Characterization of sodium/proton antiporter and betaine-homocysteine methyltransferase of a halotolerant cyanobacterium Aphanothece halophytica

Abstract

The coding region of the N a+/H+ antiporter gene from a halotolerant cyanobacterium Aphanothece halophytica, apnhaP, was isolated by Inverse Polymerase Chain Reaction (PCR) technique. The predicted gene product consists of 521 amino acids with a molecular mass of 56,881 Da. Analysis of the hydropathy plot and the transmembrane prediction program predicted 11 putative transmembrane segments in ApNhaP. It turned out that A. halophytica contains an N a+/H+ antiporter homologous to plants (SOS1 and AtNHX1 from Arabidopsis), mammalians (NHEs from human), and some bacteria (nhaP from Pseudomonas and synnhaP from Synechocystis), but with novel ion specificity. Its gene product, ApNhaP, exhibited the N a+/H + antiporter activity over a wide range of pH betw een 5 and 9 and complemented the N a+-sensitive mutant E. coli TO114 (∆nhaA∆nhaB∆chaA). The ApNhaP had virtually no activity of Li+/H + antiporter, but showed high Ca2+/H + antiporter activity at alkaline pH. The chimeric gene which encodes the N-terminal transmembrane region of apnhaP and the C-terminal cytosolic region of synnhaP and vice versa was constructed. Its gene products, ASNhaP and SANhaP , were examined for antiporter activity compared with two parental types (ApNhaP and SynNhaP). Interestingly, the Li+/H+ exchange activity which was virtually non-detectable in ApNhaP could be clearly observed in the ASNhaP chimeric protein. SANhaP chimeric protein showed reduced exchange activity of both Na+/H+ and Li+/H+ antiporter activity compared to the parental SynNhaP. These results suggest that the ion specificity of an N a+/H + antiporter is partly determined by the structural properties of the C-terminal tail which was well exemplified in the case o f A. halophytica. The degradation of glycinebetaine in A. halophytica was also investigated in this study. It was found that glycinebetaine degradation occurred when cells were subjected to hypoosmotic stress (salt downshock) and this degradation was caused by the activity of betaine-hom ocysteine methyltransferase (BHMT). The intracellular glycinebetaine level was decreased approximately 50% after salt downshock from 2.0 M to 0.5 M NaCI medium. The activity of BHM T increased from 0 to 460 nmol h-1 mg-1 after 3 h of salt downshock. Small increase of the enzyme activity was also observed when A. halophytica was deprived of carbon and nitrogen sources in the growth medium. BHMT was purified to homogeneity by hydroxyapatite, DEAE-Sepharose CL-6B and Sephadex G -200 column chromatography. A 24-fold purification and 11% overall yield were achieved with a specific activity of 595 nmol h-1 mg-1. The subunit molecular mass was determined to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS -PAGE), and the native enzyme was found to have a molecular mass of 350 kDa, suggesting an octameric structure of the enzyme. The enzyme showed optimum activity at 37℃, pH 7.5. The apparent Km values for glycinebetaine and L-homocysteine were 4.3 mM and 1.3 mM, respectively. The enzyme was 70% inactivated by 5 mM dimethylglycine whereas the same concentration of sarcosine slightly inactivated the enzyme. Two analogs of glycinebetaine were also tested for enzyme inactivation and it was found that 5 mM choline inactivated 60% of the enzym e activity and 2.5 mM betaine aldehyde completely abolished the enzyme activity. NaCI at 200 mM or higher also completely inactivated the enzyme.