Anaerobic Heme Degradation: ChuY Is an Anaerobilin Reductase That Exhibits Kinetic Cooperativity

LaMattina, Joseph W.; Delrossi, Michael; Uy, K.G.; Keul, Nicholas D.; Nix, David B.; Neelam, Anudeep; Lanzilotta, William N.

doi:10.1021/acs.biochem.6b01099

Cited by 21 publications

(36 citation statements)

References 38 publications

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“…HutZ Ep shares moderate homology (50% identity) with E. coli ChuY. However, multiple conserved amino acids in ChuY and its homologues did not appear in HutZ Ep , including some important residues buried within the ChuY dimer interface [42], such as Glu94, Gln126, Thr132, Ser136, and Thr140 (Additional file 1). Furthermore, the spatial structure of HutZ Ep is also different from that of ChuY (Additional file 1); for example, seven α-helices exist in HutZ Ep , but only six α-helices exist in ChuY [20].…”

Section: Resultsmentioning

confidence: 99%

HutZ is required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida

Shi

Fang

Huang

et al. 2019

Vet Res

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Edwardsiella piscicida is a severe fish pathogen. Haem utilization systems play an important role in bacterial adversity adaptation and pathogenicity. In this study, a speculative haem utilization protein, HutZ Ep , was characterized in E. piscicida. hutZ Ep is encoded with two other genes, hutW and hutX, in an operon that is similar to the haem utilization operon hutWXZ identified in V. cholerae. However, protein activity analysis showed that HutZ Ep is probably not related to hemin utilization. To explore the biological role of HutZ Ep , a markerless hutZ Ep in-frame mutant strain, TX01ΔhutZ, was constructed. Deletion of hutZ Ep did not significantly affect bacterial growth in normal medium, in iron-deficient conditions, or in the presence of haem but significantly retarded bacterial biofilm growth. The expression of known genes related to biofilm growth was not affected by hutZ Ep deletion, which indicated that HutZ Ep was probably a novel factor promoting biofilm formation in E. piscicida. Compared to the wild-type TX01, TX01ΔhutZ exhibited markedly compromised tolerance to acid stress and host serum stress. Pathogenicity analysis showed that inactivation of hutZ Ep significantly impaired the ability of E. piscicida to invade and reproduce in host cells and to infect host tissue. In contrast to TX01, TX01ΔhutZ was defective in blocking host macrophage activation. The expression of hutZ Ep was directly regulated by the ferric uptake regulator Fur. This study is the first functional characterization of HutZ in a fish pathogen, and these findings suggested that HutZ Ep is essential for E. piscicida biofilm formation and contributes to host infection.

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Section: Resultsmentioning

confidence: 99%

HutZ is required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida

Shi

Fang

Huang

et al. 2019

Vet Res

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“…At least one additional electron is required to complete the reaction scheme and produce the proposed tetrapyrrole structure that is consistent with the available mass spectroscopy data. 33,36 Initially, our hypothesis was that the iron atom of heme could facilitate additional electron transfer steps or even serve as the source of one electron because under physiological conditions the heme would most likely be in the ferrous state. However, given the catalytic precedent that has been set by several other RS enzymes it is also possible that the mechanism of ring opening is independent of any metal ion.…”

Section: Proposed Reaction Mechanism For Chuwmentioning

confidence: 99%

“…Hence, in a role analogous to biliverdin reductase, it has been shown that ChuY catalyzes the NADPH-dependent reduction of anaerobilin. 36 The parallels to heme oxygenase and biliverdin reductase function are striking and suggest that accumulation of anaerobilin may be toxic to the pathogen. Consistent with this hypothesis, deletion of ChuY from the enterohemorrhagic E. coli CFT073 has been shown to result in a decrease in infectivity of the pathogen.…”

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confidence: 99%

New Insight into the Mechanism of Anaerobic Heme Degradation

et al. 2019

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ChuW, ChuX, and ChuY are contiguous genes downstream from a single promoter that are expressed in the enteric pathogen Escherichia coli O157:H7 when iron is limiting. These genes, and the corresponding proteins, are part of a larger heme uptake and utilization operon that is common to several other enteric pathogens, such as Vibrio cholerae. The aerobic degradation of heme has been well characterized in humans and several pathogenic bacteria, including E. coli O157:H7, but only recently was it shown that ChuW catalyzes the anaerobic degradation of heme to release iron and produce a reactive tetrapyrrole termed "anaerobilin". ChuY has been shown to function as an anaerobilin reductase, in a role that parallels biliverdin reductase. In this work we have employed biochemical and biophysical approaches to further interrogate the mechanism of the anaerobic degradation of heme. We demonstrate that the iron atom of the heme does not participate in the catalytic mechanism of ChuW and that S-adenosyl-L-methionine binding induces conformational changes that favor catalysis. In addition, we show that ChuX and ChuY have synergistic and additive effects on the turnover rate of ChuW. Finally, we have found that ChuS is an effective source of heme or protoporphyrin IX for ChuW under anaerobic conditions. These data indicate that ChuS may have dual functionality in vivo. Specifically, ChuS serves as a heme oxygenase during aerobic metabolism of heme but functions as a cytoplasmic heme storage protein under anaerobic conditions, akin to what has been shown for PhuS (45% sequence identity) from Pseudomonas aeruginosa.

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“…This breakdown also prevents haem toxicity [21]. Under anaerobic conditions, this role is taken up by the SAMmethyltransferase ChuW and the anaerobilin reductase ChuY [22,23].The haem uptake operon is part of a larger regulatory network that allows EHEC to achieve iron homeostasis. Transcription of chuA is controlled by the iron-dependent transcriptional repressor Fur.…”

mentioning

confidence: 99%

“…This breakdown also prevents haem toxicity [21]. Under anaerobic conditions, this role is taken up by the SAMmethyltransferase ChuW and the anaerobilin reductase ChuY [22,23].…”

mentioning

confidence: 99%

Enterohaemorrhagic E. coli utilizes an AND-OR logic gate to regulate expression of an outer membrane haem receptor

Tree

2021

Preprint

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To sense the transition from environment to host, bacteria use a range of environmental cues to control expression of virulence genes. Iron is tightly sequestered in host tissues and in the human pathogen enterohaemorrhagic E. coli (EHEC) iron-limitation induces transcription of the outermembrane haem transporter encoded by chuAS. ChuA expression is post-transcriptionally activated at 37oC by a FourU RNA thermometer ensuring that the haem receptor is only expressed under low iron, high temperature conditions that indicate the host. Here we demonstrate that expression of chuA is also independently regulated by the cAMP-responsive sRNA CyaR and transcriptional terminator Rho. These results indicate that chuA expression is regulated at the transcription initiation, transcript elongation, and translational level. The natural dependence of these processes creates a hierarchy of regulatory AND and OR logic gates that integrate information about the local environment. We show that the logic of the chuA regulatory circuit is activated under conditions that satisfy low iron AND (low glucose OR high temperature). We speculate that additional sensing of a gluconeogenic environment allows further precision in determining when EHEC is at the gastrointestinal epithelium of the host. With previous studies, it appears that the chuA transcript is controlled by eight regulatory inputs that control expression through six different transcriptional and post-transcriptional mechanisms. The results highlight the ability of regulatory sRNAs to integrate multiple environmental signals into a conditional hierarchy of signal input.

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Anaerobic Heme Degradation: ChuY Is an Anaerobilin Reductase That Exhibits Kinetic Cooperativity

Cited by 21 publications

References 38 publications

HutZ is required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida

HutZ is required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida

New Insight into the Mechanism of Anaerobic Heme Degradation

Enterohaemorrhagic E. coli utilizes an AND-OR logic gate to regulate expression of an outer membrane haem receptor

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