Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen.

NAD-reducing [NiFe] hydrogenases are valuable biocatalysts for H-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O and elevated temperatures, the soluble NAD-reducing [NiFe] hydrogenase from (SH) is O-tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications.

A hydrogen-driven biocatalytic approach to recycling synthetic analogues of NAD(P)H.

We demonstrate a recycling system for synthetic nicotinamide cofactor analogues using a soluble hydrogenase with turnover number of >1000 for reduction of the cofactor analogues by H. Coupling this system to an ene reductase, we show quantitative conversion of -ethylmaleimide to -ethylsuccinimide. The biocatalyst system retained >50% activity after 7 h.

Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H-driven NAD-reduction in the presence of O.

Biocatalysts that mediate the H-dependent reduction of NAD to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD-reducing [NiFe]‑hydrogenase that sustains catalytic activity at high temperatures and in the presence of O, which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD-reducing [NiFe]‑hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1 (Ht).