The crystal structure of the NADH:quinone oxidoreductase PA1024 has been solved in complex with NAD+to 2.2 Å resolution. The nicotinamide C4 is 3.6 Å from the FMN N5 atom, with a suitable orientation for facile hydride transfer. NAD+binds in a folded conformation at the interface of the TIM‐barrel domain and the extended domain of the enzyme. Comparison of the enzyme‐NAD+structure with that of the ligand‐free enzyme revealed a different conformation of a short loop (75–86) that is part of the NAD+‐binding pocket. P78, P82, and P84 provide internal rigidity to the loop, whereas Q80 serves as an active site latch that secures the NAD+within the binding pocket. An interrupted helix consisting of two α‐helices connected by a small three‐residue loop binds the pyrophosphate moiety of NAD+. The adenine moiety of NAD+appears to π–π stack with Y261. Steric constraints between the adenosine ribose of NAD+, P78, and Q80, control the strict specificity of the enzyme for NADH. Charged residues do not play a role in the specificity of PA1024 for the NADH substrate.
Lactate monooxygenase (LMO) catalyzes the FMN‐dependent “coupled” oxidation of lactate and O2to acetate, carbon dioxide, and water, involving pyruvate and hydrogen peroxide as enzyme‐bound intermediates. Other α‐hydroxy acid oxidase family members follow an “uncoupled pathway,” wherein the α‐keto acid product quickly dissociates before the reduced flavin reacts with oxygen. Here, we report the structures of
This first crystal structure of the FMN‐dependent α‐hydroxy acid oxidase family member lactate monooxygenase (LMO) reveals it has a uniquely large active site lid that we hypothesize is stable enough to explain the slow dissociation of pyruvate that leads to its “coupled” oxidation of lactate and O2to produce acetate, carbon dioxide, and water. Also, the relatively widespread distribution of putative LMOs supports their importance and provides new motivation for their further study.
- NSF-PAR ID:
- 10076845
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 28
- Issue:
- 1
- ISSN:
- 0961-8368
- Page Range / eLocation ID:
- p. 135-149
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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