RESUMEN
A recombinant L-rhamnose isomerase (L-RhI) from probiotic Lactobacillus rhamnosus Probio-M9 (L. rhamnosus Probio-M9) was expressed. L. rhamnosus Probio-M9 was isolated from human colostrum and identified as a probiotic lactic acid bacterium, which can grow using L-rhamnose. L-RhI is one of the enzymes involved in L-rhamnose metabolism and catalyzes the reversible isomerization between L-rhamnose and L-rhamnulose. Some L-RhIs were reported to catalyze isomerization not only between L-rhamnose and L-rhamnulose but also between D-allulose and D-allose, which are known as rare sugars. Those L-RhIs are attractive enzymes for rare sugar production and have the potential to be further improved by enzyme engineering; however, the known crystal structures of L-RhIs recognizing rare sugars are limited. In addition, the optimum pH levels of most reported L-RhIs are basic rather than neutral, and such a basic condition causes non-enzymatic aldose-ketose isomerization, resulting in unexpected by-products. Herein, we report the crystal structures of L. rhamnosus Probio-M9 L-RhI (LrL-RhI) in complexes with L-rhamnose, D-allulose, and D-allose, which show enzyme activity toward L-rhamnose, D-allulose, and D-allose in acidic conditions, though the activity toward D-allose was low. In the complex with L-rhamnose, L-rhamnopyranose was found in the catalytic site, showing favorable recognition for catalysis. In the complex with D-allulose, D-allulofuranose and ring-opened D-allulose were observed in the catalytic site. However, bound D-allose in the pyranose form was found in the catalytic site of the complex with D-allose, which was unfavorable for recognition, like an inhibition mode. The structure of the complex may explain the low activity toward D-allose. KEY POINTS: ⢠Crystal structures of LrL-RhI in complexes with substrates were determined. ⢠LrL-RhI exhibits enzyme activity toward L-rhamnose, D-allulose, and D-allose. ⢠The LrL-RhI is active in acidic conditions.
Asunto(s)
Isomerasas Aldosa-Cetosa , Lacticaseibacillus rhamnosus , Humanos , Rayos X , Ramnosa , MonosacáridosRESUMEN
Emerging evidence supports that the efficacy of immune checkpoint blockade (ICB) therapy is associated with the host's gut microbiota, as prior antibiotic intake often leads to poor outcome and low responsiveness toward ICB treatment. Therefore, we hypothesized that the efficacy of ICB therapy like anti-programmed cell death protein-1 (PD-1) treatment required an intact host gut microbiota, and it was established that probiotics could enhance the recovery of gut microbiota disruption by external stimuli. Thus, the present study aimed to evaluate the effect of the probiotics, Lactobacillus rhamnosus Probio-M9, on recovering antibiotic-disrupted gut microbiota and its impact on the outcome of ICB therapy in tumor-bearing mice. We first disrupted the mouse microbiota by antibiotics and then remediated the gut microbiota by probiotics or naturally. Tumor transplantation was then performed, followed by anti-PD-1-based antitumor therapy. Changes in the fecal metagenomes and the tumor suppression effect were monitored during different stages of the experiment. Our results showed that Probio-M9 synergized with ICB therapy, significantly improving tumor inhibition compared with groups not receiving the probiotic treatment (P < 0.05 at most time points). The synergistic effect was accompanied by effective restoration of antibiotic-disrupted fecal microbiome that was characterized by a drastically reduced Shannon diversity value and shifted composition of dominating taxa. Moreover, probiotic administration significantly increased the relative abundance of beneficial bacteria (e.g., Bifidobacterium pseudolongum, Parabacteroides distasonis, and some Bacteroides species; 0.0001 < P < 0.05). The gut microbiome changes were accompanied by mild reshaping of the functional metagenomes characterized by enrichment in sugar degradation and vitamin and amino acid synthesis pathways. Collectively, this study supported that probiotic administration could enhance the efficacy and responsiveness of anti-PD-1-based immunotherapy, and Probio-M9 could be a potential candidate of microbe-based synergistic tumor therapeutics. The preclinical data obtained here would support the design of future human clinical trials for further consolidating the current findings and for safety assessment of probiotic adjunctive treatment in ICB therapy.