Thioredoxin regulates G6PDH activity by changing redox states of OpcA in the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120

Abstract

Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first reaction in the oxidative pentose phosphate pathway. In green plant chloroplasts, G6PDH is a unique redox-regulated enzyme, since it is inactivated under the reducing conditions. This regulation is accomplished using a redox-active cysteine pair, which is conserved in plant G6PDH. The inactivation of this enzyme under conditions of light must be beneficial to prevent release of CO from the photosynthetic carbon fixation cycle. In the filamentous, heterocyst-forming, nitrogen-fixing cyanobacterium sp. PCC 7120 ( 7120), G6PDH plays a pivotal role in providing reducing power for nitrogenase, and its activity is also reported to be suppressed by reduction, though G6PDH does not conserve the critical cysteines for regulation. Based on the thorough analyses of the redox regulation mechanisms of G6PDH from 7120 and its activator protein OpcA, we found that -type thioredoxin regulates G6PDH activity by changing the redox states of OpcA. Mass spectrometric analysis and mutagenesis studies indicate that Cys and Cys of OpcA are responsible for the redox regulation property of this protein. Moreover, analyses of the redox states of OpcA showed that more than half of the OpcA is present as an oxidized form, even under conditions of light, when cells are cultured under the nitrogen-fixing conditions. This redox regulation of OpcA might be necessary to provide reducing power for nitrogenase by G6PDH in heterocysts even during the day.

Characterization of TrxC, an Atypical Thioredoxin Exclusively Present in Cyanobacteria

“…Other key residues in thioredoxins are also conserved such as phenylalanines in the N-terminal part of the sequence (F39 and F54 in Figure 1 A) or an aspartate that is located opposite to the active site (D89 in Figure 1 A), although only one of the two conserved glycines in the C-terminal part of the protein are conserved (G112 in Figure 1 A) [ 1 ]. The biochemical characterization of the protein has shown that Synechocystis ’ TrxC is inactive in two classical thioredoxin activity assays ( Figure 1 B,C), in agreement with the data available for Anabaena ’s TrxC that is unable to reduce OpcA [ 21 ]. Although there are no known thioredoxin that present a similar active site to the one in TrxC, site directed mutagenesis of the proline in the active site of E. coli Trx1 (P34H) or Staphylococcus aureus Trx (P31T or P31S) changed the redox potential of these proteins to more oxidizing [ 1 , 35 , 36 , 37 ].…”

“…More recently, mutants in trxM genes have been described in Anabaena which possess two genes coding for Trx m : trxM1 and trxM2 . Both genes are dispensable although trxM1 mutant showed a pleiotropic phenotype and was unable to grow in diazotrophic conditions, while trxM2 lack any appreciable phenotype [ 20 , 21 ]. In plants trxM are involved in several functions related to fluctuating light conditions, cyclic electron flow or meristem maintenance [ 4 , 5 , 22 ].…”

“…No activity or function has been ascribed to this class of Trx in Synechocystis as mutants lacking it did not show any phenotype [ 25 ], although Anabaena trxC − mutant showed more oxidative stress than WT in nitrate grown cultures [ 20 ]. Furthermore, Anabaena TrxC seems to be inactive in reducing OpcA or G6PDH [ 21 ]. Here we describe the characterization of Synechocystis ’ TrxC protein and of mutants either lacking trxC or overexpressing it.…”

Interaction of pretilachlor with PS‐II activity of the cyanobacterium Desmonostoc muscorum PUPCCC 405.10

“…In cyanobacteria, the dissimilation of fixed carbon occurs through the oxidative pentose phosphate pathway with G6PDH as its first enzyme [18,41,42]. Pretilachlor significantly decreased the activity of G6PDH (Table 3).…”

“…Pretilachlor significantly decreased the activity of G6PDH (Table 3). The regulation of G6PDH has been suggested to be at the level of intermediate metabolites of photosynthesis such as glucose‐6‐phosphate, ribulose‐1, 5‐biphosphate, NADH, and ATP [42,43]. The inhibition of PS‐II by pretilachlor resulted in the less CO 2 fixation, leading to downregulation of G6PDH.…”