Protein histidine methylation is a rare post-translational modification of unknown biochemical importance. In vertebrates, only a few methylhistidine-containing proteins have been reported, including β-actin as an essential example. The evolutionary conserved methylation of β-actin H73 is catalyzed by an as yet unknown histidine N-methyltransferase. We report here that the protein SETD3 is the actin-specific histidine N-methyltransferase. In vitro, recombinant rat and human SETD3 methylated β-actin at H73. Knocking-out SETD3 in both human HAP1 cells and in Drosophila melanogaster resulted in the absence of methylation at β-actin H73 in vivo, whereas β-actin from wildtype cells or flies was > 90% methylated. As a consequence, we show that Setd3-deficient HAP1 cells have less cellular F-actin and an increased glycolytic phenotype. In conclusion, by identifying SETD3 as the actin-specific histidine N-methyltransferase, our work pioneers new research into the possible role of this modification in health and disease and questions the substrate specificity of SET-domain-containing enzymes.

SETD3 is a member of the SET (Su(var)3–9, Enhancer of zeste, and Trithorax) domain protein superfamily and plays important roles in hypoxic pulmonary hypertension, muscle differentiation, and carcinogenesis. Previously, we identified SETD3 as the actin-specific methyltransferase that methylates the N3 of His73 on β-actin (Kwiatkowski et al., 2018). Here, we present two structures of S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified β-actin peptide or its His-methylated variant. Structural analyses, supported by biochemical experiments and enzyme activity assays, indicate that the recognition and methylation of β-actin by SETD3 are highly sequence specific, and that both SETD3 and β-actin adopt pronounced conformational changes upon binding to each other. In conclusion, this study is the first to show a catalytic mechanism of SETD3-mediated histidine methylation on β-actin, which not only throws light on the protein histidine methylation phenomenon but also facilitates the design of small molecule inhibitors of SETD3.

Key pointsr Using recombinant DNA technology, the present study provides the first strong and direct evidence indicating that β-alanine is an efficient substrate for the mammalian transaminating enzymes 4-aminobutyrate-2-oxoglutarate transaminase and alanine-glyoxylate transaminase.r The concentration of carnosine and anserine in murine skeletal and heart muscle depends on circulating availability of β-alanine, which is in turn controlled by degradation of β-alanine in liver and kidney.r Chronic oral β-alanine supplementation is a popular ergogenic strategy in sports because it can increase the intracellular carnosine concentration and subsequently improve the performance of high-intensity exercises. The present study can partly explain why the β-alanine supplementation protocol is so inefficient, by demonstrating that exogenous β-alanine can be effectively routed toward oxidation.Abstract The metabolic fate of orally ingested β-alanine is largely unknown. Chronic β-alanine supplementation is becoming increasingly popular for improving high-intensity exercise performance because it is the rate-limiting precursor of the dipeptide carnosine (β-alanyl-L-histidine) in muscle. However, only a small fraction (3-6%) of the ingested β-alanine is used for carnosine synthesis. Thus, the present study aimed to investigate the putative contribution of two β-alanine transamination enzymes, namely 4-aminobutyrate-2-oxoglutarate transaminase (GABA-T) and alanine-glyoxylate transaminase (AGXT2), to the homeostasis of carnosine and its methylated analogue anserine. We found that, when transfected into HEK293T cells, recombinant mouse and human GABA-T and AGXT2 are able to transaminate β-alanine efficiently. The reaction catalysed by GABA-T is inhibited by vigabatrin, whereas both GABA-T and AGXT2 activity is inhibited by aminooxyacetic acid (AOA). Both GABA-T and AGXT2 are highly expressed in the mouse liver and kidney and the administration of the inhibitors effectively reduced their enzyme activity in liver (GABA-T for vigabatrin; GABA-T and AGXT2 for AOA). In vivo, injection of AOA in C57BL/6 mice placed on β-alanine (0.1% w/v in drinking water) for 2 weeks lead to a 3-fold increase in circulating β-alanine levels and to significantly higher levels of carnosine and anserine in skeletal muscle and heart. By contrast, specific inhibition of GABA-T by vigabatrin did not affect carnosine and anserine levels in either tissue. Collectively, these data demonstrate that homeostasis of carnosine and anserine in mammalian skeletal muscle and heart is controlled by circulating β-alanine levels, which are suppressed by hepatic and renal β-alanine transamination upon oral β-alanine intake.

31SETD3 is a member of SET (Su(var)3-9, Enhancer of zeste, and Trithorax) domain 32 protein superfamily and plays important roles in hypoxic pulmonary hypertension, 33 muscle differentiation, and carcinogenesis. In a previous paper (Kwiatkowski et al. 34 2018), we have identified SETD3 as the actin-specific methyltransferase that 35 methylates the N 3 of His73 on β-actin. Here we present two structures of 36 S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified 37 β-actin peptide or its His-methylated variant. Structural analyses supported by the 38 site-directed mutagenesis experiments and the enzyme activity assays indicated that 39 the recognition and methylation of β-actin by SETD3 is highly sequence specific, and 40 both SETD3 and β-actin adopt pronounce conformational changes upon binding to 41 each other. In conclusion, the structural research uncovers the molecular mechanism 42 of sequence-selective histidine methylation by SETD3, which not only throws light on 43 protein histidine methylation phenomenon, but also facilitates the design of small 44 molecule inhibitors of SETD3. 45 46 91 by SETD3. 92 93With the two solved β-actin peptide-SETD3 structures, we uncover that SETD3 94 recognizes a fragment of β-actin in a sequence-dependent manner and utilizes a 95 specific pocket to catalyze the N 3 -methylation of His73. Moreover, a comprehensive 96 structural, biochemical and enzymatic profiling of SETD3 allows us to pinpoint its 97 key residues important for substrate recognition and subsequent methylation. 98Therefore, the structural research, supplemented by biochemical and enzymatic 99 experiments, not only provides insights into the catalytic mechanism of SETD3, but 100 also will facilitate the design of specific inhibitors of SETD3 enzyme. 101 102 RESULTS 103SETD3 binds to and methylates β-actin 104 Since SETD3 was identified as a histidine methyltransferase that methylates His73 of 105 β-actin (Kwiatkowski et al., 2018, Wilkinson et al., 2019, we purified the core region 106 of SETD3 (aa 2-502) and studied by ITC its binding to a His73-containing fragment 107 of β-actin (aa 66-88) ( Figures 1A). The ITC binding experiment showed that SETD3 108 bound to the β-actin peptide with a Kd of 0.17 μM ( Figure 1B and Table 1). Given 109 that SETD3 was also reported to be a putative lysine methyltransferase that 110 methylates Lys4 and Lys36 of histone H3 (Eom et al., 2011), we also verified the 111 binding of SETD3 to two different histone peptides, H3K4(1-23) and H3K36(25-47), 112 and found that neither of them binds to SETD3 (Table 1).113 114 Furthermore, we tested the activity of SETD3 on β-actin(66-88), H3K4(1-23), and 115 H3K36(25-47) by mass spectrometry, and we found that SETD3 methylates β-actin 116 peptide (Figure 1-figure supplement 1A), but does not modify either H3K4 or H3K36 117 (Figure 1-figure supplement 1B-1C). No methylated product was detected for any of 118 above peptides in the presence of AdoMet without the addition of SETD3 (Figure 119 1-figure supple...