Identification of an Escherichia coli O157:H7 heme oxygenase with tandem functional repeats

Abstract

Heme oxygenases (HOs) catalyze the oxidation of heme to biliverdin, carbon monoxide (CO), and free iron. Iron acquisition is critical for invading microorganisms to enable survival and growth. Here we report the crystal structure of ChuS, which displays a previously uncharacterized fold and is unique compared with other characterized HOs. Despite only 19% sequence identity between the N-and C-terminal halves, these segments of ChuS represent a structural duplication, with a root-mean-square deviation of 2.1 Å between the two repeats. ChuS is capable of using ascorbic acid or cytochrome P450 reductase-NADPH as electron sources for heme oxygenation. CO detection confirmed that ChuS is a HO, and we have identified it in pathogenic Escherichia coli O157:H7. Based on sequence analysis, this HO is present in many bacteria, although not in the E. coli K-12 strain. The N-and C-terminal halves of ChuS are each a functional HO.ChuS ͉ carbon monoxide ͉ structural repeat ͉ heme degredation

Structure of the Escherichia coli O157:H7 Heme Oxygenase ChuS in Complex with Heme and Enzymatic Inactivation by Mutation of the Heme Coordinating Residue His-193

“…The spectral shifts observed for ChuS and H73A are similar to those observed for other HOs, which suggest the formation of biliverdin and free iron rather than the Fe 3ϩ -biliverdin complex formed by HO-1 (35). Because the addition of catalase and superoxide dismutase did not affect the trends of the spectral change, the observed activity of ChuS can be attributed only to specific heme degradation (6).…”

“…Since His-73 is fully conserved but does not interact with heme, it provides a good control for His-193. As previously reported, the absorbance spectrum of native ChuS exhibits a clear Soret peak at 410 nm together with the common characteristic ␣/␤-bands of HOs around 550 and 580 nm (6,15,25). The H73A mutation was not observed to instigate any spectral changes when compared with native ChuS.…”

“…Based on the complex structure and sequence conservation analysis, the mutations of H73A and H193N of ChuS were selected (6). Using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), wildtype ChuS was mutated at these two histidine positions using the following primers, where base pair changes have been underlined: H73A, sense, 5Ј-CGTAATGAATATGCAGTC-GCGGAGCAAGTTGGTACG-3Ј, antisense, 5Ј-CGTACCAA-CTTGCTCCGCGACTGCATATTCATTACG-3Ј, and H93N, sense, 5Ј-GGGCGATGACCGACGTTAATCAGTTTTTTA-CGTTGCTCAAG-3Ј, antisense, 5Ј-GAGCAACGTAAAAAA-CTGATTAACGTCGGTCATCGCCC-3Ј.…”

“…We recently reported the crystal structure of apo-ChuS and confirmed via spectral analysis and CO measurement that ChuS was an HO, 3 the first to be identified in any strain of E. coli (6). A Shigella dysenteriae chromosomal knock-out of shuS, a homologue to chuS, was shown to be defective in utilizing heme as an iron source, and expression of ShuS was up-regulated when challenged with iron-limiting conditions (7).…”

Unique coupling of mono- and dioxygenase chemistries in a single active site promotes heme degradation

“…1A) (9,10). This mechanism, which passes through both hydroxyheme and verdoheme intermediates, has been considered the "gold standard" of heme degradation even for recently identified noncanonical HO-type enzymes (11)(12)(13). We have discovered, however, that MhuD cleaves the porphyrin ring without releasing CO, which is a clear indication of a distinct mechanism, without producing the verdoheme intermediate (14).…”