Regulation of a catabolic enzyme whose substrate is always available

ABSTRACT

Certain hydrogenases are capable of converting the trace amounts of molecular hydrogen constantly present in Earth’s atmosphere to supply cells with a little energy during periods of starvation. Recently, Kropp and colleagues investigated the regulation of one of these “high-affinity” hydrogenases in response to different growth conditions for the ubiquitous soil bacterium Mycobacterium smegmatis (A. Kropp, J. D. Archer, M. Jespersen, T. D. Watts, et al., mSystems 11:e01678-25, 2026, https://doi.org/10.1128/msystems.01678-25). A mutation in the gylR gene, whose product acts as a positive transcriptional regulator of the genes involved in glycerol catabolism, causes the corresponding mutant strain to grow very slowly on glycerol, while the activity of the high-affinity hydrogenase reached levels more than 50 times higher than those of the wild-type strain of M. smegmatis. The results of this study suggest that the synthesis of the hydrogenase is subject to a regulatory mechanism similar to carbon catabolite repression, which is entirely consistent with the cellular function of this enzyme.

COMMENTARY

Most microorganisms, especially those that thrive in the soil, possess a range of catabolic enzymes that enable them to utilize the various substrates found on the rarely lavishly set lunch table as sources of energy and carbon. If more than one substrate is available, organisms typically utilize the one that provides the most energy first, while the synthesis of enzymes required to metabolize the less preferred substrate is suppressed to avoid unnecessary costs associated with gene transcription and subsequent translation. The underlying decision-making process is known as “carbon catabolite repression,” which has been well studied in Escherichia coli and Bacillus subtilis, but less so in other microorganisms (1). However, this mechanism is not limited to carbon-based energy sources; it also applies to energy-providing inorganic compounds, such as molecular hydrogen. A model organism that can grow with H₂ as the energy source is Cupriavidus necator, which expresses its H₂-oxidizing enzymes, the [NiFe]-hydrogenases, at temporarily elevated H₂ concentrations. In this organism, hydrogenase gene expression is controlled in an H₂-dependent manner by a specialized regulatory hydrogenase linked to a two-component regulatory system, whose response regulator acts as a transcriptional activator. However, transcription activation takes place only in the absence of carbon/energy sources preferred over H₂ (2, 3). Here, we see the typical two-stage regulation of catabolic enzymes, which involves a specific response to the presence of the substrate (or a downstream catabolic intermediate) and an overarching mechanism of catabolite repression.

But what about catabolic enzymes whose substrate is always present in the same amount?

One interesting example is the so-called “high-affinity” [NiFe]-hydrogenases, which, unlike “conventional” [NiFe]-hydrogenases, are capable of sustaining H₂-oxidation catalysis at very low H₂ concentration (0.4 nM in aqueous solution), as is constantly present in the Earth’s atmosphere (4). These enzymes are widespread in soil microorganisms (encoded in the genomes of 22 prokaryotic phyla) and responsible for the annual breakdown of 70 million tons of atmospheric H₂ (5). Although their catalytic turnover rates are intrinsically low, their H₂ oxidation activity provides starving and dormant microorganisms with enough energy (6) to survive in the long term under extreme conditions of deprivation.

High-affinity [NiFe]-hydrogenases occur in two different phylogenetic flavors designated as Hhy and Huc. The model soil bacterium Mycobacterium smegmatis harbors both enzymes (7). While Huc, whose impressive structure is now available (8), has been shown to have the highest catalytic activity during the transition from growth to dormancy, Hhy supports survival during long-term dormancy. The expression of both enzymes, however, is suppressed in the presence of sufficient amounts of organic carbon sources, such as glucose, glycerol, and organic acids (9), suggesting possible regulation by carbon catabolite control.

In a recent paper in mSystems, Kropp and colleagues set out to identify regulatory proteins that may control the expression of the genes encoding the Huc hydrogenase in response to carbon starvation (10). In their study, they focused on Huc synthesis during growth on glycerol, which is an excellent carbon source for M. smegmatis. The key proteins responsible for glycerol uptake and its conversion into metabolizable dihydroxyacetone phosphate are encoded by the glpFKD operon, whose expression is induced by the transcription activator GylR in the presence of glycerol-3-phosphate (11). The authors observed that in a M. smegmatis gylR mutant, not only was the growth rate on glycerol significantly decreased, but, surprisingly, Huc activity in the exponential growth phase was more than 50 times higher than in the wild-type strain. A control experiment revealed that the gylR mutant grew normally on all other carbon sources tested and that the corresponding Huc activities remained as low as in the wild-type strain, indicating that GylR plays a role only during growth on glycerol. To determine whether GylR directly regulates huc gene expression, the authors employed the classical electrophoretic mobility shift assay, in which they incubated the purified GylR regulator with a DNA fragment containing the huc promoter region. While a clear interaction between GylR and the promoter region of the glpFKD operon was observed, no binding of GylR to the huc promoter was detected, regardless of whether glycerol-3-phosphate was present or not. This result led the authors to conclude that the high Huc activity during growth on glycerol is due to a regulatory effect indirectly caused by the gylR mutation. The underlying regulatory mechanism is likely also responsible for the significant changes in the proteome pattern of the gylR mutant, as revealed by shotgun proteomics analysis. Apart from a high level of Huc hydrogenase components, proteins involved in central catabolism—such as those in the Entner–Doudoroff pathway and the pentose phosphate pathway—were significantly less abundant than in the wild type, whereas components of metabolic pathways assumed to prepare the cells for dormancy were present in higher concentrations (10). All these effects are consistent with a carbon catabolite control mechanism, which is likely triggered by the poor growth on glycerol caused by the gylR mutation.

Interestingly, Huc activity in the gylR mutant was significantly higher during exponential growth on glycerol than in the stationary phase, whereas the opposite effect was observed in the wild-type strain. Furthermore, the shotgun proteomics analysis showed that the second high-affinity hydrogenase from M. smegmatis, Hhy, appeared to be unaffected by the gylR mutation. These observations show that the regulatory processes occurring during the exponential (with low growth rates) and stationary growth phases result in different synthesis rates of high-affinity hydrogenases (10). However, the underlying signals—whether they relate to the concentrations of key metabolic intermediates, the redox status, or other factors—and the associated regulatory proteins, have yet to be identified.

In summary, the expression of the genes encoding the Huc hydrogenase of M. smegmatis is substrate-independent since H₂ is consistently present in an amount that can only be converted by high-affinity hydrogenases. However, the huc gene expression is governed by a carbon catabolite repression mechanism that prevents the energy-intensive synthesis of the enzyme when better energy sources than H₂ are available. Thus, the H₂-based energy conversion capacity of Huc comes into play when the cellular metabolic state becomes critical, and it appears to be precisely the state that prevails in the gylR mutant when growing on glycerol.

A welcome side effect of this study is that the discovery of an M. smegmatis mutant capable of synthesizing large amounts of the catalytically active Huc enzyme provides an attractive basis for enzyme production for biotechnological applications, such as enzymatic fuel cells that run on air alone (12).

The views expressed in this article do not necessarily reflect the views of the journal or of ASM.