Discovery of a gene involved in a third bacterial protoporphyrinogen oxidase activity through comparative genomic analysis and functional complementation

Abstract

Tetrapyrroles are ubiquitous molecules in nearly all living organisms. Heme, an iron-containing tetrapyrrole, is widely distributed in nature, including most characterized aerobic and facultative bacteria. A large majority of bacteria that contain heme possess the ability to synthesize it. Despite this capability and the fact that the biosynthetic pathway has been well studied, enzymes catalyzing at least three steps have remained "missing" in many bacteria. In the current work, we have employed comparative genomics via the SEED genomic platform, coupled with experimental verification utilizing Acinetobacter baylyi ADP1, to identify one of the missing enzymes, a new protoporphyrinogen oxidase, the penultimate enzyme in heme biosynthesis. COG1981 was identified by genomic analysis as a candidate protein family for the missing enzyme in bacteria that lacked HemG or HemY, two known protoporphyrinogen oxidases. The predicted amino acid sequence of COG1981 is unlike those of the known enzymes HemG and HemY, but in some genomes, the gene encoding it is found neighboring other heme biosynthetic genes. When the COG1981 gene was deleted from the genome of A. baylyi, a bacterium that lacks both hemG and hemY, the organism became auxotrophic for heme. Cultures accumulated porphyrin intermediates, and crude cell extracts lacked protoporphyrinogen oxidase activity. The heme auxotrophy was rescued by the presence of a plasmid-borne protoporphyrinogen oxidase gene from a number of different organisms, such as hemG from Escherichia coli, hemY from Myxococcus xanthus, or the human gene for protoporphyrinogen oxidase.

Fig. 1.

Comparison of A. baylyi ADP1 wild-type and ACN1054 cultures. (A) Growth characteristics of wild-type and ACN1054 mutant A. baylyi ADP1. Shown are data for ACN1054 cultures without added heme (▾) and supplemented with exogenous heme (○) and wild-type cells without heme supplementation (•). (B) Growth analysis of ACN1054 with expression of known PPO enzymes. Shown are data on the growth of ACN1054 cells alone (▪) and with plasmid-encoded expression of human PPOX (•), M. xanthus HemY (○), and E. coli HemG (▵). Data for wild-type A. baylyi ADP1 are shown for comparison (▾).

Fig. 2.

Porphyrin accumulation in mutant and wild-type A. baylyi cells. After stimulation of the heme biosynthetic pathway, porphyrin accumulation is clearly seen in ACN1054 cells (left) but not in wild-type cells (right). Fluorescence is visible with exposure to long-wavelength UV light.

Fig. 3.

PPO activity in Acinetobacter crude cell extracts as measured by continuous fluorescence spectroscopy. (A) In vitro PPO assay results are shown for wild-type (•) and ACN1054 (○) extracts, as well as an extract-free control (▾). (B) PPO activity in membrane and soluble fractions of Acinetobacter extracts. Activity levels in the soluble fraction (○), the membrane fraction (▾), an extract-free control (•), and the membrane and soluble fractions together (▵) are shown. Approximately 0.1 mg/ml protein was present in each extract. Proto, protoporphyrin.