A Highly Stable Plastidic-Type Ferredoxin-NADP(H) Reductase in the Pathogenic Bacterium Leptospira interrogans

Abstract

Leptospira interrogans is a bacterium that is capable of infecting animals and humans, and its infection causes leptospirosis with a range of symptoms from flu-like to severe illness and death. Despite being a bacteria, Leptospira interrogans contains a plastidic class ferredoxin-NADP(H) reductase (FNR) with high catalytic efficiency, at difference from the bacterial class FNRs. These flavoenzymes catalyze the electron transfer between NADP(H) and ferredoxins or flavodoxins. The inclusion of a plastidic FNR in Leptospira metabolism and in its parasitic life cycle is not currently understood. Bioinformatic analyses of the available genomic and proteins sequences showed that the presence of this enzyme in nonphotosynthetic bacteria is restricted to the Leptospira genus and that a [4Fe-4S] ferredoxin (LB107) encoded by the Leptospira genome may be the natural substrate of the enzyme. Leptospira FNR (LepFNR) displayed high diaphorase activity using artificial acceptors and functioned as a ferric reductase. LepFNR displayed cytochrome c reductase activity with the Leptospira LB107 ferredoxin with an optimum at pH 6.5. Structural stability analysis demonstrates that LepFNR is one of the most stable FNRs analyzed to date. The persistence of a native folded LepFNR structure was detected in up to 6 M urea, a condition in which the enzyme retains 38% activity. In silico analysis indicates that the high LepFNR stability might be due to robust interactions between the FAD and the NADP+ domains of the protein. The limited bacterial distribution of plastidic class FNRs and the biochemical and structural properties of LepFNR emphasize the uniqueness of this enzyme in the Leptospira metabolism. Our studies show that in L. interrogans a plastidic-type FNR exchanges electrons with a bacterial-type ferredoxin, process which has not been previously observed in nature.

Conversion of fatty aldehydes into alk (a/e)nes by in vitroreconstituted cyanobacterial aldehyde-deformylating oxygenase with the cognate electron transfer system

“…These results indicated that FNRs from the evolutionarily diverse classes of cyanobacteria had different binding affinity for NADPH and catalytic efficiency towards the ferricyanide reduction. In addition, it was observed that the specific activity of FNR using NADPH (375 μM) was 23-fold higher than NADH (2 mM), demonstrating that, like other FNRs [31,34], FNR from PCC7942 also prefers NADPH over NADH.…”

“…Therefore FNR from PCC7942 should have the characteristic FAD absorption spectrum. The UV/vis spectrum of FNR clearly showed two peaks at 385 and 455 nm, and a shoulder at 480 nm, demonstrating that FNR is certainly a flavoprotein (Figure 3A) [31,32]. …”

A new catalytic mechanism of bacterial ferredoxin‐NADP+ reductases due to a particular NADP+ binding mode

“…The NADP + content was determined as the μmoles of NADPH produced by the reduction of NADP + by the dehydrogenase in the presence of glucose‐6‐phosphate (G6P) using a standard NADP + curve. The same analyses were performed with other bacterial reductases as FPR from P. carotovorum (PcFPR) and B. abortus (BaFPR) and with the plastidic type FNR from L. interrogans (LepFNR) 16 and P.sativum (PeaFNR). These FPRs and FNRs were obtained as recombinant proteins in E. coli with N‐terminal polyhistidine tags.…”

“…The nucleotide remains attached to the enzyme after extensive dialysis and gel filtration. To elucidate if this also occurs with other FPRs or FNRs, we analyzed another subclass II FPR from Pectobacterium carotovorum , a subclass IA FPR from Brucella abortus and two different plastidic type FNRs, from P. sativum and Leptospira interrogans 16 . We observed that althought with different extent, all FPRs contain the nucleotide bound.…”

GspD, The Type II Secretion System Secretin of Leptospira, Protects Hamsters against Lethal Infection with a Virulent L. interrogans Isolate

“…Such secreted products include acyltransferases, amylases, kinases, celluloses, pectinases, ADP-ribosylation enzymes, proteases, lipases, and phosphatases, which break down complex biochemical components, thus conferring survival advantages to both pathogenic as well as environmental bacteria [ 17 , 49 , 50 ]. It has been shown that Leptospira synthetizes diverse hydrolytic enzymes, such as hyaluronidase [ 51 ], catalase [ 52 ], lipases [ 53 ], putative cytochrome oxidases [ 54 , 55 ], and others like the virulence modifying proteins (VMPs) [ 56 ] and the putative Quorum sensing (QS) proteins [ 57 ], which might well be secreted by the leptospiral T2S. The presence of a T2S in Leptospira implies its role in metabolism, but it also might well be related to pathogenesis, as it has been reported for other Gram-negatives, such as the Vibrio cholerae CLT toxin [ 58 ], the E. coli LT toxin [ 59 ], the Pseudomonas aeruginosa elastase, exotoxin A, phospholipase C, alkaline phosphatase, and lipases [ 41 , 60 ], as well as other proteins in plant pathogens [ 17 ], which are all secreted through their T2S.…”