Title: Modification of Rhizopus lactate dehydrogenase for improved resistance to fructose 1,6-bisphosphate Authors
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 3, 2012
Publication Date: May 3, 2012
Citation: Skory, C.D., Rich, J.O. 2012. Nodification of Rhizopus lactate dehydrogenase for improved resistance to fructose 1,6-bisphosphate [abstract]. Symposium on Biotechnology for Fuels and Chemicals. Paper #9-22. Technical Abstract: Rhizopus oryzae is frequently used for fermentative production of lactic acid. We determined that one of the key enzymes, lactate dehydrogenase (LDH), involved in synthesis of lactic acid by R. oryzae was significantly inhibited by fructose 1,6-bisphosphate (FBP) at physiological concentrations. This glycolytic intermediate accumulates in the presence of excess glucose and serves as an allosteric regulator to stimulate LDH function in certain bacterial isolates; however, it is generally accepted that most LDH are indifferent to the presence of FBP. Allosteric activation involves the formation of salt bridges with phosphate moieties of bound FBP with amino acids H188 and R173, which are highly conserved in most LDH. These amino acids are present in R. oryzae LDH, but a closely related LDH from R. delemar containing an R173C substitution had more than a 24-fold increased resistance to FBP. We hypothesized that performing a similar replacement in R. oryzae LDH may confer this same resistance to FBP and result in improved activity. While this modification increased Ki for FBP >10-fold, it also decreased Vmax of the enzyme by 65%. Therefore, we performed several other site-directed changes of amino acids involved in stability of the P-axis interface of the LDH tetramer in the presence of FBP. Modified enzymes R173Q, H188D, H188Y, and H188Y/R173Q (double mutation) all showed >10-fold improved resistance to FBP compared to wild type. R173Q was chosen for in vivo studies because it showed <10% decrease in Vmax with >20-fold increase in Ki. Results of fermentation studies will be presented.