Aminofutalosine Deaminase in the Menaquinone Pathway of Helicobacter pylori
Mu Feng1, Rajesh K Harijan1, Lawrence D Harris2
1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States.
Abstract:
Helicobacter pylori is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. H. pylori uses the unusual mqn pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (HpMTAN) is the direct path for producing downstream metabolites in the mqn pathway. However, genomic analysis indicates jhp0252 is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (HpAFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S-adenosylhomocysteine. The kcat/Km value for AFL is 6.8 × 104 M-1 s-1, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for HpAFLDA and demonstrated inhibitory effects on H. pylori growth. Supplementation with futalosine partially restored H. pylori growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-HpAFLDA and with MTCF at the catalytic sites show a catalytic site Zn2+ or Fe2+ as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp3 hydroxyl group of the transition state analogue. Metabolomics analysis revealed that HpAFLDA has intracellular activity and is inhibited by MTCF. The mqn pathway in H. pylori bifurcates at aminofutalosine with HpMTAN producing adenine and depurinated futalosine and HpAFLDA producing futalosine. Inhibition of cellular HpMTAN or HpAFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.
Insights
Helicobacter pylori uses a unique pathway for menaquinone biosynthesis. Researchers identified a novel enzyme, HpAFLDA, crucial for this pathway, and its inhibitor shows potential for H. pylori growth inhibition.
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Helicobacter pylori, a Gram-negative bacterium, is a primary cause of gastric and duodenal ulcers.
- H. pylori employs the mqn pathway for menaquinone biosynthesis, utilizing aminofutalosine (AFL) as a key intermediate.
- Previously, 5'-methylthioadenosine nucleosidase (HpMTAN) was considered the sole enzyme for AFL hydrolysis in this pathway.
Purpose of the Study:
- To investigate the role of the putative aminofutalosine deaminase (AFLDA) encoded by jhp0252 in H. pylori menaquinone biosynthesis.
- To characterize the enzymatic activity, substrate specificity, and structural properties of HpAFLDA.
- To evaluate the potential of HpAFLDA as a therapeutic target by assessing the effects of its inhibition on H. pylori growth.
Main Methods:
- Recombinant expression and biochemical characterization of HpAFLDA.
- Determination of kinetic parameters (kcat/Km) for AFL and related substrates.
- Crystallographic analysis of apo-HpAFLDA and its complex with the inhibitor MTCF.
- Metabolomics analysis to assess intracellular enzyme activity and pathway flux under inhibitor treatment.
Main Results:
- jhp0252 encodes an active aminofutalosine deaminase (HpAFLDA) with significant catalytic efficiency for AFL.
- HpAFLDA exhibits broad substrate specificity, including 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S-adenosylhomocysteine.
- The inhibitor 5'-methylthiocoformycin (MTCF) effectively inhibits HpAFLDA and H. pylori growth, with futalosine partially restoring growth.
- Crystal structures reveal a catalytic metal ion (Zn2+ or Fe2+) essential for enzyme activity.
- Metabolomics confirm intracellular HpAFLDA activity and inhibition by MTCF, leading to decreased menaquinone-6 levels.
Conclusions:
- The mqn pathway in H. pylori bifurcates at AFL, with both HpMTAN and HpAFLDA playing significant roles in menaquinone biosynthesis.
- HpAFLDA is a validated target, and its inhibition, along with HpMTAN, impacts menaquinone-6 production.
- Inhibitors of HpAFLDA, such as MTCF, demonstrate potential as therapeutic agents against H. pylori infections.
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