Aminofutalosine Deaminase in the Menaquinone Pathway of Helicobacter pylori

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 k cat/K m 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.

MAT Gain of Activity Mutation in Helicobacter pylori Is Associated with Resistance to MTAN Transition State Analogues

“…coli and characterization for FL hydrolysis showed weak catalytic activity ( k cat / K m = 19 M –1 s –1 ) relative to MTAN activity for AFL with a k cat / K m = 1.4 × 10 4 M –1 s –1 (Table S4). Hp MqnB is therefore unlikely to play a catalytic role in menaquinone biosynthesis in H. pylori, confirmed with whole-cell metabolism studies .…”

“…18 However, expression of recombinant HpMqnB in E. coli and character-ization for FL hydrolysis showed weak catalytic activity (k cat / K m = 19 M −1 s −1 ) relative to MTAN activity for AFL with a k cat /K m = 1.4 × 10 4 M −1 s −1 (Table S4). 17 HpMqnB is therefore unlikely to play a catalytic role in menaquinone biosynthesis in H. pylori, confirmed with whole-cell metabolism studies. 17 The AFL deaminase, MqnF, is highly active but has an unknown function in H. pylori and is not induced (Figure 9).…”

“…17 HpMqnB is therefore unlikely to play a catalytic role in menaquinone biosynthesis in H. pylori, confirmed with whole-cell metabolism studies. 17 The AFL deaminase, MqnF, is highly active but has an unknown function in H. pylori and is not induced (Figure 9). 18 Increased mRNA expression from both mqnE and mqnC in BTDIA-resistant H. pylori is significant, as both enzymes catalyze radical SAM-dependent reactions.…”

“…16,20 The addition of DHFL into BTDIA-inhibited H. pylori culture partially reversed growth inhibition, supporting MTAN as a growth inhibition target. 17 However, the incomplete restoration of growth by DHFL indicated that BTDIA or inhibition of MTAN may act at secondary sites. The intracellular concentrations of menaquinone pathway intermediates AFL, FL, and DHFL were compared in native and E99K HpMAT cells cultured at near-IC 50 values for BTDIA (14 nM; Figure 7).…”

“…The metabolic role of MTAN in menaquinone synthesis is a focus here, as supplementation with DHFL only partially overcomes growth inhibition of H. pylori with MTAN inhibitors. 17 Inhibition of MTAN causes accumulation of 5′-dAdo, suggesting a second mechanism of action, the welldocumented product inhibition of 5′-dAdo and methionine on SAM radical reactions, for example, those involved in biotin synthase (BioB), lipoyl synthase (LipA), and tyrosine lyase (ThiH). 25 To these can be added the more recently described radical SAM reactions of MqnE and MqnC in the menaquinone pathway, although these enzymes have not yet been fully characterized.…”