PDXK mutations cause polyneuropathy responsive to pyridoxal 5'-phosphate supplementation.

OBJECTIVE: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. METHODS: We performed genome-wide sequencing, homozygosity mapping, and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification. RESULTS: We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP-binding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5'-phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization. INTERPRETATION: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels. ANN NEUROL 2019;86:225-240.

Bacillus subtilis CBR05 induces Vitamin B6 biosynthesis in tomato through the de novo pathway in contributing disease resistance against Xanthomonas campestris pv. vesicatoria.

Expression profiling for genes involved in Vitamin B6 (VitB6) biosynthesis was undertaken to delineate the involvement of de novo and salvage pathway induced by Bacillus subtilis CBR05 against, Xanthomonas campestris pv. vesicatoria in tomato. Pyridoxine biosynthesis (PDX) genes such as PDX1.2 and PDX1.3, were found to be overexpressed significantly at 72 hpi in B. subtilis and pyridoxine inoculated plants. Most significant upregulation was observed in the transcript profile of PDX1.3, which showed more than 12- fold increase in expression. Unfortunately, salt sensitive overlay4 (SOS4) profiling showed irregular expression which corroborates that SOS4 role in VitB6 biosynthesis needs further studies for deciphering a clear notion about their role in tomato. Antioxidant enzymes i.e., superoxide dismutase, catalase, polyphenol oxidase, and peroxidase activities clearly demonstrate escalation till 48 hpi and gets reduced in 72 hpi. Pot trials also confirm that B. subtilis compared to pyridoxine supplementation alone show plant disease resistance and elongated roots. The present study confirms that B. subtilis, as a versatile agent in eliciting induced systemic resistance regulated by de novo pathway as a model for plant defense against X. campestris pv. vesicatoria substantiated by VitB6 biosynthesis. Nevertheless, the study is preliminary and needs further evidence for affirming this phenomenon.

Expression analysis of a novel pyridoxal kinase messenger RNA splice variant, PKL, in oil rape suffering abiotic stress and phytohormones.

Pyridoxal kinase is key enzyme for the biosynthesis of pyridoxal 5'-phosphate, the biologically active form of vitamin B6, in the salvage pathway. A pyridoxal kinase gene, BnPKL (GenBank accession No. DQ463962), was isolated from oilseed rape (Brassica napus L.) following water stress through rapid amplification of complementary DNA (cDNA) ends. The results showed that the gene had two splice variants: PKL and PKL2. PKL, the long cDNA, encodes a 334 amino acid protein with a complete ATP-binding site, pyridoxal kinase-binding site and dimer interface site of a pyridoxal kinase, while PKL2, the short cDNA, lacked a partial domain. Southern blot showed that there were two copies in Brassica napus. The expression of BnPKL cDNA could rescue the mutant phenotype of Escherichia coli defective in pyridoxal kinase. Real-time reverse transcription-polymerase chain reaction revealed that the relative abundance of two transcripts are modulated by development and environmental stresses. Abscisic acid and NaCl were inclined to decrease PKL expression, but H2O2 and cold temperatures induced the PKL expression. In addition, the PKL expression could be transiently induced by jasmonate acid at an early stage, abscisic acid, salicylic acid and jasmonate acid enhanced the PKL expression in roots. Our results demonstrated that BnPKL was a pyridoxal kinase involved in responses to biotic and abiotic stresses.

Bombyx mori pyridoxal kinase cDNA cloning and enzymatic characterization.

Pyridoxal kinase (PLK) (EC 2.7.1.35) catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5.-phosphate (PLP), an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method (GenBank accession number: DQ452397). The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 kDa. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.

The pyridoxal kinase gene TaPdxK from wheat complements vitamin B6 synthesis-defective Escherichia coli.

Pyridoxal kinase (EC 2.7.1.35) is a key enzyme in the conversion of vitamin B6 to pyridoxal 5'-phosphate (PLP). PLP is the crucial cofactor required by numerous enzymes involved in amino acids metabolism. Recently, studies with Arabidopsis salt overly sensitive 4 mutants demonstrated that pyridoxal kinase is a novel salt tolerance determinant important for the regulation of Na+ and K+ homeostasis in plants. We describe here the TaPdxK gene which encodes a pyridoxal kinase, cloned from Triticum aestivum by RACE PCR method. The putative amino acid sequence of TaPdxK is 78% identical to Arabidopsis AtSOS4. Southern analysis suggests that there are at least two copies of pyridoxal kinase genes in wheat genome. The expression of TaPdxK cDNAs complements an Escherichia coli mutant defective in pyridoxal kinase. TaPdxK transcripts were detected in roots, shoots, spikes and anthers by RT-PCR analysis. TaPdxK expression level was not regulated by salt, ABA, and osmotic stress.

Expression of a novel pyridoxal kinase mRNA splice variant, PKH-T, in human testis.

AIM: To identify the genes specifically expressed in human adult and fetal testes and spermatozoa. METHODS: A human testis cDNA microarray was established. Then mRNAs of human adult and fetal testis and spermatozoa were purified and probes were prepared by a reverse transcription reaction with mRNA as the template. The microarray was hybridized with probes of adult and fetal testes and spermatozoa. The nucleic acid sequences of differentially expressed genes were determined and homologies were searched in the databases of GenBank. RESULTS: A novel human testis-specific gene, PKH-T, was identified by hybridizing adult and fetal testis and spermatozoa probes with a human testis cDNA microarray. The cDNA of PKH-T was 1 069 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AY303972) and PKH-T was also determined as Interim GenSymbol (Unigene, HS.38041). PKH-T contained most PKH conserved motif. The 239 amino acid sequences deduced from the 719 bp open reading frame (ORF) had a homology with the gene PKH (U89606). PKH-T was specifically and strongly expressed in the testis. Comparison of the differential expressions of PKH and PKH-T in testes of different developmental stages indicated that PKH-T was expressed in the adult testis and spermatozoa, while PKH, in the adult, fetal and aged testes. PKH-T had no expression in the testis of Sertoli cell only and partially spermatogenic arrest patients. CONCLUSION: PKH-T is a gene highly expressed in adult human testis and spermatozoa. It may play an important role in spermatogenesis and could be related to male infertility.

The Arabidopsis salt overly sensitive 4 mutants uncover a critical role for vitamin B6 in plant salt tolerance.

Salt stress is a major environmental factor influencing plant growth and development. To identify salt tolerance determinants, a genetic screen for salt overly sensitive (sos) mutants was performed in Arabidopsis. We present here the characterization of sos4 mutants and the positional cloning of the SOS4 gene. sos4 mutant plants are hypersensitive to Na(+), K(+), and Li(+) ions. Under NaCl stress, sos4 plants accumulate more Na(+) and retain less K(+) compared with wild-type plants. SOS4 encodes a pyridoxal kinase that is involved in the biosynthesis of pyridoxal-5-phosphate, an active form of vitamin B6. The expression of SOS4 cDNAs complements an Escherichia coli mutant defective in pyridoxal kinase. Supplementation of pyridoxine but not pyridoxal in the growth medium can partially rescue the sos4 defect in salt tolerance. SOS4 is expressed ubiquitously in all plant tissues. As a result of alternative splicing, two transcripts are derived from the SOS4 gene, the relative abundance of which is modulated by development and environmental stresses. Besides being essential cofactors for numerous enzymes, as shown by pharmacological studies in animal cells, pyridoxal-5-phosphate and its derivatives are also ligands for P2X receptor ion channels. Our results demonstrate that pyridoxal kinase is a novel salt tolerance determinant important for the regulation of Na(+) and K(+) homeostasis in plants. We propose that pyridoxal-5-phosphate regulates Na(+) and K(+) homeostasis by modulating the activities of ion transporters.

Porcine pyridoxal kinase c-DNA cloning, expression and confirmation of its primary sequence.

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.