Rhodobacter sphaeroides has a family II pyrophosphatase: comparison with other species of photosynthetic bacteria

Abstract

The cytoplasmic pyrophosphatase from Rhodobacter sphaeroides was purified and characterized. The enzyme is a homodimer of 64 kDa. The N-terminus was sequenced and used to obtain the complete pyrophosphatase sequence from the preliminary genome sequence of Rba. sphaeroides, showing extensive sequence similarity to family II or class C pyrophosphatases. The enzyme hydrolyzes only Mg-PP(i) and Mn-PP(i) with a K(m) of 0.35 mM for both substrates. It is not activated by free Mg (2+), in contrast to the cytoplasmic pyrophosphatase from Rhodospirillum rubrum, and it is not inhibited by NaF, methylendiphosphate, or imidodiphosphate. This work shows that Rba. sphaeroides and Rhodobacter capsulatus cytoplasmic pyrophosphatases belong to family II, in contrast to Rsp. rubrum, Rhodopseudomonas palustris, Rhodopseudomonas gelatinosa, and Rhodomicrobium vannielii cytoplasmic pyrophosphatases which should be classified as members of family I. This is the first report of family II cytoplasmic pyrophosphatases in photosynthetic bacteria and in a gram-negative organism.

The inorganic pyrophosphatases of microorganisms: a structural and functional review

“…Additionally, the authors discuss the possibility that the enzyme is enclosed in an organelle. In a follow-up study, McLaughlin, Lindmark & Müller (1978b) demonstrated that the enzyme is heat stable, which is consistent with other family I c-PPases and the temperature sensitivity observed in some family II examples ( Klemme & Gest, 1971 ; Romero, García-Contreras & Celis, 2003 ; Celis et al, 2003 ). However, the presence of this enzyme in the membrane was intriguing.…”

Comparative biochemical and functional studies of family I soluble inorganic pyrophosphatases from photosynthetic bacteria

“…capsulatus and Rhodop. sphaeroides sPPases have already been described as family II enzymes [21]. It can be speculated that this could be a singular case of horizontal gene transfer in photosynthetic prokaryotes, as it has been shown that marine unicellular cyanobacteria possess two paralogous ppa genes of different phylogeny; one of them, similar to the proteobacterial homologs, was probably obtained by horizontal gene transfer [22] (see below).…”

Family II pyrophosphatases from photosynthetic bacteria can hydrolyze free pyrophosphate

“…capsulatus, and Rdv. sulfidophillus family II pyrophosphatases were partially purified as reported by Celis et al 10 Construction of an Escherichia coli strain that overproduces his 6 -sppase from Rba. sphaeroides…”

“…rubrum cannot hydrolyze pyrophosphate with Mn 2+ , expressing their maximal hydrolytic rate with Mg 2+ , and they certainly cannot function if the reaction medium does not contain divalent cations. 10 The amount of hydrolyzed pyrophosphate by the partially purified Rs-sPPase when divalent cations were not added to the reaction medium (1,080.56 nmol Pi -1 ⋅min -1 [mg of protein] -1 ) was equivalent to the amount obtained when Mg 2+ was added (1,100.5 nmol Pi -1 ⋅min -1 [mg of protein] -1 ; Table 1); this suggests the existence of divalent cations bound to the enzyme that may form the metal-substrate complex. Based on the above assumption, another two partially purified family II pyrophosphatases from the photosynthetic bacteria Rba.…”