Raw starch-degrading α-amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme.

AIMS: The aims were to isolate a raw starch-degrading α-amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli. METHODS AND RESULTS: A 1539 complete open reading frame of a starch-degrading α-amylase gene baqA from B. aquimaris MKSC 6·2 has been determined by employing PCR and inverse PCR techniques. Bioinformatics analysis revealed that B. aquimaris MKSC 6.2 α-amylase (BaqA) has no starch-binding domain, and together with a few putative α-amylases from bacilli may establish a novel GH13 subfamily most closely related to GH13_1. Two consecutive tryptophans (Trp201 and Trp202, BaqA numbering) were identified as a sequence fingerprint of this novel GH13 subfamily. Escherichia coli cells produced the recombinant BaqA protein as inclusion bodies. The refolded recombinant BaqA protein degraded raw cassava and corn starches, but exhibited no activity with soluble starch. CONCLUSIONS: A novel raw starch-degrading B. aquimaris MKSC 6.2 α-amylase BaqA is proposed to be a member of new GH13 subfamily. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has contributed to the overall knowledge and understanding of amylolytic enzymes that are able to bind and digest raw starch directly.

Mitochondrial DNA polymorphism in disease: a possible contributor to respiratory dysfunction.

Intergenomic variation in the human mitochondrial genome was examined in 27 mtDNA sequences using a pairwise analysis technique. Analysis of 16 of these mtDNA sequences from patients with mitochondrial cytopathies indicated a wide range between different mitochondrial genes in the degree of nucleotide variation from the standard Cambridge sequence. Mean complex I polymorphic frequencies in cytopathic (CPEO, MERRF, MELAS and LHON collectively) patients and in LHON patients differed significantly from controls (P < or = 0.05, t). Total mean sequence divergence (mean number of diverging nucleotides between two sequences per 100 bp) over the entire mtDNA coding region was 0.21% for cytopathies (n = 16) as opposed to 0.18% for a control group (n = 4). Within the cytopathy group, the greatest pairwise divergence was observed in ND3 and ND6 subunits of complex I (0.46 and 0.70% respectively) and the magnitude of specific gene divergences differed considerably from those observed for the corresponding genes in the control population. The extent to which the increased variation in ND3 and ND6 is a general phenomenon applicable to all subjects rather than a finding specific to cytopathies cannot be stated with certainty given the small control group. Regardless as to which of these suggestions is correct, the possibility exists that increased nucleotide variation in certain mitochondrial ND subunits may contribute to respiratory inefficiency through a cumulative effect of a series of polymorphisms of minor individual mutagenic potential.

Tissue segregation of a heteroplasmic mtDNA mutation in MERRF (myoclonic epilepsy with ragged red fibers) encephalomyopathy.

The distribution of the causal 8344A-->G mtDNA mutation has been examined in six tissues of a patient with myoclonic epilepsy with ragged red fibers (MERRF), to study the developmental genetics of this type of mitochondrial disorder, and to determine the pathophysiological importance of the mtDNA heteroplasmy generally observed in such patients. Heteroplasmy of the mtDNA was observed in all six tissues (cerebellum, cerebrum, pancreas, liver, muscle, and heart) suggesting that, whereas the mtDNA mutation is relatively new, the mutated population must have existed before the formation of the three primary embryonic layers. The tissue distribution reveals significant variations in the ratio between the mutated and the normal mtDNA species, indicating the randomness of mtDNA segregation during developmental cell division and differentiation events. The result suggests the existence of tissue-specific nuclear factor(s) that determines the expression of the 8344A-->G mutation in various tissues; in MERRF syndrome, expression is mainly in the central nervous system.

A new disease-related mutation for mitochondrial encephalopathy lactic acidosis and strokelike episodes (MELAS) syndrome affects the ND4 subunit of the respiratory complex I.

The molecular lesions in two patients exhibiting classical clinical manifestations of MELAS (mitochondrial encephalopathy, lactic acidosis, and strokelike episodes) syndrome have been investigated. A recently reported disease-related A----G base substitution at nt 3243 of the mtDNA, in the DHU loop of tRNA(Leu), was detected by restriction-enzyme analysis of the relevant PCR-amplified segment of the mtDNA of one patient but was not observed, by either restriction-enzyme analysis or nucleotide sequencing, in the other. To define the molecular lesion in the patient who does not have the A----G base substitution at nt 3243, the total mitochondrial genome of the patient has been sequenced. An A----G base substitution at nt 11084, leading to a Thr-to-Ala amino acid replacement in the ND4 subunit of the respiratory complex I, is suggested to be a disease-related mutation.

Antipeptide antibodies to the carboxy terminal and the DCCD binding region of the human mitochondrial ATP synthase beta-subunit.

Antibodies to defined epitopes on the human ATP synthase would provide a powerful tool in the definition of the subunit composition of the enzyme complex and in the characterization of any defect in its assembly in diseases associated with mitochondrial disorders. Antibodies have been thus raised against synthetic peptides, corresponding to two regions on the human ATP synthase beta-subunit: the C-terminal region, and a region which includes the two dicyclohexylcarbodiimide (DCCD)-reactive glutamic acid residues suggested to be involved in the enzyme catalytic activity. The antibodies to the C-terminal peptide reacted with the ATP synthase beta-subunit in ELISA, in Western immunoblotting and in immunohistochemical experiments, and had the ability to immunoprecipitate the enzyme complex. The antibodies to the DCCD-binding region peptide did not react to the ATP synthase beta-subunit in its native configuration, although reacted well under Western immunoblotting conditions.

Normal variants of human mitochondrial DNA and translation products: the building of a reference data base.

A good standard reference for the highly polymorphic human mitochondrial DNA (mtDNA) sequence is essential for studies of normal and disease-related nucleotide variants in the mitochondrial genome. A consensus sequence for the human mitochondrial genome has been derived from thirteen unrelated mtDNA sequences. We report 128 nucleotide variants of the human mtDNA sequence, and 62 amino acid variants of the human mitochondrial translation products, observed in the coding region of these mtDNA sequences.

A tRNA(Lys) mutation in the mtDNA is the causal genetic lesion underlying myoclonic epilepsy and ragged-red fiber (MERRF) syndrome.

Skeletal muscle mtDNA of three patients with mitochondrial encephalomyopathy, characterized clinically by myoclonic epilepsy and ragged-red fiber (MERRF) syndrome, has been sequenced to determine the underlying molecular defect(s). An A-to-G substitution of nt 8344 in the tRNA(Lys) gene, a substitution suggested to be associated with MERRF encephalomyopathy, was detected in these patients. Abnormal patterns of mitochondrial translation products were observed in the skeletal muscle of patients, consistent with the expected consequential defect in protein synthesis. The genealogical studies of the three patients, as well as mtDNA from one published MERRF patient and from nine other normal and disease controls, revealed that the tRNA(Lys) mutations in the MERRF patients have arisen independently. These observations provided evidence that the base substitution is a causal mutation for MERRF.

The association of the autoantigens of primary biliary cirrhosis with the mitochondrial H+-ATPase--a reassessment.

The claimed association between the M2 autoantigens of primary biliary cirrhosis (PBC) and the mitochondrial H+-ATPase has been re-examined in view of the recent reports that PBC autoantibodies react specifically with the lipoate acetyl transferases of 2-oxo acid dehydrogenases. Study of F0F1-ATPase purified from human and yeast mitochondria, and the comparison between immunoprecipitates obtained with antibodies against the H+-ATPase beta subunit and anti-M2 antibodies of PBC, established that the M2 antigens are not associated with the H+-ATPase complex. The M2 antigens did copurify with a crude bovine heart F1-ATPase preparation, but not with F1-ATPase from yeast, human heart or human liver.