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Salicylaldehyde dehydrogenase (SALD) catalyses the last reaction in the upper pathway of naphthalene degradation: the oxidation of salicylaldehyde to salicylate. This enzyme has been isolated and studied from a few organisms that belong to the betaproteobacteria and gammaproteobacteria, predominantly Pseudomonas putida. Furthermore, there is only one crystal structure of this enzyme, which was obtained from P. putida G7. Here, crystallographic studies and analysis of the crystal structure of an Alpine soil metagenome-derived SALD (SALDAP) from an alphaproteobacterium are presented. The SALDAP gene was discovered using gene-targeted sequence assembly and it was cloned into a pLATE51 vector. The recombinant protein was overexpressed in Escherichia coli BL21 (DE3) cells and the soluble protein was purified to homogeneity. The protein crystallized at 20°C and diffraction data from the crystals were collected at a resolution of 1.9 Å. The crystal belonged to the orthorhombic space group C2221, with unit-cell parameters a = 116.8, b = 121.7, c = 318.0 Å. Analysis of the crystal structure revealed its conformation to be similar to the organization of the aldehyde dehydrogenase superfamily with three domains: the catalytic, NAD+-binding and bridging domains. The crystal structure of NahF from P. putida G7 was found to be the best structural homologue of SALDAP, even though the enzymes share only 48% amino-acid identity. Interestingly, a carboxylic acid (protocatechuic acid) was found to be a putative ligand of the enzyme and differential scanning fluorimetry was employed to confirm ligand binding. These findings open up the possibility of studying the mechanism(s) of product inhibition and biocatalysis of carboxylic acids using this enzyme and other related aldehyde dehydrogenases.

This research was funded by the ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency (Grant RTI2018-096246-B-I00, to A.L.P., PID2019-110900GBI00 to M.M. and SAF2015-69796 to E.S.), Consejeriia de Economiia, Conocimiento, Empresas y Universidad, Junta de Andalucia (Grant P18-RT-2413, to A.L.P.), and the Government of AragonFEDER (Grant E35-20R to M.M.)., HIF-1 alpha is a master regulator of oxygen homeostasis involved in different stages of cancer development. Thus, HIF-1 alpha inhibition represents an interesting target for anti-cancer therapy. It was recently shown that the HIF-1 alpha interaction with NQO1 inhibits proteasomal degradation of the former, thus suggesting that targeting the stability and/or function of NQO1 could lead to the destabilization of HIF-1 alpha as a therapeutic approach. Since the molecular interactions of NQO1 with HIF-1 alpha are beginning to be unraveled, in this review we discuss: (1) Structure-function relationships of HIF-1 alpha; (2) our current knowledge on the intracellular functions and stability of NQO1; (3) the pharmacological modulation of NQO1 by small ligands regarding function and stability; (4) the potential effects of genetic variability of NQO1 in HIF-1 alpha levels and function; (5) the molecular determinants of NQO1 as a chaperone of many different proteins including cancer-associated factors such as HIF-1 alpha, p53 and p73 alpha. This knowledge is then further discussed in the context of potentially targeting the intracellular stability of HIF-1 alpha by acting on its chaperone, NQO1. This could result in novel anti-cancer therapies, always considering that the substantial genetic variability in NQO1 would likely result in different phenotypic responses among individuals., ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency RTI2018-096246-B-I00 PID2019-110900GBI00 SAF2015-69796, Junta de Andalucia P18-RT-2413, Government of AragonFEDER E35-20R