Characterization of the first angiotensin-converting like enzyme in bacteria: Ancestor ACE is already active

Abstract

Angiotensin-converting enzyme (ACE) is a metallopeptidase that converts angiotensin I into angiotensin II. ACE is crucial in the control of cardiovascular and renal homeostasis and fertility in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two active sites, have been characterized. So far, only soluble, single domain ACEs from invertebrates have been cloned, and these have been implicated in reproduction in insects. Furthermore, an ACE-related carboxypeptidase was recently characterized in Leishmania, a unicellular eukaryote, suggesting the existence of ACE in more distant organisms. Interestingly, in silico databank analysis revealed that bacterial DNA sequences could encode putative ACE-like proteins, strikingly similar to vertebrates' enzymes. To gain more insight into the bacterial enzymes, we cloned the putative ACE from the phytopathogenic bacterium, Xanthomonas axonopodis pv. citri, named XcACE. The 2 kb open reading frame encodes a 672-amino-acid soluble protein containing a single active site. In vitro expression and biochemical characterization revealed that XcACE is a functional 72 kDa dipeptidyl-carboxypeptidase. As in mammals, this metalloprotease hydrolyses angiotensin I into angiotensin II. XcACE is sensitive to ACE inhibitors and chloride ions concentration. Variations in the active site residues, highlighted by structural modelling, can account for the different substrate selectivity and inhibition profile compared to human ACE. XcACE characterization demonstrates that ACE is an ancestral enzyme, provoking questions about its appearance and structure/activity specialisation during the course of evolution.

Fig. 1

Homology of the bacterial ACE related to various ACEs in the phylogenetic tree. (A): Percentage of identity in amino acids between XcACE and various ACE-like enzymes primary sequences described or deduced from genomic sequences: Ndom, human N-domain ACE (gi: 4732026, residues 1–583); Cdom, human C-domain ACE (gi: 4732026, residues 641–1306); ACE2, human ACE2 (gi: 71658783); ACEr, Drosophila melanogaster ACEr (gi: 73921650); X camp, Xanthomonas campestris pv. campestris predicted dipeptidyl-carboxypeptidase (gi: 66769433); E lito, Erythrobacter litoralis predicted dipeptidyl-carboxypeptidase (gi: 61100845); G viol, Gloeobacter violaceus predicted dipeptidyl-carboxypeptidase (gi: 37522712). (B): unrooted cladogram indicating evolutionary relationships between the different ACE homologues among the living kingdom.

Fig. 2

RT-PCR assay of the XcACE transcript. cDNA was generated and submitted to PCR (cycling parameters: 94 °C, 2 min; 94 °C, 45 s, 61 °C, 30 s, 72 °C, 1 min, for 25, 30 or 40 cycles, 72 °C, 4 min).Treatments of the different samples are indicated above the corresponding lane. The size of the expected band is indicated. L: DNA molecular weight marker, W: water control. The number of PCR cycles is indicated under each sample.

Fig. 3

Semi-purification and Western blot of XcACE. (A): FPLC profile of the last purification step (see Section 2.8.1), absorbance at 280 nm (full line), the gradient (dashed line) was from 0 to 50 mS/cm, arbitrary units of ACE activity (dotted line), the horizontal bar indicates the fractions pooled for further analysis. (B): Native and recombinant XcACE were analysed by SDS-PAGE followed by Western blot with the antiserum HKCE. Lane a, molecular-mass standards; lane b, human somatic ACE; lane c, native XcACE; lane d, recombinant XcACE. The band corresponding to XcACE is indicated (arrow).

Fig. 4

Biochemical characterization of XcACE. Hydrolysis of various ACE substrates by native (nXcACE) (A–D) and recombinant (rXcACE) (E). Hydrolysis of angiotensin I (A), hydrolysis of HHL (B), AcSDAcKP (C), [Leu5]-enkephalin (D) and [Leu5]-enkephalinamide (E) by native XcACE. Hydrolysis of angiotensin I by recombinant XcACE (F). The specificity of the reactions was assessed in the presence of 10 μM captopril (dashed line). AngI, angiotensin I; AngII, angiotensin II; AH, hippuric acid; YGG, tyrosyl-glycyl-glycine. Chloride sensitivity of XcACE (F). Effect of chloride ions concentration on HHL hydrolysis by native (◇) XcACE, C-(ж) and N-domain (▲) of human somatic ACE.

Fig. 5

Comparison of the charge of the active site between tACE and XcACE. ACE is represented as a surface with the positive charge indicated in blue and the negative in red. The zinc atom is represented (green), and some of the residues are indicated. Lisinopril (in yellow) is shown to orientate the active site. A, The active site viewed as a surface from the lysyl pocket for tACE (left) and XcACE (right). B, The active site cavity viewed from the outside for tACE (left) and XcACE (right). tACE is shown to have a higher negative charge in the lysyl binding pocket.

Fig. 6

XcACE activity during bacterial growth. Follow-up of bacterial growth by O.D. at 600 nm (◇) and ACE activity (nmoles hippuric acid generated) in culture medium (□) and within bacteria (○). Error bars were omitted for clarity.