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Background: NADPH-dependent ␣-keto acid reductase, belonging to the short-chain dehydrogenase/reductase family, is involved in bacterial alginate metabolism. Results: A novel NADH-dependent ␣-keto acid reductase was identified, and its tertiary structure was determined by x-ray crystallography. Conclusion: Two short and long loops are structural determinants for coenzyme specificity. Significance: A method for structure-based conversion of a coenzyme requirement was established.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes implicated in several dominant and recessive disease phenotypes. The canonical function of ARSs is to couple an amino acid to a cognate transfer RNA (tRNA). We identified three novel disease-associated missense mutations in the alanyl-tRNA synthetase (AARS) gene in three families with dominant axonal Charcot-Marie-Tooth (CMT) disease. Two mutations (p.Arg326Trp and p.Glu337Lys) are located near a recurrent pathologic change in AARS, p.Arg329His. The third (p.Ser627Leu) is in the editing domain of the protein in which hitherto only mutations associated with recessive encephalopathies have been described. Yeast complementation assays demonstrated that two mutations (p.Ser627Leu and p.Arg326Trp) represent loss-of-function alleles, while the third (p.Glu337Lys) represents a hypermorphic allele. Further, aminoacylation assays confirmed that the third mutation (p.Glu337Lys) increases tRNA charging velocity. To test the effect of each mutation in the context of a vertebrate nervous system, we developed a zebrafish assay. Remarkably, all three mutations caused a pathological phenotype of neural abnormalities when expressed in zebrafish, while expression of the human wild-type messenger RNA (mRNA) did not. Our data indicate that not only functional null or hypomorphic alleles, but also hypermorphic AARS alleles can cause dominantly inherited axonal CMT disease.

(2016) A novel HSD17B10 mutation impairing the activities of the mitochondrial RNase P complex causes X-linked intractable epilepsy and neurodevelopmental regression, RNA Biology, 13:5, 477-485, DOI: 10.1080/15476286.2016 ABSTRACTWe report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene HSD17B10 for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in HSD17B10 cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5 0 -processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes that ligate amino acids onto tRNA molecules. Genes encoding ARSs have been implicated in myriad dominant and recessive disease phenotypes. Glycyl-tRNA synthetase (GARS) is a bi-functional ARS that charges tRNAGly in the cytoplasm and mitochondria. GARS variants have been associated with dominant Charcot-Marie-Tooth disease but have not been convincingly implicated in recessive phenotypes. Here we describe a patient from the NIH Undiagnosed Diseases Program with a multi-system, developmental phenotype. Whole-exome sequence analysis revealed that the patient is compound heterozygous for one frameshift (p.Glu83Ilefs*6) and one missense (p.Arg310Gln) GARS variant. Using in vitro and in vivo functional studies, we show that both GARS variants cause a loss-of-function effect: the frameshift variant results in depleted protein levels and the missense variant reduces GARS tRNA charging activity. In support of GARS variant pathogenicity, our patient shows striking phenotypic overlap with other patients having ARS-related recessive diseases, including features associated with variants in both cytoplasmic and mitochondrial ARSs; this observation is consistent with the essential function of GARS in both cellular locations. In summary, our clinical, genetic, and functional analyses expand the phenotypic spectrum associated with GARS variants.

Although abnormal increases in the level or activity of cyclin-dependent kinase 4 (CDK4) occur frequently in cancer, the underlying mechanism is not fully understood. Here, we show that methionyl-tRNA synthetase (MRS) specifically stabilizes CDK4 by enhancing the formation of the complex between CDK4 and a chaperone protein. Knockdown of MRS reduced the CDK4 level, resulting in G0/G1 cell cycle arrest. The effects of MRS on CDK4 stability were more prominent in the tumor suppressor p16 INK4anegative cancer cells because of the competitive relationship of the two proteins for binding to CDK4. Suppression of MRS reduced cell transformation and the tumorigenic ability of a p16 INK4a-negative breast cancer cell line in vivo. Further, the MRS levels showed a positive correlation with those of CDK4 and the downstream signals at high frequency in p16 INK4a-negative human breast cancer tissues. This work revealed an unexpected functional connection between the two enzymes involving protein synthesis and the cell cycle.

As opposed to typical bacteria exhibiting chemotaxis towards low-molecular-weight substances, such as amino acids and mono/oligosaccharides, gram-negative Sphingomonas sp. strain A1 shows chemotaxis towards alginate and pectin polysaccharides. To identify the mechanism of chemotaxis towards macromolecules, a genomic fragment was isolated from the wild-type strain A1 through complementation with the mutant strain A1-M5 lacking chemotaxis towards pectin. This fragment contained several genes including sph1118. Through whole-genome sequencing of strain A1-M5, sph1118 was found to harbour a mutation. In fact, sph1118 disruptant lost chemotaxis towards pectin, and this deficiency was recovered by complementation with wild-type sph1118. Interestingly, the gene disruptant also exhibited decreased pectin assimilation. Furthermore, the gene product SPH1118 was expressed in recombinant E. coli cells, purified and characterised. Differential scanning fluorimetry and UV absorption spectroscopy revealed that SPH1118 specifically binds to pectin with a dissociation constant of 8.5 μM. Using binding assay and primary structure analysis, SPH1118 was predicted to be a periplasmic pectin-binding protein associated with an ATP-binding cassette transporter. This is the first report on the identification and characterisation of a protein triggering chemotaxis towards the macromolecule pectin as well as its assimilation.