Lysyl oxidase (LOX) is an extracellular cuproenzyme essential in catalyzing the crosslinkages of collagen and elastin in the extracellular matrix (ECM). The enzyme's localization to the ECM, particularly in cancer cells, makes it a potential target for activated prodrugs that tend to accumulate in the tumor stroma. High LOX expression typically indicates a poor prognosis in colon, breast, prostate, and lung cancer patients. LOX is synthesized as a 50 kDa inactive proenzyme (Pro-LOX) which undergoes extracellular proteolytic processing to form a 29 kDa mature enzyme (LOX) and an 18 kDa propeptide. The LOX propeptide has been shown to inhibit the transforming activity of the H - Ras oncogene in NIH 3T3 fibroblasts; however, only the active and mature form of the LOX enzyme in the ECM is responsible for metastasis in hypoxic cells. This work aims to compare the structure and function of mature LOX and the full-length 50 kDa enzyme. The mature form of lysyl oxidase was overexpressed to a final yield of 28.5 mg and concentrated to 4.39 mg/mL. Enzyme activity was measured at 0.11 U/mg which was 10X higher than previously published LOX activity data. The enzyme incorporated copper to 65%, consistent with published data. Enzyme was crystalized in malic acid with 30-micron crystals being obtained. Efforts are underway to obtain larger crystals. If they diffract to a reasonable resolution, these could lead to the first crystal structure of this enzyme. The full-length enzyme was synthesized by Bio Basic Inc. and cloned into pET21b. This construct was used to overexpress the enzyme to a final yield of 25 mg which was concentrated to 10 mg/mL. Characterization efforts are underway and crystal plates have been set up.

Comparison of mature and full-length lysyl oxidase

ACS Fall 2020 Virtual Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.