, we extensively evaluated the peptide formation of C-domain mutants in a bimodular NRPS system. Thus, we identified three key mutations that govern the preference for stereoconfiguration and side-chain identity. These mutations show similar effects in three different C-domains (GrsB1, TycB1, and SrfAC) when di- or pentapeptides are synthesized in vitro or in vivo. Strikingly, mutation E386L allows the stereopreference to be switched from d- to l-configured donor substrates. Our findings provide valuable insights into how cryptic specificity filters in C-domains can be re-engineered to clear roadblocks for NRPS engineering and enable the production of novel bioactive compounds.

Recent progress in the reprogramming of nonribosomal peptide synthetases.

Nonribosomal peptide synthetases (NRPSs) biosynthesize nonribosomal peptide (NRP) natural products, which belong to the most promising resources for drug discovery and development because of their wide range of therapeutic applications. The results of genetic, biochemical, and bioinformatics analyses have enhanced our understanding of the mechanisms of the NRPS machinery. A major goal in NRP biosynthesis is to reprogram the NRPS machinery to enable the biosynthetic production of designed peptides. Reprogramming strategies for the NRPS machinery have progressed considerably in recent years, thereby increasing the yields and generating modified peptides. Here, the recent progress in NRPS reprogramming and its application in peptide synthesis are described.

The Lrp transcriptional factor of an entomopathogenic bacterium, Xenorhabdus hominickii, activates non-ribosomal peptide synthetases to suppress insect immunity.

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. The infective juveniles deliver the symbiotic bacteria to the hemocoel of target insects, in which the bacteria proliferate and help the development of the host nematode. The successful parasitism of the nematode-bacterial complex depends on host immunosuppression by the bacteria via their secondary metabolites. Leucine-responsive regulatory protein (Lrp) is a global bacterial transcriptional factor that plays a crucial role in parasitism. However, its regulatory targets to suppress insect immunity are not clearly understood. This study investigated the bacterial genes regulated by Lrp and the subsequent production of secondary metabolites in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage of the bacteria in a target insect, Spodoptera exigua. A preliminary in silico screening indicated that 3.7% genes among 4075 predicted genes encoded in X. hominickii had the Lrp-response element on their promoters, including two non-ribosomal peptide synthetases (NRPSs). Eight NRPS (NRPS1-NRPS8) genes were predicted in the bacterial genome, in which six NRPS (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites and the NRPS expression levels. The immunosuppressive activities of X. hominickii were dependent on the Lrp expression level. The metabolites produced by Lrp expression included the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (=cPF) was produced by the bacteria at high Lrp expression and inhibited the phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays a crucial role in insect immunosuppression by modulating NRPS expression.

Characterization and catalytic investigation of fungal single-module nonribosomal peptide synthetase in terpene-amino acid meroterpenoid biosynthesis.

Hybrid natural products are compounds that originate from diverse biosynthetic pathways and undergo a conjugation process, which enables them to expand their chemical diversity and biological functionality. Terpene-amino acid meroterpenoids have garnered increasing attention in recent years, driven by the discovery of noteworthy examples such as the anthelmintic CJ-12662, the insecticidal paeciloxazine, and aculene A (1). In the biosynthesis of terpene-amino acid natural products, single-module nonribosomal peptide synthetases (NRPSs) have been identified to be involved in the esterification step, catalyzing the fusion of modified terpene and amino acid components. Despite prior investigations into these NRPSs through gene deletion or in vivo experiments, the enzymatic basis and mechanistic insights underlying this family of single-module NRPSs remain unclear. In this study, we performed biochemical characterization of AneB by in vitro characterization, molecular docking, and site-directed mutagenesis. The enzyme reaction analyses, performed with L-proline and daucane/nordaucane sesquiterpene substrates, revealed that AneB specifically esterifies the C10-OH of aculenes with L-proline. Notably, in contrast to ThmA in CJ-12662 biosynthesis, which exclusively recognizes oxygenated amorpha-4,11-diene sesquiterpenes for L-tryptophan transfer, AneB demonstrates broad substrate selectivity, including oxygenated amorpha-4,11-diene and 2-phenylethanol, resulting in the production of diverse unnatural prolyl compounds. Furthermore, site-directed mutagenesis experiments indicated the involvement of H794 and D798 in the esterification catalyzed by AneB. Lastly, domain swapping between AneB and ThmA unveiled that the A‒T domains of ThmA can be effectively harnessed by the C domain of AneB for L-tryptophan transfer, thus highlighting the potential of the C domain of AneB for generating various terpene-amino acid meroterpenoid derivatives. ONE-SENTENCE SUMMARY: The enzymatic basis and mechanistic insights into AneB, a single-module NRPS, highlight its capacity to generate various terpene-amino acid meroterpenoid derivatives.

High-Throughput Engineering of Nonribosomal Extension Modules.

Nonribosomal peptides constitute an important class of natural products that display a wide range of bioactivities. They are biosynthesized by large assembly lines called nonribosomal peptide synthetases (NRPSs). Engineering NRPS modules represents an attractive strategy for generating customized synthetases for the production of peptide variants with improved properties. Here, we explored the yeast display of NRPS elongation and termination modules as a high-throughput screening platform for assaying adenylation domain activity and altering substrate specificity. Depending on the module, display of A-T bidomains or C-A-T tridomains, which also include an upstream condensation domain, proved to be most effective. Reprograming a tyrocidine synthetase elongation module to accept 4-propargyloxy-phenylalanine, a noncanonical amino acid that is not activated by the native protein, illustrates the utility of this approach for altering NRPS specificity at internal sites.

Structural Basis of Amide-Forming Adenylation Enzyme VinM in Vicenistatin Biosynthesis.

Adenylation enzymes activate amino acid substrates to aminoacyl adenylates and generally transfer this moiety onto the thiol group of the phosphopantetheine arm of a carrier protein for the selective incorporation of aminoacyl building blocks in natural product biosynthesis. In contrast to the canonical thioester-forming adenylation enzymes, the amide-forming adenylation enzyme VinM transfers an l-alanyl group onto the amino group of the aminoacyl unit attached to the phosphopantetheine arm of the carrier protein VinL to generate dipeptidyl-VinL in vicenistatin biosynthesis. It is unclear how VinM distinguishes aminoacyl-VinL from VinL for amide bond formation. Herein we describe structural and biochemical analyses of VinM. We determined the crystal structure of VinM in complex with VinL using a designed pantetheine-type cross-linking probe. The VinM-VinL complex structure in combination with site-directed mutagenesis analysis revealed that the interactions with both the phosphopantetheine arm and VinL are critical for the amide-forming activity of VinM.

Two distinct non-ribosomal peptide synthetase-independent siderophore synthetase gene clusters identified in Armillaria and other species in the Physalacriaceae.

Siderophores are important for ferric iron solubilization, sequestration, transportation, and storage, especially under iron-limiting conditions such as aerobic conditions at high pH. Siderophores are mainly produced by non-ribosomal peptide synthetase-dependent siderophore pathway, non-ribosomal peptide synthetase-independent siderophore synthetase pathway, or the hybrid non-ribosomal peptide synthetases/non-ribosomal peptide synthetases-independent siderophore pathway. Outcompeting or inhibition of plant pathogens, alteration of host defense mechanisms, and alteration of plant-fungal interactions have been associated with fungal siderophores. To understand these mechanisms in fungi, studies have been conducted on siderophore biosynthesis by ascomycetes with limited focus on the basidiomycetes. Armillaria includes several species that are pathogens of woody plants and trees important to agriculture, horticulture, and forestry. The aim of this study was to investigate the presence of non-ribosomal peptide synthetases-independent siderophore synthetase gene cluster(s) in genomes of Armillaria species using a comparative genomics approach. Iron-dependent growth and siderophore biosynthesis in strains of selected Armillaria spp. were also evaluated in vitro. Two distinct non-ribosomal peptide synthetases-independent siderophore synthetase gene clusters were identified in all the genomes. All non-ribosomal peptide synthetases-independent siderophore synthetase genes identified putatively encode Type A' non-ribosomal peptide synthetases-independent siderophore synthetases, most of which have IucA_IucC and FhuF-like transporter domains at their N- and C-terminals, respectively. The effect of iron on culture growth varied among the strains studied. Bioassays using the CAS assay on selected Armillaria spp. revealed in vitro siderophore biosynthesis by all strains irrespective of added FeCl3 concentration. This study highlights some of the tools that Armillaria species allocate to iron homeostasis. The information generated from this study may in future aid in developing molecular based methods to control these phytopathogens.

Advances on structure, bioactivity, and biosynthesis of amino acid-containing trans-AT polyketides.

Trans-AT polyketides represent a class of natural compounds utilizing independent acyltransferase during their biosynthesis. They are well known for their diverse chemical structures and potent bioactivities. Trans-AT polyketides are synthesized through biosynthetic gene clusters predominantly composed of polyketide synthases (PKS), but often found in hybrid with non-ribosomal peptide synthetases (NRPS). This genetic hybridization results in the incorporation of amino acid residues into polyketide structures, significantly enhancing their structural diversity. Numerous amino acid-containing trans-AT polyketides have been identified, drawing significant attention to the mechanisms underlying amino acid incorporation and their impact on the biological activity of polyketides. Here, we discussed their origins, structures, biological activities, and the specific roles of amino acids in modulating both the bioactivity and biosynthesis of 38 trans-AT polyketides containing amino acids for the first time. This comprehensive analysis will serve as a crucial reference for the exploration of novel compounds and the improvement of structures and activities.

Biosynthesis and engineering of the nonribosomal peptides with a C-terminal putrescine.

The broad bioactivities of nonribosomal peptides rely on increasing structural diversity. Genome mining of the Burkholderiales strain Schlegelella brevitalea DSM 7029 leads to the identification of a class of dodecapeptides, glidonins, that feature diverse N-terminal modifications and a uniform putrescine moiety at the C-terminus. The N-terminal diversity originates from the wide substrate selectivity of the initiation module. The C-terminal putrescine moiety is introduced by the unusual termination module 13, the condensation domain directly catalyzes the assembly of putrescine into the peptidyl backbone, and other domains are essential for stabilizing the protein structure. Swapping of this module to another two nonribosomal peptide synthetases leads to the addition of a putrescine to the C-terminus of related nonribosomal peptides, improving their hydrophilicity and bioactivity. This study elucidates the mechanism for putrescine addition and provides further insights to generate diverse and improved nonribosomal peptides by introducing a C-terminal putrescine.

Mur ligase F as a new target for the flavonoids quercitrin, myricetin, and (-)-epicatechin.

MurC, D, E, and F are ATP-dependent ligases involved in the stepwise assembly of the tetrapeptide stem of forming peptidoglycan. As highly conserved targets found exclusively in bacterial cells, they are of significant interest for antibacterial drug discovery. In this study, we employed a computer-aided molecular design approach to identify potential inhibitors of MurF. A biochemical inhibition assay was conducted, screening twenty-four flavonoids and related compounds against MurC-F, resulting in the identification of quercitrin, myricetin, and (-)-epicatechin as MurF inhibitors with IC50 values of 143 µM, 139 µM, and 92 µM, respectively. Notably, (-)-epicatechin demonstrated mixed type inhibition with ATP and uncompetitive inhibition with D-Ala-D-Ala dipeptide and UM3DAP substrates. Furthermore, in silico analysis using Sitemap and subsequent docking analysis using Glide revealed two plausible binding sites for (-)-epicatechin. The study also investigated the crucial structural features required for activity, with a particular focus on the substitution pattern and hydroxyl group positions, which were found to be important for the activity. The study highlights the significance of computational approaches in targeting essential enzymes involved in bacterial peptidoglycan synthesis.

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics.

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

Streptomyces lividans 66 produces a protease inhibitor via a tRNA-utilizing enzyme interacting with a C-minus NRPS.

Small peptide aldehydes (SPAs) with protease inhibitory activity are naturally occurring compounds shown to be synthesized by non-ribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology and have been utilized as therapeutic agents. They are also physiologically relevant and have been postulated to regulate the development of their producing microorganisms. Previously, we identified an NRPS-like biosynthetic gene cluster (BGC) in Streptomyces lividans 66 that lacked a condensation (C) domain but included a tRNA-utilizing enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA, which we named livipeptin. Using evolutionary genome mining approaches, here, we confirm the presence of L/F transferase tRUEs within the genomes of diverse Streptomyces and related organisms, including fusions with the anticipated C-minus NRPS-like protein. We then demonstrate genetic functional cooperation between the identified L/F-transferase divergent tRUE homolog with the C-minus NRPS, leading to the synthesis of a metabolic fraction with protease inhibitory activity. Semisynthetic assays in the presence of RNAse revealed that the productive interaction between the tRUE and the C-minus NRPS enzymes is indeed tRNA dependent. We expect our findings to boost the discovery of SPAs, as well as the development of protease-mediated biotechnologies, by exploiting the uncovered genetic basis for synthesizing putative acetyl-leu/phe-arginine protease inhibitors. Furthermore, these results will facilitate the purification and structural elucidation of livipeptin, which has proven difficult to chemically characterize. SIGNIFICANCE: The discovery of natural products biosynthetic genes marks a significant advancement in our understanding of these metabolites, for example of their evolution, activity, and biosynthesis, but also opens biotechnological opportunities and knowledge to advance genome mining approaches. We made this possible by uncovering a new biosynthetic pathway in Streptomyces lividans 66 shown to direct the synthesis of a strong protease inhibitor, termed livipeptin, following unprecedented biosynthetic rules and genes. Thus, by shedding light on the genetic mechanisms predicted to govern the production of acetyl-leu/phe-arginine protease inhibitors, including the elusive livipeptin, this study enables novel protease-mediated biotechnologies as well as approaches for discovering protease inhibitors from genome data.

Abi Family Protein, DidK, Is Involved in the Maturation of Anticancer Depsipeptide, Didemnin B.

Didemnin B is a marine-derived depsipeptide with potent antiviral and anticancer activities. A prodrug activation mechanism was previously proposed for the biosynthesis of didemnin B by the nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) assembly line, but the enzyme involved in the maturation process remained unknown. Herein, we demonstrated that DidA, a dimodular NRPS predicted with unrelated activity to didemnin B structure assembly, was indispensable to produce didemnin B, confirming the prodrug mechanism in didemnin B biosynthesis. We further identified an Abi family transmembrane protease, DidK, that functioned as an esterase in the maturation step of didemnin B by in vivo gene knockout and heterologous expression. DidK is structurally distinct from other known hydrolytic enzymes involved in the maturation of bacterial nonribosomal peptides and is the first Abi family protein known to participate in NRPS/PKS-derived natural product production. Further bioinformatic analysis revealed more than 20 DidK homologues encoded in bacterial NRPS/PKS BGCs, suggesting that the involvement of Abi family proteins in natural product biosynthesis might not be rare. These results not only clarify the priming and maturation steps of didemnin B biosynthesis but also expand the function scope of Abi family proteins.

Analysis of Rhizonin Biosynthesis Reveals Origin of Pharmacophoric Furylalanine Moieties in Diverse Cyclopeptides.

Rhizonin A and B are hepatotoxic cyclopeptides produced by bacterial endosymbionts (Mycetohabitans endofungorum) of the fungus Rhizopus microsporus. Their toxicity critically depends on the presence of 3-furylalanine (Fua) residues, which also occur in pharmaceutically relevant cyclopeptides of the endolide and bingchamide families. The biosynthesis and incorporation of Fua by non-ribosomal peptide synthetases (NRPS), however, has remained elusive. By genome sequencing and gene inactivation we elucidated the gene cluster responsible for rhizonin biosynthesis. A suite of isotope labeling experiments identified tyrosine and l-DOPA as Fua precursors and provided the first mechanistic insight. Bioinformatics, mutational analysis and heterologous reconstitution identified dioxygenase RhzB as necessary and sufficient for Fua formation. RhzB is a novel type of heme-dependent aromatic oxygenases (HDAO) that enabled the discovery of the bingchamide biosynthesis gene cluster through genome mining.

Expression of blue pigment synthetase a from Streptomyces lavenduale reveals insights on the effects of refactoring biosynthetic megasynthases for heterologous expression in Escherichia coli.

High GC bacteria from the genus Streptomyces harbor expansive secondary metabolism. The expression of biosynthetic proteins and the characterization and identification of biological "parts" for synthetic biology purposes from such pathways are of interest. However, the high GC content of proteins from actinomycetes in addition to the large size and multi-domain architecture of many biosynthetic proteins (such as non-ribosomal peptide synthetases; NRPSs, and polyketide synthases; PKSs often called "megasynthases") often presents issues with full-length translation and folding. Here we evaluate a non-ribosomal peptide synthetase (NRPS) from Streptomyces lavenduale, a multidomain "megasynthase" gene that comes from a high GC (72.5%) genome. While a preliminary step in revealing differences, to our knowledge this presents the first head-to-head comparison of codon-optimized sequences versus a native sequence of proteins of streptomycete origin heterologously expressed in E. coli. We found that any disruption in co-translational folding from codon mismatch that reduces the titer of indigoidine is explainable via the formation of more inclusion bodies as opposed to compromising folding or posttranslational modification in the soluble fraction. This result supports that one could apply any refactoring strategies that improve soluble expression in E. coli without concern that the protein that reaches the soluble fraction is differentially folded.

Identification of a novel lipoic acid biosynthesis pathway reveals the complex evolution of lipoate assembly in prokaryotes.

Lipoic acid is an essential biomolecule found in all domains of life and is involved in central carbon metabolism and dissimilatory sulfur oxidation. The machineries for lipoate assembly in mitochondria and chloroplasts of higher eukaryotes, as well as in the apicoplasts of some protozoa, are all of prokaryotic origin. Here, we provide experimental evidence for a novel lipoate assembly pathway in bacteria based on a sLpl(AB) lipoate:protein ligase, which attaches octanoate or lipoate to apo-proteins, and 2 radical SAM proteins, LipS1 and LipS2, which work together as lipoyl synthase and insert 2 sulfur atoms. Extensive homology searches combined with genomic context analyses allowed us to precisely distinguish between the new and established pathways and map them on the tree of life. This not only revealed a much wider distribution of lipoate biogenesis systems than expected, in particular, the novel sLpl(AB)-LipS1/S2 pathway, and indicated a highly modular nature of the enzymes involved, with unforeseen combinations, but also provided a new framework for the evolution of lipoate assembly. Our results show that dedicated machineries for both de novo lipoate biogenesis and scavenging from the environment were implemented early in evolution and that their distribution in the 2 prokaryotic domains was shaped by a complex network of horizontal gene transfers, acquisition of additional genes, fusions, and losses. Our large-scale phylogenetic analyses identify the bipartite archaeal LplAB ligase as the ancestor of the bacterial sLpl(AB) proteins, which were obtained by horizontal gene transfer. LipS1/S2 have a more complex evolutionary history with multiple of such events but probably also originated in the domain archaea.

The distinct initiation sites and processing activities of TTLL4 and TTLL7 in glutamylation of brain tubulin.

Mammalian brain tubulins undergo a reversible posttranslational modification-polyglutamylation-which attaches a secondary polyglutamate chain to the primary sequence of proteins. Loss of its erasers can disrupt polyglutamylation homeostasis and cause neurodegeneration. Tubulin tyrosine ligase like 4 (TTLL4) and TTLL7 were known to modify tubulins, both with preference for the ß-isoform, but differently contribute to neurodegeneration. However, differences in their biochemical properties and functions remain largely unknown. Here, using an antibody-based method, we characterized the properties of a purified recombinant TTLL4 and confirmed its sole role as an initiator, unlike TTLL7, which both initiates and elongates the side chains. Unexpectedly, TTLL4 produced stronger glutamylation immunosignals for α-isoform than ß-isoform in brain tubulins. Contrarily, the recombinant TTLL7 raised comparable glutamylation immunoreactivity for two isoforms. Given the site selectivity of the glutamylation antibody, we analyzed modification sites of two enzymes. Tandem mass spectrometry analysis revealed their incompatible site selectivity on synthetic peptides mimicking carboxyl termini of α1- and ß2-tubulins and a recombinant tubulin. Particularly, in the recombinant α1A-tubulin, a novel region was found glutamylated by TTLL4 and TTLL7, that again at distinct sites. These results pinpoint different site specificities between two enzymes. Moreover, TTLL7 exhibits less efficiency to elongate microtubules premodified by TTLL4, suggesting possible regulation of TTLL7 elongation activity by TTLL4-initiated sites. Finally, we showed that kinesin behaves differentially on microtubules modified by two enzymes. This study underpins the different reactivity, site selectivity, and function of TTLL4 and TTLL7 on brain tubulins and sheds light on their distinct role in vivo.