The metalloprotein YhcH is an anomerase providing N-acetylneuraminate aldolase with the open form of its substrate

Takfarinas Kentache,Leopold Thabault,Gladys Deumer,Vincent Haufroid,Raphaël Frédérick,Carole L Linster,Alessio Peracchi,Maria Veiga-Da-Cunha,Guido T Bommer,Emile Van Schaftingen

doi:10.1016/j.jbc.2021.100699

Abstract

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alpha-anomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate.

Highlights

Most of the enzymes of the classical metabolic pathways are molecularly identified
To verify that αNeu5Ac is the true substrate of Neu5Ac aldolase (NanA), we used a spectrophotometric assay in which the pyruvate formed by this enzyme is reduced by lactate dehydrogenase to Llactate with concomitant consumption of NADH [6] for the assay
Adding E. coli recombinant E. coli Neu5Ac aldolase (NanA) to such an assay containing 200 μM Neu5Ac did not lead to the expected rapid consumption of ≈14 μM NADH (ΔA340 = 0.084 AU), if α-Neu5Ac was rapidly consumed by the aldolase (Fig. 1B)

Summary

Introduction

Most of the enzymes of the classical metabolic pathways are molecularly identified. Yet even in the case of widely studied. Enzymes that are likely to have been understudied (to the major exception of carbonic anhydrases) are those that accelerate spontaneous reactions. Sialoconjugates are abundant in the gastrointestinal and respiratory tracts, where they may interact with bacteria Many of these bacteria have acquired the capacity to metabolize sialoconjugates and/or sialic acids. The first enzymatic step in the degradation of sialic acid is its cleavage by N-acetylneuraminate lyase, an enzyme catalyzing an aldolase type of reaction leading to pyruvate and Nacetylmannosamine formation [4]. The latter is phosphorylated to N-acetylmannosamine-6-phosphate, which is further metabolized by an epimerase. N-acetylneuraminate is metabolized by a similar pathway in mammalian cells [5]

Results

Discussion

Conclusion