Another unusual type of citric acid cycle enzyme in Helicobacter pylori: the malate:quinone oxidoreductase

Abstract

The only enzyme of the citric acid cycle for which no open reading frame (ORF) was found in the Helicobacter pylori genome is the NAD-dependent malate dehydrogenase. Here, it is shown that in this organism the oxidation of malate to oxaloacetate is catalyzed by a malate:quinone oxidoreductase (MQO). This flavin adenine dinucleotide-dependent membrane-associated enzyme donates electrons to quinones of the electron transfer chain. Similar to succinate dehydrogenase, it is part of both the electron transfer chain and the citric acid cycle. MQO activity was demonstrated in isolated membranes of H. pylori. The enzyme is encoded by the ORF HP0086, which is shown by the fact that expression of the HP0086 sequence from a plasmid induces high MQO activity in mqo deletion mutants of Escherichia coli or Corynebacterium glutamicum. Furthermore, this plasmid was able to complement the phenotype of the C. glutamicum mqo deletion mutant. Interestingly, the protein predicted to be encoded by this ORF is only distantly related to known or postulated MQO sequences from other bacteria. The presence of an MQO shown here and the previously demonstrated presence of a 2-ketoglutarate:ferredoxin oxidoreductase and a succinyl-coenzyme A (CoA):acetoacetyl-CoA transferase indicate that H. pylori possesses a complete citric acid cycle, but one which deviates from the standard textbook example in three steps.

FIG. 1

Tree reflecting the similarity between MQOs from different organisms. Bar, expected change of 0.1 per amino acid. For details of the analysis, see Materials and Methods.

FIG. 2

Complementation of the phenotype of an mqo deletion mutant of C. glutamicum by ORF HP0086 expressed from a plasmid. The C. glutamicum wild-type (Wt), Δmqo, and Δmqo/pHp-mqo strains were plated on minimal medium agar containing 1% (wt/vol) glucose. In the case of strain Δmqo/pHp-mqo the plate also contained 25 μg of kanamycin ml⁻¹. The plates were incubated for 60 h at 30°C.

FIG. 3

Tentative scheme for the citric acid cycle of H. pylori based on genome sequence data and biochemical data. Unusual enzymes are labeled with an asterisk. Pyr, pyruvate; AcCoA, acetyl-CoA; Cit, citrate; Icit, isocitrate; Kg, 2-ketoglutarate; Suc-CoA, succinyl-CoA; Suc, succinate; Fum, fumarate; Mal, malate; Oaa, oxaloacetate; Fd_ox and Fd_red: oxidized and reduced ferredoxin, respectively; MQ and MQH₂, oxidized and reduced menaquinone, respectively. Enzymes: 1, pyruvate:ferredoxin oxidoreductase; 2, citrate synthase; 3, aconitase; 4, isocitrate dehydrogenase; 5, α-ketoglutarate:ferredoxin oxidoreductase; 6, succinyl-CoA acetoacetyl-CoA transferase; 7, fumarate reductase (SDH); 8, fumarase; 9, malate:quinone oxidoreductase. EC numbers and corresponding H. pylori genes of all enzymes except the MQO can be found elsewhere (19).