Substrate Specificity, Inhibitor Selectivity and Structure-Function Relationships of Aldo-Keto Reductase 1B15: A Novel Human Retinaldehyde Reductase

Abstract

Human aldo-keto reductase 1B15 (AKR1B15) is a newly discovered enzyme which shares 92% amino acid sequence identity with AKR1B10. While AKR1B10 is a well characterized enzyme with high retinaldehyde reductase activity, involved in the development of several cancer types, the enzymatic activity and physiological role of AKR1B15 are still poorly known. Here, the purified recombinant enzyme has been subjected to substrate specificity characterization, kinetic analysis and inhibitor screening, combined with structural modeling. AKR1B15 is active towards a variety of carbonyl substrates, including retinoids, with lower k cat and Km values than AKR1B10. In contrast to AKR1B10, which strongly prefers all-trans-retinaldehyde, AKR1B15 exhibits superior catalytic efficiency with 9-cis-retinaldehyde, the best substrate found for this enzyme. With ketone and dicarbonyl substrates, AKR1B15 also shows higher catalytic activity than AKR1B10. Several typical AKR inhibitors do not significantly affect AKR1B15 activity. Amino acid substitutions clustered in loops A and C result in a smaller, more hydrophobic and more rigid active site in AKR1B15 compared with the AKR1B10 pocket, consistent with distinct substrate specificity and narrower inhibitor selectivity for AKR1B15.

Characterization of AKR1B16, a novel mouse aldo-keto reductase

“…When AKR1B16 was expressed in conventional bacterial systems it formed inclusion bodies and partitioned to the insoluble fraction of cell lysates (data not shown). This was similar to what Salabei et al [10] had reported previously and to what we had observed for AKR1B15.1 [16]. Because AKR1B15.1 could be successfully purified as soluble protein when co-expressed together with three chaperone systems (DnaK-DnaJ-GrpE, ClpB and GroEL-GroES) in E. coli BL21(DE3) [16,34,45], we used the same procedure here for AKR1B16.…”

“…The only unambiguous orthology in the AKR1B subfamily among human and murine species is existing between AKR1B3 (mouse), AKR1B4 (rat) and human aldose reductase (AKR1B1), based on their similar expression pattern and catalytic activity with glucose as a substrate [4,[12][13][14]. AKR1B10 and the closely related protein AKR1B15.1 (92% identity) are not functionally orthologous to either mouse AKR1B7 or AKR1B8 (or their rat orthologs), according to their different tissue distribution and catalytic properties [15,16]. This imposes a limitation on the use of the knockout technology for studying the physiological function of AKR1B10 and AKR1B15 in rodent models.…”

“…The conversion of steroids was analyzed by an HPLC-based method [15]. Briefly, up to 15 µg AKR1B16 or up to 1.5 µg AKR1B15.1, purified as reported [16], were incubated with 10-20 nM 3 H-labeled steroid (estrone…”

Aldo-Keto Reductase Family 1 Member B10 Inhibitors: Potential Drugs for Cancer Treatment

“…AKR1B10 is a 36-kDa cytosolic reductase with a high amino acid sequence identity to AKR1B15 (92%) [3] and AKR1B1 (71%), referred to as aldose reductase (AR) [4]. AKR1B10 displays enzymatic activity for substrates such as retinaldehyde [5, 6], lipid peroxidation products [7-9], and xenobiotics [10, 11].…”

Aldo-Keto Reductase Family 1 Member B10 Inhibitors: Potential Drugs for Cancer Treatment

“…AKR1B10 is a 36-kDa cytosolic reductase with a high amino acid sequence identity to AKR1B15 (92%) [3] and AKR1B1 (71%), referred to as aldose reductase (AR) [4]. AKR1B10 displays enzymatic activity for substrates such as retinaldehyde [5,6], lipid peroxidation products [7][8][9], and xenobiotics [10,11].…”